Nuova ricerca

Serena CARRA

Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Home | Curriculum(pdf) | Didattica |


Pubblicazioni

2024 - SerpinA1 levels in amyotrophic lateral sclerosis patients: An exploratory study [Articolo su rivista]
Martinelli, Ilaria; Zucchi, Elisabetta; Simonini, Cecilia; Gianferrari, Giulia; Bedin, Roberta; Biral, Chiara; Ghezzi, Andrea; Fini, Nicola; Carra, Serena; Mandrioli, Jessica
abstract

Background: SerpinA1, a serine protease inhibitor, is involved in the modulation of microglial-mediated inflammation in neurodegenerative diseases. We explored SerpinA1 levels in cerebrospinal fluid (CSF) and serum of amyotrophic lateral sclerosis (ALS) patients to understand its potential role in the pathogenesis of the disease. Methods: SerpinA1, neurofilament light (NfL) and heavy (NfH) chain, and chitinase-3-like protein-1 (CHI3L1) were determined in CSF and serum of ALS patients (n = 110) and healthy controls (n = 10) (automated next-generation ELISA), and correlated with clinical parameters, after identifying three classes of progressors (fast, intermediate, slow). Biomarker levels were analyzed for diagnostic power and association with progression and survival. Results: SerpinA1serum was significantly decreased in ALS (median: 1032 μg/mL) compared with controls (1343 μg/mL) (p = 0.02). SerpinA1CSF was elevated only in fast progressors (8.6 μg/mL) compared with slow (4.43 μg/mL, p = 0.01) and intermediate (4.42 μg/mL, p = 0.03) progressors. Moreover, SerpinA1CSF correlated with neurofilament and CHI3L1 levels in CSF. Contrarily to SerpinA1CSF , neurofilament and CHI3L1 concentrations in CSF correlated with measures of disease progression in ALS, while SerpinA1serum mildly related with time to generalization (rho = 0.20, p = 0.04). In multivariate analysis, the ratio between serum and CSF SerpinA1 (SerpinA1 ratio) and NfHCSF were independently associated with survival. Conclusions: Higher SerpinA1CSF levels are found in fast progressors, suggesting SerpinA1 is a component of the neuroinflammatory mechanisms acting upon fast-progressing forms of ALS. Both neurofilaments or CHI3L1CSF levels outperformed SerpinA1 at predicting disease progression rate in our cohort, and so the prognostic value of SerpinA1 alone as a measure remains inconclusive.

2023 - A shared fate for nuclear and cytosolic inclusions [Articolo su rivista]
Alberti, Simon; Carra, Serena
abstract

Coordination of protein quality control processes across organelles is poorly understood. A study now shows that the cytosolic juxtanuclear quality control (JUNQ) and intranuclear quality control (INQ) compartments face each other on opposite sides of the nuclear envelope before their vacuolar degradation, promoting proteostasis.

2023 - Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis [Articolo su rivista]
Gönczi, Mónika; Teixeira, João M C; Barrera-Vilarmau, Susana; Mediani, Laura; Antoniani, Francesco; Nagy, Tamás Milán; Fehér, Krisztina; Ráduly, Zsolt; Ambrus, Viktor; Tőzsér, József; Barta, Endre; Kövér, Katalin E; Csernoch, László; Carra, Serena; Fuxreiter, Monika
abstract

: During muscle cell differentiation, the alternatively spliced, acidic β-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the β-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid β-domain variants, as well as by a disordered β-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the β-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that β-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process.

2023 - Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process [Articolo su rivista]
Choudhary, Dhawal; Mediani, Laura; Avellaneda, Mario J; Bjarnason, Sveinn; Alberti, Simon; Boczek, Edgar E; Heidarsson, Pétur O; Mossa, Alessandro; Carra, Serena; Tans, Sander J; Cecconi, Ciro
abstract

: Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

2023 - Tardigrade small heat shock proteins can limit desiccation-induced protein aggregation [Articolo su rivista]
Hibshman, Jonathan D; Carra, Serena; Goldstein, Bob
abstract

Small heat shock proteins (sHSPs) are chaperones with well-characterized roles in heat stress, but potential roles for sHSPs in desiccation tolerance have not been as thoroughly explored. We identified nine sHSPs from the tardigrade Hypsibius exemplaris, each containing a conserved alpha-crystallin domain flanked by disordered regions. Many of these sHSPs are highly expressed. Multiple tardigrade and human sHSPs could improve desiccation tolerance of E. coli, suggesting that the capacity to contribute to desicco-protection is a conserved property of some sHSPs. Purification and subsequent analysis of two tardigrade sHSPs, HSP21 and HSP24.6, revealed that these proteins can oligomerize in vitro. These proteins limited heat-induced aggregation of the model enzyme citrate synthase. Heterologous expression of HSP24.6 improved bacterial heat shock survival, and the protein significantly reduced heat-induced aggregation of soluble bacterial protein. Thus, HSP24.6 likely chaperones against protein aggregation to promote heat tolerance. Furthermore, HSP21 and HSP24.6 limited desiccation-induced aggregation and loss of function of citrate synthase. This suggests a mechanism by which tardigrade sHSPs promote desiccation tolerance, by limiting desiccation-induced protein aggregation, thereby maintaining proteostasis and supporting survival. These results suggest that sHSPs provide a mechanism of general stress resistance that can also be deployed to support survival during anhydrobiosis.Small heat shock proteins from the tardigrade Hypsibius exemplaris are shown to provide a mechanism of stress resistance that can support not just heat tolerance but desiccation tolerance as well.

2023 - Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly [Articolo su rivista]
Kassouf, Toufic; Shrivastava, Rohit; Meszka, Igor; Bailly, Aymeric; Polanowska, Jolanta; Trauchessec, Helene; Mandrioli, Jessica; Carra, Serena; Xirodimas, Dimitris P
abstract

The elimination of aberrant inclusions is regarded as a therapeutic approach in neurodegeneration. In amyotro-phic lateral sclerosis (ALS), mutations in proteins found within cytoplasmic condensates called stress granules (SGs) are linked to the formation of pathological SGs, aberrant protein inclusions, and neuronal toxicity. We found that inhibition of NEDP1, the enzyme that processes/deconjugates the ubiquitin-like molecule NEDD8, promotes the disassembly of physiological and pathological SGs. Reduction in poly(ADP-ribose) polymerase1 activity through hyper-NEDDylation is a key mechanism for the observed phenotype. These effects are related to improved cell survival in human cells, and in C. elegans, nedp1 deletion ameliorates ALS phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, correlated to the disassembly of pathological SGs.

2023 - The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins [Articolo su rivista]
Ecroyd, Heath; Bartelt-Kirbach, Britta; Ben-Zvi, Anat; Bonavita, Raffaella; Bushman, Yevheniia; Casarotto, Elena; Cecconi, Ciro; Lau, Wilson Chun Yu; Hibshman, Jonathan D; Joosten, Joep; Kimonis, Virginia; Klevit, Rachel; Liberek, Krzysztof; Mcmenimen, Kathryn A; Miwa, Tsukumi; Mogk, Axel; Montepietra, Daniele; Peters, Carsten; Rocchetti, Maria Teresa; Saman, Dominik; Sisto, Angela; Secco, Valentina; Strauch, Annika; Taguchi, Hideki; Tanguay, Morgan; Tedesco, Barbara; Toth, Melinda E; Wang, Zihao; Benesch, Justin L P; Carra, Serena
abstract

: The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.

2022 - Case report: p.Glu134del SOD1 mutation in two apparently unrelated ALS patients with mirrored phenotype [Articolo su rivista]
Gianferrari, Giulia; Martinelli, Ilaria; Simonini, Cecilia; Zucchi, Elisabetta; Fini, Nicola; Carra, Serena; Moglia, Cristina; Mandrioli, Jessica
abstract

: With upcoming personalized approaches based on genetics, it is important to report new mutations in amyotrophic lateral sclerosis (ALS) genes in order to understand their pathogenicity and possible patient responses to specific therapies. SOD1 mutations are the second most frequent genetic cause of ALS in European populations. Here, we describe two seemingly unrelated Italian patients with ALS carrying the same SOD1 heterozygous c.400_402 deletion (p.Glu134del). Both patients had spinal onset in their lower limbs, progressive muscular weakness with respiratory involvement, and sparing bulbar function. In addition to the clinical picture, we discuss the possible pathogenic role of this unfamiliar SOD1 mutation.

2022 - Missense mutation in ATXN2 gene (c.2860C > T) in an amyotrophic lateral sclerosis patient with aggressive disease phenotype [Articolo su rivista]
Ghezzi, A.; Martinelli, I.; Carra, S.; Mediani, L.; Zucchi, E.; Simonini, C.; Gianferrari, G.; Fini, N.; Cereda, C.; Gellera, C.; Pensato, V.; Mandrioli, J.
abstract

Background: ALS symptoms have been previously described only in the context of ATXN2 CAG expansions, whereas missense mutations of the gene have never been described in ALS patients. Case presentation: We identified a novel missense mutation (c.2860C > T) of ATXN2, for which in silico analysis showed a possible pathogenic effect on protein expression, in a patient presenting an aggressive disease phenotype. Discussion: Our findings raise the possibility for unknown genetic factors interacting with ATXN2 mutations, or for an autonomous pathogenic role for this specific point mutation in ATXN2 gene in driving the clinical phenotype toward ALS. We also found that stress granules in the fibroblasts from the patient entrapped higher amounts of defective ribosomal products compared to fibroblasts from three healthy subjects, suggesting that ATXN2 mutation-related toxicity may have implication in protein quality control.

2022 - Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells [Articolo su rivista]
Ferrari, V.; Cristofani, R.; Cicardi, M. E.; Tedesco, B.; Crippa, V.; Chierichetti, M.; Casarotto, E.; Cozzi, M.; Mina, F.; Galbiati, M.; Piccolella, M.; Carra, S.; Vaccari, T.; Nalbandian, A.; Kimonis, V.; Fortuna, T. R.; Pandey, U. B.; Gagliani, M. C.; Cortese, K.; Rusmini, P.; Poletti, A.
abstract

Aim: Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. Methods: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. Results: We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. Conclusion: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

2022 - RNA Molecular Signature Profiling in PBMCs of Sporadic ALS Patients: HSP70 Overexpression Is Associated with Nuclear SOD1 [Articolo su rivista]
Garofalo, M.; Pandini, C.; Bordoni, M.; Jacchetti, E.; Diamanti, L.; Carelli, S.; Raimondi, M. T.; Sproviero, D.; Crippa, V.; Carra, S.; Poletti, A.; Pansarasa, O.; Gagliardi, S.; Cereda, C.
abstract

Superoxide dismutase 1 (SOD1) is one of the causative genes associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder. SOD1 aggregation contributes to ALS patho-genesis. A fraction of the protein is localized in the nucleus (nSOD1), where it seems to be involved in the regulation of genes participating in the oxidative stress response and DNA repair. Peripheral blood mononuclear cells (PBMCs) were collected from sporadic ALS (sALS) patients (n = 18) and healthy controls (n = 12) to perform RNA-sequencing experiments and differential expression analysis. Patients were stratified into groups with “high” and “low” levels of nSOD1. We obtained different gene expression patterns for high-and low-nSOD1 patients. Differentially expressed genes in high nSOD1 form a cluster similar to controls compared to the low-nSOD1 group. The pathways activated in high-nSOD1 patients are related to the upregulation of HSP70 molecular chaperones. We demonstrated that, in this condition, the DNA damage is reduced, even under oxidative stress conditions. Our findings highlight the importance of the nuclear localization of SOD1 as a protective mechanism in sALS patients.

2022 - Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report [Articolo su rivista]
Cable, J.; Weber-Ban, E.; Clausen, T.; Walters, K. J.; Sharon, M.; Finley, D. J.; Gu, Y.; Hanna, J.; Feng, Y.; Martens, S.; Simonsen, A.; Hansen, M.; Zhang, H.; Goodwin, J. M.; Reggio, A.; Chang, C.; Ge, L.; Schulman, B. A.; Deshaies, R. J.; Dikic, I.; Harper, J. W.; Wertz, I. E.; Thoma, N. H.; Slabicki, M.; Frydman, J.; Jakob, U.; David, D. C.; Bennett, E. J.; Bertozzi, C. R.; Sardana, R.; Eapen, V. V.; Carra, S.
abstract

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome–lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium “Targeting protein degradation: from small molecules to complex organelles.” The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

2021 - Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) [Articolo su rivista]
Klionsky, D. J.; Abdel-Aziz, A. K.; Abdelfatah, S.; Abdellatif, M.; Abdoli, A.; Abel, S.; Abeliovich, H.; Abildgaard, M. H.; Abudu, Y. P.; Acevedo-Arozena, A.; Adamopoulos, I. E.; Adeli, K.; Adolph, T. E.; Adornetto, A.; Aflaki, E.; Agam, G.; Agarwal, A.; Aggarwal, B. B.; Agnello, M.; Agostinis, P.; Agrewala, J. N.; Agrotis, A.; Aguilar, P. V.; Ahmad, S. T.; Ahmed, Z. M.; Ahumada-Castro, U.; Aits, S.; Aizawa, S.; Akkoc, Y.; Akoumianaki, T.; Akpinar, H. A.; Al-Abd, A. M.; Al-Akra, L.; Al-Gharaibeh, A.; Alaoui-Jamali, M. A.; Alberti, S.; Alcocer-Gomez, E.; Alessandri, C.; Ali, M.; Alim Al-Bari, M. A.; Aliwaini, S.; Alizadeh, J.; Almacellas, E.; Almasan, A.; Alonso, A.; Alonso, G. D.; Altan-Bonnet, N.; Altieri, D. C.; Alvarez, E. M. C.; Alves, S.; Alves da Costa, C.; Alzaharna, M. M.; Amadio, M.; Amantini, C.; Amaral, C.; Ambrosio, S.; Amer, A. O.; Ammanathan, V.; An, Z.; Andersen, S. U.; Andrabi, S. A.; Andrade-Silva, M.; Andres, A. M.; Angelini, S.; Ann, D.; Anozie, U. C.; Ansari, M. Y.; Antas, P.; Antebi, A.; Anton, Z.; Anwar, T.; Apetoh, L.; Apostolova, N.; Araki, T.; Araki, Y.; Arasaki, K.; Araujo, W. L.; Araya, J.; Arden, C.; Arevalo, M. -A.; Arguelles, S.; Arias, E.; Arikkath, J.; Arimoto, H.; Ariosa, A. R.; Armstrong-James, D.; Arnaune-Pelloquin, L.; Aroca, A.; Arroyo, D. S.; Arsov, I.; Artero, R.; Asaro, D. M. L.; Aschner, M.; Ashrafizadeh, M.; Ashur-Fabian, O.; Atanasov, A. G.; Au, A. K.; Auberger, P.; Auner, H. W.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avalos, Y.; Aveic, S.; Aveleira, C. A.; Avin-Wittenberg, T.; Aydin, Y.; Ayton, S.; Ayyadevara, S.; Azzopardi, M.; Baba, M.; Backer, J. M.; Backues, S. K.; Bae, D. -H.; Bae, O. -N.; Bae, S. H.; Baehrecke, E. H.; Baek, A.; Baek, S. -H.; Baek, S. H.; Bagetta, G.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X.; Bai, Y.; Bairagi, N.; Baksi, S.; Balbi, T.; Baldari, C. T.; Balduini, W.; Ballabio, A.; Ballester, M.; Balazadeh, S.; Balzan, R.; Bandopadhyay, R.; Banerjee, S.; Banerjee, S.; Banreti, A.; Bao, Y.; Baptista, M. S.; Baracca, A.; Barbati, C.; Bargiela, A.; Barila, D.; Barlow, P. G.; Barmada, S. J.; Barreiro, E.; Barreto, G. E.; Bartek, J.; Bartel, B.; Bartolome, A.; Barve, G. R.; Basagoudanavar, S. H.; Bassham, D. C.; Bast, R. C.; Basu, A.; Batoko, H.; Batten, I.; Baulieu, E. E.; Baumgarner, B. L.; Bayry, J.; Beale, R.; Beau, I.; Beaumatin, F.; Bechara, L. R. G.; Beck, G. R.; Beers, M. F.; Begun, J.; Behrends, C.; Behrens, G. M. N.; Bei, R.; Bejarano, E.; Bel, S.; Behl, C.; Belaid, A.; Belgareh-Touze, N.; Bellarosa, C.; Belleudi, F.; Bello Perez, M.; Bello-Morales, R.; Beltran, J. S. D. O.; Beltran, S.; Benbrook, D. M.; Bendorius, M.; Benitez, B. A.; Benito-Cuesta, I.; Bensalem, J.; Berchtold, M. W.; Berezowska, S.; Bergamaschi, D.; Bergami, M.; Bergmann, A.; Berliocchi, L.; Berlioz-Torrent, C.; Bernard, A.; Berthoux, L.; Besirli, C. G.; Besteiro, S.; Betin, V. M.; Beyaert, R.; Bezbradica, J. S.; Bhaskar, K.; Bhatia-Kissova, I.; Bhattacharya, R.; Bhattacharya, S.; Bhattacharyya, S.; Bhuiyan, M. S.; Bhutia, S. K.; Bi, L.; Bi, X.; Biden, T. J.; Bijian, K.; Billes, V. A.; Binart, N.; Bincoletto, C.; Birgisdottir, A. B.; Bjorkoy, G.; Blanco, G.; Blas-Garcia, A.; Blasiak, J.; Blomgran, R.; Blomgren, K.; Blum, J. S.; Boada-Romero, E.; Boban, M.; Boesze-Battaglia, K.; Boeuf, P.; Boland, B.; Bomont, P.; Bonaldo, P.; Bonam, S. R.; Bonfili, L.; Bonifacino, J. S.; Boone, B. A.; Bootman, M. D.; Bordi, M.; Borner, C.; Bornhauser, B. C.; Borthakur, G.; Bosch, J.; Bose, S.; Botana, L. M.; Botas, J.; Boulanger, C. M.; Boulton, M. E.; Bourdenx, M.; Bourgeois, B.; Bourke, N. M.; Bousquet, G.; Boya, P.; Bozhkov, P. V.; Bozi, L. H. M.; Bozkurt, T. O.; Brackney, D. E.; Brandts, C. H.; Braun, R. J.; Braus, G. H.; Bravo-Sagua, R.; Bravo-San Pedro, J. M.; Brest, P.; Bringer, M. -A.; Briones-Herrera, A.; Broaddus, V. C.; Brodersen, P.; Brodsky, J. L.; Brody, S. L.; Bronson, P. G.; Bronstein, J. M.; Brown, C. N.; Brown, R. E.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bruno, D.;
abstract

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

2021 - Hsp90-mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling [Articolo su rivista]
Mediani, L.; Antoniani, F.; Galli, V.; Vinet, J.; Carra, A. D.; Bigi, I.; Tripathy, V.; Tiago, T.; Cimino, M.; Leo, G.; Amen, T.; Kaganovich, D.; Cereda, C.; Pansarasa, O.; Mandrioli, J.; Tripathi, P.; Troost, D.; Aronica, E.; Buchner, J.; Goswami, A.; Sterneckert, J.; Alberti, S.; Carra, S.
abstract

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.

2021 - HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain [Articolo su rivista]
Boczek, E. E.; Fursch, J.; Niedermeier, M. L.; Jawerth, L.; Jahnel, M.; Ruer-Gruss, M.; Kammer, K. -M.; Heid, P.; Mediani, L.; Wang, J.; Yan, X.; Pozniakovski, A.; Poser, I.; Mateju, D.; Hubatsch, L.; Carra, S.; Alberti, S.; Hyman, A. A.; Stengel, F.
abstract

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.

2021 - Protein products of nonstop mRNA disrupt nucleolar homeostasis [Articolo su rivista]
Davis, Z. H.; Mediani, L.; Antoniani, F.; Vinet, J.; Li, S.; Alberti, S.; Lu, B.; Holehouse, A. S.; Carra, S.; Brandman, O.
abstract

Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.

2021 - Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor [Articolo su rivista]
Tiago, T.; Hummel, B.; Morelli, F. F.; Basile, V.; Vinet, J.; Galli, V.; Mediani, L.; Antoniani, F.; Pomella, S.; Cassandri, M.; Garone, M. G.; Silvestri, B.; Cimino, M.; Cenacchi, G.; Costa, R.; Mouly, V.; Poser, I.; Yeger-Lotem, E.; Rosa, A.; Alberti, S.; Rota, R.; Ben-Zvi, A.; Sawarkar, R.; Carra, S.
abstract

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

2021 - The landscape of molecular chaperones across human tissues reveals a layered architecture of core and variable chaperones [Articolo su rivista]
Shemesh, N.; Jubran, J.; Dror, S.; Simonovsky, E.; Basha, O.; Argov, C.; Hekselman, I.; Abu-Qarn, M.; Vinogradov, E.; Mauer, O.; Tiago, T.; Carra, S.; Ben-Zvi, A.; Yeger-Lotem, E.
abstract

The sensitivity of the protein-folding environment to chaperone disruption can be highly tissue-specific. Yet, the organization of the chaperone system across physiological human tissues has received little attention. Through computational analyses of large-scale tissue transcriptomes, we unveil that the chaperone system is composed of core elements that are uniformly expressed across tissues, and variable elements that are differentially expressed to fit with tissue-specific requirements. We demonstrate via a proteomic analysis that the muscle-specific signature is functional and conserved. Core chaperones are significantly more abundant across tissues and more important for cell survival than variable chaperones. Together with variable chaperones, they form tissue-specific functional networks. Analysis of human organ development and aging brain transcriptomes reveals that these functional networks are established in development and decline with age. In this work, we expand the known functional organization of de novo versus stress-inducible eukaryotic chaperones into a layered core-variable architecture in multi-cellular organisms.

2020 - ALS and FTD: Where RNA metabolism meets protein quality control [Articolo su rivista]
Mandrioli, J.; Mediani, L.; Alberti, S.; Carra, S.
abstract

Recent genetic and biochemical evidence has improved our understanding of the pathomechanisms that lead to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative diseases with overlapping symptoms and causes. Impaired RNA metabolism, enhanced aggregation of protein-RNA complexes, aberrant formation of ribonucleoprotein (RNP) granules and dysfunctional protein clearance via autophagy are emerging as crucial events in ALS/FTD pathogenesis. Importantly, these processes interact at the molecular level, converging on a common pathogenic cascade. In this review, we summarize key principles underlying ALS and FTD, and we discuss how mutations in genes involved in RNA metabolism, protein quality control and protein degradation meet mechanistically to impair the functionality and dynamics of RNP granules, and how this leads to cellular toxicity and death. Finally, we describe recent advances in understanding signaling pathways that become dysfunctional in ALS/FTD, partly due to altered RNP granule dynamics, but also with stress granule-independent mechanisms and, thus could be promising targets for future therapeutic intervention.

2020 - BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes [Articolo su rivista]
Adriaenssens, E.; Tedesco, B.; Mediani, L.; Asselbergh, B.; Crippa, V.; Antoniani, F.; Carra, S.; Poletti, A.; Timmerman, V.
abstract

Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.

2020 - BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes [Articolo su rivista]
Mediani, L.; Galli, V.; Carra, A. D.; Bigi, I.; Vinet, J.; Ganassi, M.; Antoniani, F.; Tiago, T.; Cimino, M.; Mateju, D.; Cereda, C.; Pansarasa, O.; Alberti, S.; Mandrioli, J.; Carra, S.
abstract

Stress granules (SGs) are dynamic ribonucleoprotein granules induced by environmental stresses. They play an important role in the stress response by integrating mRNA stability, translation, and signaling pathways. Recent work has connected SG dysfunction to neurodegenerative diseases. In these diseases, SG dynamics are impaired because of mutations in SG proteins or protein quality control factors. Impaired SG dynamics and delayed SG dissolution have also been observed for SGs that accumulate misfolding-prone defective ribosomal products (DRiPs). DRiP accumulation inside SGs is controlled by a surveillance system referred to as granulostasis and encompasses the molecular chaperones VCP and the HSPB8-BAG3-HSP70 complex. BAG3 is a member of the BAG family of proteins, which includes five additional members. One of these proteins, BAG6, is functionally related to BAG3 and able to assist degradation of DRiPs. However, whether BAG6 is involved in granulostasis is unknown. We report that BAG6 is not recruited into SGs induced by different types of stress, nor does it affect SG dynamics. BAG6 also does not replace BAG3’s function in SG granulostasis. We show that BAG3 and BAG6 target different subsets of DRiPs, and BAG3 binding to DRiPs is mediated by HSPB8 and HSP70. Our data support the idea that SGs are sensitive to BAG3-HSP70-bound DRiPs but not to BAG6-bound DRiPs. Additionally, only BAG3 is strongly upregulated in the stress recovery phase, when SGs dissolve. These data exclude a role for BAG6 in granulostasis and point to a more specialized function in the clearance of a specific subset of DRiPs.

2020 - Studying heat shock proteins through single-molecule mechanical manipulation [Articolo su rivista]
Choudhary, D.; Mediani, L.; Carra, S.; Cecconi, C.
abstract

Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as “sponges” that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed.

2019 - Autophagic and proteasomal mediated removal of mutant androgen receptor in muscle models of spinal and bulbar muscular atrophy [Articolo su rivista]
Cicardi, M. E.; Cristofani, R.; Crippa, V.; Ferrari, V.; Tedesco, B.; Casarotto, E.; Chierichetti, M.; Galbiati, M.; Piccolella, M.; Messi, E.; Carra, S.; Pennuto, M.; Rusmini, P.; Poletti, A.
abstract

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates.

2019 - Defective ribosomal products challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin [Articolo su rivista]
Mediani, L.; Guillen-Boixet, J.; Vinet, J.; Franzmann, T. M.; Bigi, I.; Mateju, D.; Carra, A. D.; Morelli, F. F.; Pinheiro Tiago, Tatiana Sofia; Poser, I.; Alberti, S.; Carra, S.
abstract

Nuclear protein aggregation has been linked to genome instability and disease. The main source of aggregation-prone proteins in cells is defective ribosomal products (DRiPs), which are generated by translating ribosomes in the cytoplasm. Here, we report that DRiPs rapidly diffuse into the nucleus and accumulate in nucleoli and PML bodies, two membraneless organelles formed by liquid–liquid phase separation. We show that nucleoli and PML bodies act as dynamic overflow compartments that recruit protein quality control factors and store DRiPs for later clearance. Whereas nucleoli serve as constitutive overflow compartments, PML bodies are stress-inducible overflow compartments for DRiPs. If DRiPs are not properly cleared by chaperones and proteasomes due to proteostasis impairment, nucleoli undergo amyloidogenesis and PML bodies solidify. Solid PML bodies immobilize 20S proteasomes and limit the recycling of free ubiquitin. Ubiquitin depletion, in turn, compromises the formation of DNA repair compartments at fragile chromosomal sites, ultimately threatening cell survival.

2019 - Nucleoli and Promyelocytic Leukemia Protein (PML) bodies are phase separated nuclear protein quality control compartments for misfolded proteins [Articolo su rivista]
Mediani, L.; Guillen-Boixet, J.; Alberti, S.; Carra, S.
abstract

We uncovered a role for nucleoli and PML-bodies as phase-separated protein quality control organelles that compartmentalize protein quality control factors and misfolded proteins for their efficient clearance. Failure to dispose misfolded proteins converts nucleoli and PML-bodies into a solid state that immobilizes ubiquitin, limiting its recycling for genome integrity maintenance.

2019 - Nucleolus: A Liquid Droplet Compartment for Misbehaving Proteins [Articolo su rivista]
Alberti, S.; Carra, S.
abstract

A new study reports an unexpected function of the nucleolus as a protein quality control compartment for misfolded and aggregation-prone proteins. These findings have important implications for protein misfolding diseases.

2019 - Proteostasis and ALS: Protocol for a phase II, randomised, double-blind, placebo-controlled, multicentre clinical trial for colchicine in ALS (Co-ALS) [Articolo su rivista]
Mandrioli, J.; Crippa, V.; Cereda, C.; Bonetto, V.; Zucchi, E.; Gessani, A.; Ceroni, M.; Chio, A.; D'Amico, R.; Monsurro, M. R.; Riva, Nicoletta; Sabatelli, M.; Silani, V.; Simone, I. L.; Soraru, G.; Provenzani, A.; D'Agostino, V. G.; Carra, S.; Poletti, Arcadio
abstract

Introduction: Disruptions of proteasome and autophagy systems are central events in amyotrophic lateral sclerosis (ALS) and support the urgent need to find therapeutic compounds targeting these processes. The heat shock protein B8 (HSPB8) recognises and promotes the autophagy-mediated removal of misfolded mutant SOD1 and TDP-43 fragments from ALS motor neurons (MNs), as well as aggregating species of dipeptides produced in C9ORF72-related diseases. In ALS-SOD1 mice and in human ALS autopsy specimens, HSPB8 is highly expressed in spinal cord MNs that survive at the end stage of disease. Moreover, the HSPB8-BAG3-HSP70 complex maintains granulostasis, which avoids conversion of dynamic stress granules (SGs) into aggregation-prone assemblies. We will perform a randomised clinical trial (RCT) with colchicine, which enhances the expression of HSPB8 and of several autophagy players, blocking TDP-43 accumulation and exerting crucial activities for MNs function. Methods and analysis: Colchicine in amyotrophic lateral sclerosis (Co-ALS) is a double-blind, placebo-controlled, multicentre, phase II RCT. ALS patients will be enrolled in three groups (placebo, colchicine 0.01 mg/day and colchicine 0.005 mg/day) of 18 subjects treated with riluzole; treatment will last 30 weeks, and follow-up will last 24 weeks. The primary aim is to assess whether colchicine decreases disease progression as measured by ALS Functional Rating Scale - Revised (ALSFRS-R) at baseline and at treatment end. Secondary aims include assessment of (1) safety and tolerability of Colchicine in patiets with ALS; (2) changes in cellular activity (autophagy, protein aggregation, and SG and exosome secretion) and in biomarkers of disease progression (neurofilaments); (3) survival and respiratory function and (4) quality of life. Preclinical studies with a full assessment of autophagy and neuroinflammation biomarkers in fibroblasts, peripheral blood mononuclear cells and lymphoblasts will be conducted in parallel with clinic assessment to optimise time and resources. Ethics and dissemination: The study protocol was approved by the Ethics Committee of Area Vasta Emilia Nord and by Agenzia Italiana del Farmaco (EUDRACT N.2017-004459-21) based on the Declaration of Helsinki. This research protocol was written without patient involvement. Patients' association will be involved in disseminating the study design and results. Results: will be presented during scientific symposia or published in scientific journals.

2019 - Small heat shock proteins: multifaceted proteins with important implications for life [Articolo su rivista]
Carra, Serena; Alberti, Simon; Benesch, Justin L. P.; Boelens, Wilbert; Buchner, Johannes; Carver, John A.; Cecconi, Ciro; Ecroyd, Heath; Gusev, Nikolai; Hightower, Lawrence E.; Klevit, Rachel E.; Lee, Hyun O.; Liberek, Krzysztof; Lockwood, Brent; Poletti, Angelo; Timmerman, Vincent; Toth, Melinda E.; Vierling, Elizabeth; Wu, Tangchun; Tanguay, Robert M.
abstract

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world’s experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26–29 August 2018.

2019 - The Regulation of the Small Heat Shock Protein B8 in Misfolding Protein Diseases Causing Motoneuronal and Muscle Cell Death [Articolo su rivista]
Cristofani, Riccardo; Rusmini, Paola; Galbiati, Mariarita; Cicardi, Maria Elena; Ferrari, Veronica; Tedesco, Barbara; Casarotto, Elena; Chierichetti, Marta; Messi, Elio; Piccolella, Margherita; Carra, Serena; Crippa, Valeria; Poletti, Angelo
abstract

Misfolding protein diseases are a wide class of disorders in which the aberrantly folded protein aggregates accumulate in affected cells. In the brain and in the skeletal muscle, misfolded protein accumulation induces a variety of cell dysfunctions that frequently lead to cell death. In motoneuron diseases (MNDs), misfolded proteins accumulate primarily in motoneurons, glial cells and/or skeletal muscle cells, altering motor function. The deleterious effects of misfolded proteins can be counteracted by the activity of the protein quality control (PQC) system, composed of chaperone proteins and degradative systems. Here, we focus on a PQC system component: heat shock protein family B (small) member 8 (HSPB8), a chaperone induced by harmful stressful events, including proteotoxicity. In motoneuron and muscle cells, misfolded proteins activate HSPB8 transcription and enhance HSPB8 levels, which contributes to prevent aggregate formation and their harmful effects. HSPB8 acts not only as a chaperone, but also facilitates the autophagy process, to enable the efficient clearance of the misfolded proteins. HSPB8 acts as a dimer bound to the HSP70 co-chaperone BAG3, a scaffold protein that is also capable of binding to HSP70 (associated with the E3-ligase CHIP) and dynein. When this complex is formed, it is transported by dynein to the microtubule organization center (MTOC), where aggresomes are formed. Here, misfolded proteins are engulfed into nascent autophagosomes to be degraded via the chaperone-assisted selective autophagy (CASA). When CASA is insufficient or impaired, HSP70 and CHIP associate with an alternative co-chaperone, BAG1, which routes misfolded proteins to the proteasome for degradation. The finely tuned equilibrium between proteasome and CASA activity is thought to be crucial for maintaining the functional cell homeostasis during proteotoxic stresses, which in turn is essential for cell survival. This fine equilibrium seems to be altered in MNDs, like Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), contributing to the onset and the progression of disease. Here, we will review how misfolded proteins may affect the PQC system and how the proper activity of this system can be restored by boosting or regulating HSPB8 activity, with the aim to ameliorate disease progression in these two fatal MNDs.

2018 - 234th ENMC International Workshop: Chaperone dysfunction in muscle disease Naarden, The Netherlands, 8–10 December 2017 [Articolo su rivista]
Weihl, Conrad C.; Udd, Bjarne; Hanna, Michael; Ben-Zvi, Anat; Blaettler, Thomas; Bryson-Richardson, Robert; Carra, Serena; Dimachkie, Mazen; Findlay, Andrew; Greensmith, Linda; Greenspan, Stephen; Hanna, Michael; Höhfled, Jörg; Jonson, Per Harald; Kampinga, Harm; Larsson, Lars; Linke, Wolfgang; Lynch, Gonrdon; Machado, Pedro; Orlando, Lianna; Richard, Isabelle; Roos, Andreas; Sarparanta, Jaakko; Timmerman, Vincent; Udd, Bjarne; Weihl, Conrad; Zah, Laura
abstract

No Abstract Available

2018 - Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks [Articolo su rivista]
Meister-Broekema, Melanie; Freilich, Rebecca; Jagadeesan, Chandhuru; Rauch, Jennifer N.; Bengoechea, Rocio; Motley, William W.; Kuiper, E. F. Elsiena; Minoia, Melania; Furtado, Gabriel V.; van Waarde, Maria A. W. H.; Bird, Shawn J.; Rebelo, Adriana; Zuchner, Stephan; Pytel, Peter; Scherer, Steven S.; Morelli, Federica F.; Carra, Serena; Weihl, Conrad C.; Bergink, Steven; Gestwicki, Jason E.; Kampinga, Harm H.
abstract

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.

2018 - Quality Control of Membraneless Organelles [Articolo su rivista]
Alberti, Simon; Carra, Serena
abstract

The formation of membraneless organelles (MLOs) by phase separation has emerged as a new way of organizing the cytoplasm and nucleoplasm of cells. Examples of MLOs forming via phase separation are nucleoli in the nucleus and stress granules in the cytoplasm. The main components of these MLOs are macromolecules such as RNAs and proteins. In order to assemble by phase separation, these proteins and RNAs have to undergo many cooperative interactions. These cooperative interactions are supported by specific molecular features within phase-separating proteins, such as multivalency and the presence of disordered domains that promote weak and transient interactions. However, these features also predispose phase-separating proteins to aberrant behavior. Indeed, evidence is emerging for a strong link between phase-separating proteins, MLOs, and age-related diseases. In this review, we discuss recent progress in understanding the formation, properties, and functions of MLOs. We pay special attention to the emerging link between MLOs and age-related diseases, and we explain how changes in the composition and physical properties of MLOs promote their conversion into an aberrant state. Furthermore, we discuss the key role of the protein quality control machinery in regulating the properties and functions of MLOs and thus in preventing age-related diseases.

2018 - Tdp-25 Routing to Autophagy and Proteasome Ameliorates its Aggregation in Amyotrophic Lateral Sclerosis Target Cells [Articolo su rivista]
Cicardi, Maria Elena; Cristofani, Riccardo; Rusmini, Paola; Meroni, Marco; Ferrari, Veronica; Vezzoli, Giulia; Tedesco, Barbara; Piccolella, Margherita; Messi, Elio; Galbiati, Mariarita; Boncoraglio, Alessandra; Carra, Serena; Crippa, Valeria; Poletti, Angelo
abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motoneurons, while non-neuronal cells may contribute to disease onset and progression. Most ALS cases are characterized by the mislocalization and aggregation of the TAR DNA binding protein 43 (TDP-43) in affected cells. TDP-43 aggregates contain C-terminal TDP-43 fragments of 35 kDa (TDP-35) and 25 kDa (TDP-25) and have been mainly studied in motoneurons, while little is currently known about their rate of accumulation and clearance in myoblasts. Here, we performed a comparative study in immortalized motoneuronal like (NSC34; i-motoneurons) cells and stabilized myoblasts (C2C12; s-myoblasts) to evaluate if these two cell types differentially accumulate and clear TDP forms. The most aggregating specie in i-motoneurons is the TDP-25 fragment, mainly constituted by the “prion-like” domain of TDP-43. To a lower extent, TDP-25 also aggregates in s-myoblasts. In both cell types, all TDP species are cleared by proteasome, but TDP-25 impairs autophagy. Interestingly, the routing of TDP-25 fragment to proteasome, by overexpressing BAG1, or to autophagy, by overexpressing HSPB8 or BAG3 decreased its accumulation in both cell types. These results demonstrate that promoting the chaperone-assisted clearance of ALS-linked proteins is beneficial not only in motoneurons but also in myoblasts.

2018 - The small heat shock protein B8 (HSPB8) efficiently removes aggregating species of dipeptides produced in C9ORF72-related neurodegenerative diseases [Articolo su rivista]
Cristofani, Riccardo; Crippa, Valeria; Vezzoli, Giulia; Rusmini, Paola; Galbiati, Mariarita; Cicardi, Maria Elena; Meroni, Marco; Ferrari, Veronica; Tedesco, Barbara; Piccolella, Margherita; Messi, Elio; Carra, Serena; Poletti, Angelo
abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not “classical” misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.

2017 - Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function [Articolo su rivista]
Morelli F., F.; Verbeek D., S.; Bertacchini, Jessika; Vinet, Jonathan; Mediani, Laura; Marmiroli, Sandra; Cenacchi, G.; Nasi, Milena; DE BIASI, Sara; Brunsting J., F.; Lammerding, J.; Pegoraro, E.; Angelini, C.; Tupler, Rossella; Alberti, S.; Carra, Serena
abstract

Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy.

2017 - An aberrant phase transition of stress granules triggered by misfolded protein and prevented by chaperone function [Articolo su rivista]
Mateju, Daniel; Franzmann, Titus M.; Patel, Avinash; Kopach, Andrii; Boczek, Edgar E.; Maharana, Shovamayee; Lee, Hyun O.; Carra, Serena; Hyman, Anthony A.; Alberti, Simon
abstract

Stress granules (SG) are membrane-less compartments involved in regulating mRNAs during stress. Aberrant forms of SGs have been implicated in age-related diseases, such as amyotrophic lateral sclerosis (ALS), but the molecular events triggering their formation are still unknown. Here, we find that misfolded proteins, such as ALS-linked variants of SOD1, specifically accumulate and aggregate within SGs in human cells. This decreases the dynamics of SGs, changes SG composition, and triggers an aberrant liquid-to-solid transition of in vitro reconstituted compartments. We show that chaperone recruitment prevents the formation of aberrant SGs and promotes SG disassembly when the stress subsides. Moreover, we identify a backup system for SG clearance, which involves transport of aberrant SGs to the aggresome and their degradation by autophagy. Thus, cells employ a system of SG quality control to prevent accumulation of misfolded proteins and maintain the dynamic state of SGs, which may have relevance for ALS and related diseases.

2017 - An interaction study in mammalian cells demonstrates weak binding of HSPB2 to BAG3, which is regulated by HSPB3 and abrogated by HSPB8 [Articolo su rivista]
Morelli, FEDERICA FRANCESCA; Mediani, Laura; Heldens, Lonneke; Bertacchini, Jessika; Bigi, Ilaria; Carrà, Arianna Dorotea; Vinet, Jonathan; Carra, Serena
abstract

The ten mammalian small heat shock proteins (sHSPs/HSPBs) show a different expression profile, although the majority of them are abundant in skeletal and cardiac muscles. HSPBs form hetero-oligomers and homo-oligomers by interacting together and complexes containing, e.g., HSPB2/HSPB3 or HSPB1/HSPB5 have been documented in mammalian cells and muscles. Moreover, HSPB8 associates with the Hsc70/Hsp70 co-chaperone BAG3, in mammalian, skeletal, and cardiac muscle cells. Interaction of HSPB8 with BAG3 regulates its stability and function. Weak association of HSPB5 and HSPB6 with BAG3 has been also reported upon overexpression in cells, supporting the idea that BAG3 might indirectly modulate the function of several HSPBs. However, it is yet unknown whether other HSPBs highly expressed in muscles such as HSPB2 and HSPB3 also bind to BAG3. Here, we report that in mammalian cells, upon overexpression, HSPB2 binds to BAG3 with an affinity weaker than HSPB8. HSPB2 competes with HSPB8 for binding to BAG3. In contrast, HSPB3 negatively regulates HSPB2 association with BAG3. In human myoblasts that express HSPB2, HSPB3, HSPB8, and BAG3, the latter interacts selectively with HSPB8. Combining these data, it supports the interpretation that HSPB8-BAG3 is the preferred interaction.

2017 - Granulostasis: Protein Quality Control of RNP Granules [Articolo su rivista]
Alberti, Simon; Mateju, Daniel; Mediani, Laura; Carra, Serena
abstract

Ribonucleoprotein (RNP) granules transport, store, or degrade messenger RNAs, thereby indirectly regulating protein synthesis. Normally, RNP granules are highly dynamic compartments. However, because of aging or severe environmental stress, RNP granules, in particular stress granules (SGs), convert into solid, aggregate-like inclusions. There is increasing evidence that such RNA-protein inclusions are associated with several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD) and Alzheimer's disease (AD). Thus, understanding what triggers the conversion of RNP granules into aggregates and identifying the cellular players that control RNP granules will be critical to develop treatments for these diseases. In this review article, we discuss recent insight into RNP and SG formation. More specifically, we examine the evidence for liquid-liquid phase separation (LLPS) as an organizing principle of RNP granules and the role of aggregation-prone RNA-binding proteins (RBPs) in this process. We further discuss recent findings that liquid-like SGs can sequester misfolded proteins, which promote an aberrant conversion of liquid SGs into solid aggregates. Importantly, very recent studies show that a specific protein quality control (PQC) process prevents the accumulation of misfolding-prone proteins in SGs and, by doing so, maintains the dynamic state of SGs. This quality control process has been referred to as granulostasis and it relies on the specific action of the HSPB8-BAG3-HSP70 complex. Additional players such as p97/valosin containing protein (VCP) and other molecular chaperones (e.g., HSPB1) participate, directly or indirectly, in granulostasis, and ensure the timely elimination of defective ribosomal products and other misfolded proteins from SGs. Finally, we discuss recent findings that, in the stress recovery phase, SGs are preferentially disassembled with the assistance of chaperones, and we discuss evidence for a back-up system that targets aberrant SGs to the aggresome for autophagy-mediated clearance. Altogether the findings discussed here provide evidence for an intricate network of interactions between RNP granules and various components of the PQC machinery. Molecular chaperones in particular are emerging as key players that control the composition and dynamics of RNP granules, which may be important to protect against age-related diseases.

2017 - Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases [Articolo su rivista]
Cristofani, Riccardo; Crippa, Valeria; Rusmini, Paola; Cicardi, Maria Elena; Meroni, Marco; Licata, Nausicaa V.; Sala, Gessica; Giorgetti, Elisa; Grunseich, Christopher; Galbiati, Mariarita; Piccolella, Margherita; Messi, Elio; Ferrarese, Carlo; Carra, Serena; Poletti, Angelo
abstract

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.

2017 - The growing world of small heat shock proteins: from structure to functions [Articolo su rivista]
Carra, Serena; Alberti, Simon; Arrigo, Patrick A.; Benesch, Justin L.; Benjamin, Ivor J.; Boelens, Wilbert; Bartelt-Kirbach, Britta; Brundel, Bianca J. J. M.; Buchner, Johannes; Bukau, Bernd; Carver, John A.; Ecroyd, Heath; Emanuelsson, Cecilia; Finet, Stephanie; Golenhofen, Nikola; Goloubinoff, Pierre; Gusev, Nikolai; Haslbeck, Martin; Hightower, Lawrence E.; Kampinga, Harm H.; Klevit, Rachel E.; Liberek, Krzysztof; Mchaourab, Hassane S.; Mcmenimen, Kathryn A.; Poletti, Angelo; Quinlan, Roy; Strelkov, Sergei V.; Toth, Melinda E.; Vierling, Elizabeth; Tanguay, Robert M.
abstract

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).

2017 - The role of the heat shock protein B8 (HSPB8) in motoneuron diseases [Articolo su rivista]
Rusmini, Paola; Cristofani, Riccardo; Galbiati, Mariarita; Cicardi, Maria E.; Meroni, Marco; Ferrari, Veronica; Vezzoli, Giulia; Tedesco, Barbara; Messi, Elio; Piccolella, Margherita; Carra, Serena; Crippa, Valeria; Poletti, Angelo
abstract

Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA) are two motoneuron diseases (MNDs) characterized by aberrant protein behavior in affected cells. In familial ALS (fALS) and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS) even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC) system. The small heat shock protein B8 (HSPB8) is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

2017 - The small heat shock protein B8 (HSPB8) modulates proliferation and migration of breast cancer cells [Articolo su rivista]
Piccolella, Margherita; Crippa, Valeria; Cristofani, Riccardo; Rusmini, Paola; Galbiati, Mariarita; Cicardi, Maria Elena; Meroni, Marco; Ferri, Nicola; Morelli, FEDERICA FRANCESCA; Carra, Serena; Messi, Elio; Poletti, Angelo
abstract

Breast cancer (BC) is one of the major causes of cancer death in women and is closely related to hormonal dysregulation. Estrogen receptor (ER)-positive BCs are generally treated with anti hormone therapy using antiestrogens or aromatase inhibitors. However, BC cells may become resistant to endocrine therapy, a process facilitated by autophagy, which may either promote or suppress tumor expansion. The autophagy facilitator HSPB8 has been found overexpressed in some BC. Here we found that HSPB8 is highly expressed and differentially modulated by natural or synthetic selective ER modulators (SERMs), in the triple-positive hormone-sensitive BC (MCF-7) cells, but not in triple-negative MDA-MB-231 BC cells. Specific SERMs induced MCF-7 cells proliferation in a HSPB8 dependent manner whereas, did not modify MDA-MB-231 cell growth. ER expression was unaffected in HSPB8-depleted MCF-7 cells. HSPB8 over-expression did not alter the distribution of MCF-7 cells in the various phases of the cell cycle. Conversely and intriguingly, HSPB8 downregulation resulted in an increased number of cells resting in the G0/G1 phase, thus possibly reducing the ability of the cells to pass through the restriction point. In addition, HSPB8 downregulation reduced the migratory ability of MCF-7 cells. None of these modifications were observed, when another small HSP (HSPB1), also expressed in MCF-7 cells, was downregulated. In conclusion, our data suggest that HSPB8 is involved in the mechanisms that regulate cell cycle and cell migration in MCF-7 cells.

2016 - A Surveillance Function of the HSPB8-BAG3-HSP70 Chaperone Complex Ensures Stress Granule Integrity and Dynamism [Articolo su rivista]
Ganassi, Massimo; Mateju, Daniel; Bigi, Ilaria; Mediani, Laura; Poser, Ina; Lee, Hyun O.; Seguin, SAMUEL JOSEPH ANDRE'; Morelli, FEDERICA FRANCESCA; Vinet, Jonathan; Leo, Giuseppina; Pansarasa, Orietta; Cereda, Cristina; Poletti, Angelo; Alberti, Simon; Carra, Serena
abstract

Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS.

2016 - Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356 [Articolo su rivista]
Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Arozena, A. A.; Adachi, H.; Adams, C. M.; Adams, P. D.; Adeli, K.; Adhihetty, P. J.; Adler, S. G.; Agam, G.; Agarwal, R.; Aghi, M. K.; Agnello, M.; Agostinis, P.; Aguilar, P. V.; Aguirre-Ghiso, J.; Airoldi, E. M.; Ait-Si-Ali, S.; Akematsu, T.; Akporiaye, E. T.; Al-Rubeai, M.; Albaiceta, G. M.; Albanese, C.; Albani, D.; Albert, M. L.; Aldudo, J.; Algul, H.; Alirezaei, M.; Alloza, I.; Almasan, A.; Almonte-Beceril, M.; Alnemri, E. S.; Alonso, C.; Altan-Bonnet, N.; Altieri, D. C.; Alvarez, S.; Alvarez-Erviti, L.; Alves, S.; Amadoro, G.; Amano, A.; Amantini, C.; Ambrosio, S.; Amelio, I.; Amer, A. O.; Amessou, M.; Amon, A.; An, Z.; Anania, F. A.; Andersen, S. U.; Andley, U. P.; Andreadi, C. K.; Andrieu-Abadie, N.; Anel, A.; Ann, D. K.; Anoopkumar-Dukie, S.; Antonioli, M.; Aoki, H.; Apostolova, N.; Aquila, S.; Aquilano, K.; Araki, K.; Arama, E.; Aranda, A.; Araya, J.; Arcaro, A.; Arias, E.; Arimoto, H.; Ariosa, A. R.; Armstrong, J. L.; Arnould, T.; Arsov, I.; Asanuma, K.; Askanas, V.; Asselin, E.; Atarashi, R.; Atherton, S. S.; Atkin, J. D.; Attardi, L. D.; Auberger, P.; Auburger, G.; Aurelian, L.; Autelli, R.; Avagliano, L.; Avantaggiati, M. L.; Avrahami, L.; Azad, N.; Awale, S.; Bachetti, T.; Backer, J. M.; Bae, D. -H.; Bae, J. -S.; Bae, O. -N.; Bae, S. H.; Baehrecke, E. H.; Baek, S. -H.; Baghdiguian, S.; Bagniewska-Zadworna, A.; Bai, H.; Bai, J.; Bai, X. -Y.; Bailly, Y.; Balaji, K. N.; Balduini, W.; Ballabio, A.; Balzan, R.; Banerjee, R.; Banhegyi, G.; Bao, H.; Barbeau, B.; Barrachina, M. D.; Barreiro, E.; Bartel, B.; Bartolome, A.; Bassham, D. C.; Bassi, M. T.; Bast, R. C.; Basu, A.; Batista, M. T.; Batoko, H.; Battino, M.; Bauckman, K.; Baumgarner, B. L.; Bayer, K. U.; Beale, R.; Beaulieu, J. -F.; Beck, G. R.; Becker, C.; Beckham, J. D.; Bedard, P. -A.; Bednarski, P. J.; Begley, T. J.; Behl, C.; Behrends, C.; Behrens, G. M. N.; Behrns, K. E.; Bejarano, E.; Belaid, A.; Belleudi, F.; Benard, G.; Berchem, G.; Bergamaschi, D.; Bergami, M.; Berkhout, B.; Berliocchi, L.; Bernard, A.; Bernard, M.; Bernassola, F.; Bertolotti, A.; Bess, A. S.; Besteiro, S.; Bettuzzi, S.; Bhalla, S.; Bhattacharyya, S.; Bhutia, S. K.; Biagosch, C.; Bianchi, M. W.; Biard-Piechaczyk, M.; Billes, V.; Bincoletto, C.; Bingol, B.; Bird, S. W.; Bitoun, M.; Bjedov, I.; Blackstone, C.; Blanc, L.; Blanco, G. A.; Blomhoff, H. K.; Boada-Romero, E.; Bockler, S.; Boes, M.; Boesze-Battaglia, K.; Boise, L. H.; Bolino, A.; Boman, A.; Bonaldo, P.; Bordi, M.; Bosch, J.; Botana, L. M.; Botti, J.; Bou, G.; Bouche, M.; Bouchecareilh, M.; Boucher, M. -J.; Boulton, M. E.; Bouret, S. G.; Boya, P.; Boyer-Guittaut, M.; Bozhkov, P. V.; Brady, N.; Braga, V. M. M.; Brancolini, C.; Braus, G. H.; Bravo-San-Pedro, J. M.; Brennan, L. A.; Bresnick, E. H.; Brest, P.; Bridges, D.; Bringer, M. -A.; Brini, M.; Brito, G. C.; Brodin, B.; Brookes, P. S.; Brown, E. J.; Brown, K.; Broxmeyer, H. E.; Bruhat, A.; Brum, P. C.; Brumell, J. H.; Brunetti-Pierri, N.; Bryson-Richardson, R. J.; Buch, S.; Buchan, A. M.; Budak, H.; Bulavin, D. V.; Bultman, S. J.; Bultynck, G.; Bumbasirevic, V.; Burelle, Y.; Burke, R. E.; Burmeister, M.; Butikofer, P.; Caberlotto, L.; Cadwell, K.; Cahova, M.; Cai, D.; Cai, J.; Cai, Q.; Calatayud, S.; Camougrand, N.; Campanella, M.; Campbell, G. R.; Campbell, M.; Campello, S.; Candau, R.; Caniggia, I.; Cantoni, L.; Cao, L.; Caplan, A. B.; Caraglia, M.; Cardinali, C.; Cardoso, S. M.; Carew, J. S.; Carleton, L. A.; Carlin, C. R.; Carloni, S.; Carlsson, S. R.; Carmona-Gutierrez, D.; Carneiro, L. A. M.; Carnevali, O.; Carra, S.; Carrier, A.; Carroll, B.; Casas, C.; Casas, J.; Cassinelli, G.; Castets, P.; Castro-Obregon, S.; Cavallini, G.; Ceccherini, I.; Cecconi, F.; Cederbaum, A. I.; Cena, V.; Cenci, S.; Cerella, C.; Cervia, D.; Cetrullo, S.; Chaachouay, H.; Chae, H. -J.; Chagin, A. S.; Chai, C. -Y.; Chakrabarti, G.; Chamilos, G.; Chan, E. Y. W.; Chan, M. T. V.; Chandra, D.; Chandra, P.; Chang, C. -P.; Chang,
abstract

2016 - Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) [Articolo su rivista]
Klionsky, Daniel J; Abdelmohsen, Kotb; Abe, Akihisa; Abedin, Md Joynal; Abeliovich, Hagai; Acevedo Arozena, Abraham; Adachi, Hiroaki; Adams, Christopher M; Adams, Peter D; Adeli, Khosrow; Adhihetty, Peter J; Adler, Sharon G; Agam, Galila; Agarwal, Rajesh; Aghi, Manish K; Agnello, Maria; Agostinis, Patrizia; Aguilar, Patricia V; Aguirre Ghiso, Julio; Airoldi, Edoardo M; Ait Si Ali, Slimane; Akematsu, Takahiko; Akporiaye, Emmanuel T; Al Rubeai, Mohamed; Albaiceta, Guillermo M; Albanese, Chris; Albani, Diego; Albert, Matthew L; Aldudo, Jesus; Algül, Hana; Alirezaei, Mehrdad; Alloza, Iraide; Almasan, Alexandru; Almonte Beceril, Maylin; Alnemri, Emad S; Alonso, Covadonga; Altan Bonnet, Nihal; Altieri, Dario C; Alvarez, Silvia; Alvarez Erviti, Lydia; Alves, Sandro; Amadoro, Giuseppina; Amano, Atsuo; Amantini, Consuelo; Ambrosio, Santiago; Amelio, Ivano; Amer, Amal O; Amessou, Mohamed; Amon, Angelika; An, Zhenyi; Anania, Frank A; Andersen, Stig U; Andley, Usha P; Andreadi, Catherine K; Andrieu Abadie, Nathalie; Anel, Alberto; Ann, David K; Anoopkumar Dukie, Shailendra; Antonioli, Manuela; Aoki, Hiroshi; Apostolova, Nadezda; Aquila, Saveria; Aquilano, Katia; Araki, Koichi; Arama, Eli; Aranda, Agustin; Araya, Jun; Arcaro, Alexandre; Arias, Esperanza; Arimoto, Hirokazu; Ariosa, Aileen R; Armstrong, Jane L; Arnould, Thierry; Arsov, Ivica; Asanuma, Katsuhiko; Askanas, Valerie; Asselin, Eric; Atarashi, Ryuichiro; Atherton, Sally S; Atkin, Julie D; Attardi, Laura D; Auberger, Patrick; Auburger, Georg; Aurelian, Laure; Autelli, Riccardo; Avagliano, Laura; Avantaggiati, Maria Laura; Avrahami, Limor; Awale, Suresh; Azad, Neelam; Bachetti, Tiziana; Backer, Jonathan M; Bae, Dong Hun; Bae, Jae Sung; Bae, Ok Nam; Bae, Soo Han; Baehrecke, Eric H; Baek, Seung Hoon; Baghdiguian, Stephen; Bagniewska Zadworna, Agnieszka; Bai, Hua; Bai, Jie; Bai, Xue Yuan; Bailly, Yannick; Balaji, Kithiganahalli Narayanaswamy; Balduini, Walter; Ballabio, Andrea; Balzan, Rena; Banerjee, Rajkumar; Bánhegyi, Gábor; Bao, Haijun; Barbeau, Benoit; Barrachina, Maria D; Barreiro, Esther; Bartel, Bonnie; Bartolomé, Alberto; Bassham, Diane C; Bassi, Maria Teresa; Bast, Robert C; Basu, Alakananda; Batista, Maria Teresa; Batoko, Henri; Battino, Maurizio; Bauckman, Kyle; Baumgarner, Bradley L; Bayer, K. Ulrich; Beale, Rupert; Beaulieu, Jean François; Beck, George R; Becker, Christoph; Beckham, J. David; Bédard, Pierre André; Bednarski, Patrick J; Begley, Thomas J; Behl, Christian; Behrends, Christian; Behrens, Georg Mn; Behrns, Kevin E; Bejarano, Eloy; Belaid, Amine; Belleudi, Francesca; Bénard, Giovanni; Berchem, Guy; Bergamaschi, Daniele; Bergami, Matteo; Berkhout, Ben; Berliocchi, Laura; Bernard, Amélie; Bernard, Monique; Bernassola, Francesca; Bertolotti, Anne; Bess, Amanda S; Besteiro, Sébastien; Bettuzzi, Saverio; Bhalla, Savita; Bhattacharyya, Shalmoli; Bhutia, Sujit K; Biagosch, Caroline; Bianchi, Michele Wolfe; Biard Piechaczyk, Martine; Billes, Viktor; Bincoletto, Claudia; Bingol, Baris; Bird, Sara W; Bitoun, Marc; Bjedov, Ivana; Blackstone, Craig; Blanc, Lionel; Blanco, Guillermo A; Blomhoff, Heidi Kiil; Boada Romero, Emilio; Böckler, Stefan; Boes, Marianne; Boesze Battaglia, Kathleen; Boise, Lawrence H; Bolino, Alessandra; Boman, Andrea; Bonaldo, Paolo; Bordi, Matteo; Bosch, Jürgen; Botana, Luis M; Botti, Joelle; Bou, German; Bouché, Marina; Bouchecareilh, Marion; Boucher, Marie Josée; Boulton, Michael E; Bouret, Sebastien G; Boya, Patricia; Boyer Guittaut, Michaël; Bozhkov, Peter V; Brady, Nathan; Braga, Vania Mm; Brancolini, Claudio; Braus, Gerhard H; Bravo San Pedro, José M; Brennan, Lisa A; Bresnick, Emery H; Brest, Patrick; Bridges, Dave; Bringer, Marie Agnès; Brini, Marisa; Brito, Glauber C; Brodin, Bertha; Brookes, Paul S; Brown, Eric J; Brown, Karen; Broxmeyer, Hal E; Bruhat, Alain; Brum, Patricia Chakur; Brumell, John H; Brunetti Pierri, Nicola; Bryson Richardson, Robert J; Buch, Shilpa; Buchan, Alastair M; Budak
abstract

Guidelines for studying autophagy

2016 - Specific protein homeostatic functions of small heat-shock proteins increase lifespan [Articolo su rivista]
Vos, Michel J.; Carra, Serena; Kanon, Bart; Bosveld, Floris; Klauke, Karin; Sibon, Ody C. M.; Kampinga, Harm H.
abstract

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.

2016 - The Role of the Protein Quality Control System in SBMA [Articolo su rivista]
Rusmini, Paola; Crippa, Valeria; Cristofani, Riccardo; Rinaldi, Carlo; Cicardi, Maria Elena; Galbiati, Mariarita; Carra, Serena; Bilal, Malik; Greensmith, Linda; Poletti, Angelo
abstract

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked disease associated with the expansion of the CAG triplet repeat present in exon 1 of the androgen receptor (AR) gene. This results in the production of a mutant AR containing an elongated polyglutamine tract (polyQ) in its N-terminus. Interestingly, the ARpolyQ becomes toxic only after its activation by the natural androgenic ligands, possibly because of aberrant androgen-induced conformational changes of the ARpolyQ, which generate misfolded species. These misfolded ARpolyQ species must be cleared from motoneurons and muscle cells, and this process is mediated by the protein quality control (PQC) system. Experimental evidence suggested that failure of the PQC pathways occurs in disease, leading to ARpolyQ accumulation and toxicity in the target cells. In this review, we summarized the overall impact of mutant and misfolded ARpolyQ on the PQC system and described how molecular chaperones and the degradative pathways (ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and the unfolded protein response (UPR), which activates the endoplasmic reticulum-associated degradation (ERAD)) are differentially affected in SBMA. We also extensively and critically reviewed several molecular and pharmacological approaches proposed to restore a global intracellular activity of the PQC system. Collectively, these data suggest that the fine and delicate equilibrium existing among the different players of the PQC system could be restored in a therapeutic perspective by the synergic/additive activities of compounds designed to tackle sequential or alternative steps of the intracellular defense mechanisms triggered against proteotoxic misfolded species.

2016 - The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity [Articolo su rivista]
Crippa, Valeria; Cicardi, Maria Elena; Ramesh, Nandini; Seguin, SAMUEL JOSEPH ANDRE'; Ganassi, Massimo; Bigi, Ilaria; Diacci, Chiara; Zelotti, Elena; Baratashvili, Madina; Gregory, Jenna M.; Dobson, Christopher M.; Cereda, Cristina; Pandey, Udai Bhan; Poletti, Angelo; Carra, Serena
abstract

Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.

2016 - Transcriptional induction of the heat shock protein B8 mediates the clearance of misfolded proteins responsible for motor neuron diseases [Articolo su rivista]
Crippa, Valeria; D'Agostino, Vito G.; Cristofani, Riccardo; Rusmini, Paola; Cicardi, Maria E.; Messi, Elio; Loffredo, Rosa; Pancher, Michael; Piccolella, Margherita; Galbiati, Mariarita; Meroni, Marco; Cereda, Cristina; Carra, Serena; Provenzani, Alessandro; Poletti, Angelo
abstract

Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.

2015 - CHARACTERIZATION OF THE R7S MUTATION OF HEAT SHOCK PROTEIN HSPB3 AND TWO NOVEL MUTATIONS FOUND IN PATIENTS SUFFERING OF MYOPATHY: UNDERSTANDING THE MECHANISMS LEADING TO DISEASE. [Poster]
Morelli, FEDERICA FRANCESCA; Heldens, Lonneke; Verbeek, Dineke; Angelini, Corrado; Cenacchi, Giovanna; Tupler, Rossella; Carra, Serena
abstract

HSPB3 is a poorly characterized member of the small HSPB family that forms a complex with HSPB2. The complex is induced in differentiated muscle cells and might play a role in muscle maintenance. R7S mutation was associated with distal hereditary motor neuropathy type 2C. We identified in myopathic patients two novel mutations of HSPB3: a) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs causes neuromuscular diseases; b) p.A33AfsX50-HSPB3, a missense mutation, which leads to a premature stop codon. We characterized the subcellular localization of HSPB2-HSPB3 and the effects of the HSPB3 mutants on protein localization, stability and complex formation. While p.A33AfsX50-HSPB3 is degraded after synthesis, R7S and R116P are stable. Unlike of R116P, R7S still interacts with HSPB2. HSPB2 and HSPB3 are enriched in the nuclei, where they form intranuclear (IN) and perinuclear (PN) aggregates. Aggregation tendency of HSPB3 is increased by its mutants. These IN and PN aggregates influence nuclear envelope and lamin A/C distribution. Lamins regulate not only nuclear shape, but also transcription. Moreover, mutations on lamin A/C are associated with neuromuscular disease. In addition, lamin A/C is recruited in nuclear speckles that contain splicing factors such as SC35. HSPB2-HSPB3 do not colocalize with speckles and do not alter the recruitment in speckles of lamin A/C; instead, they alter speckles shape, which became round (mimicking transcription inhibition). Combined our results show that HSPB2-HSPBB3 affect nuclear structure; this in turn may deregulate remodeling of nuclear lamina and RNA transcription. Intriguingly, the muscle biopsy from patient with R116P mutation shows nuclear aggregation and morphological alterations. This further suggests that HSPB3 (in complex with HSPB2) may modulate nuclear structure/functions and that alteration thereof may contribute to disease.

2015 - Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease. [Poster]
Morelli, FEDERICA FRANCESCA; Heldens, Lonneke; Verbeek, Dineke; Angelini, Corrado; Cenacchi, Giovanna; Tupler, Rossella; Carra, Serena
abstract

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2. Expression of HSPB2 and HSPB3 is restricted to differentiated skeletal and cardiac muscle cells, where HSPB2-HSPB3 participates in muscle maintenance. Recently the R7S mutation in HSPB3 was associated with distal hereditary motor neuropathy type 2C (dHMN 2C). We identified in myopathic patients two novel mutations of HSPB3: 1) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs also causes neuromuscular diseases; 2) p.A33AfsX50-HSPB3, which disrupts the reading frame leading to a premature stop codon at amino acid 50. Here we first characterized in cells the subcellular distribution of HSPB2 and HSPB3. Next, we studied whether/how HSPB3 mutations affect: a) HSPB3 stability and subcellular localization; b) complex formation. While p.A33AfsX50-HSPB3 is immediately degraded after synthesis and cannot be detected, R7S and R116P are rather stable. Concerning interaction with HSPB2, R7S still interacted with HSPB2, while R116 disrupted complex formation. Concerning subcellular distribution, in two cell types (HEK293T and HeLa cells), HSPB2 and HSPB3 were enriched in the nuclei, where they formed intranuclear and perinuclear aggregates. Aggregation propensity was increased by R7S and R116P mutations. Intriguingly, nuclear aggregation and alteration of nuclear morphology were also found in the muscle biopsy from the patient with the R116P mutation. Next, we found in cells that HSPB2 and HSPB3 (wt and mutants) alter nuclear envelope (NE) integrity and lamin A/C distribution. Interestingly, DMPK, which has been shown to interact with HSPB2, also affects NE. Lamins regulate not only nuclear shape, but also gene expression, transcription and mutations in NE components cause neuromuscular and muscular diseases. Lamin A/C has been associated with intranuclear speckles, where it can colocalize with HSPB1 or HSPB5. Speckles contain splicing factors such as SC35. We thus analyzed speckles in HSPB2-B3 expressing cells. HSPB2-B3 did not inhibit Lamin A/C recruitment into speckles. Also, we did not find any major colocalization of HSPB2 and HSPB3 with speckles. Instead, HSPB2 and HSPB3 altered the shape of speckles, which became round (mimicking a treatment with the RNA transcription inhibitor actinomycin D). In summary, our results show that HSPB2-HSPB3 affect nuclear architecture, which may in turn deregulate RNA transcription. Future studies will identify the molecular mechanisms leading to the observed effects on NE and speckles. Since remodeling of nuclear lamina is required for muscle differentiation, it is tempting to speculate that HSPB2-B3 may modulate muscle differentiation/maintenance by regulating lamin functions and NE stability. Alteration of such functions due to HSPB3 mutations may be detrimental for motor neuron and muscle cells, contributing to disease progression.

2015 - Characterization of the interplay between the protein quality control and the stress granule response: implication in neurodegenerative diseases [Relazione in Atti di Convegno]
Carra, Serena
abstract

Stress granules (SGs) are transient heterogeneous mRNA-protein complexes induced during stress, which exert cellular pro-survival functions. Recent data implicate SGs and deregulated proteostasis in amyotrophic lateral sclerosis, frontotemporal lobar degeneration and multisystem proteinopathy, which are also associated with mutations of valosin-containing protein (VCP) and where protein aggregates that contain components of SGs accumulate. This suggests that inappropriate SG dynamics may be relevant to pathogenesis. Interestingly, SG formation is driven by the reversible self-aggregation of mRNA-binding proteins that contain prion-like domains. Unlike prionogenic fibrillar aggregates, SGs are dynamic structures, which disassemble within few hours after their formation, even if the stress persists. Due to the heterogeneous composition of SGs and to the crowded molecular environment, SGs may, indirectly, require protein quality control (PQC) assistance for proper assembly and disassembly. Previously, the autophagy-lysosome pathway and VCP, key players of the PQC, were shown to regulate SG degradation (Buchan et al., Cell 2013). Here we investigated whether impairment of PQC, autophagy and lysosome-mediated degradation may affect SG response. We provide evidence supporting that inhibition of VCP, autophagy or lysosomes affects SG formation, morphology and composition. In particular, defective ribosomal products (DRIPs) and the large ribosome subunit 60S, which are released from disassembling polysomes, are normally excluded from SGs. Instead, we observed that both DRIPs and 60S were significantly retained within and/or adjacent to SGs in cells with impaired autophagy, lysosome, or VCP function (Seguin et al., CDD 2014). Next, we found that depletion of other chaperones and co-chaperones involved in the degradation of ubiquitinated proteins and DRIPs also affect SG dynamics and translation restoration after stress. These findings further reinforce the hypothesis that PQC and SGs are tightly interconnected and work in concert to maintain and restore protein and RNA homeostasis. Deregulation of the PQC would thus render the cells particularly vulnerable under challenging/disease conditions, thereby participating to disease progression.

2015 - Impairment of the Protein Quality Control System Affects Stress Granule Response and Dynamics [Relazione in Atti di Convegno]
Carra, Serena
abstract

Stress granules (SGs) are transient heterogeneous mRNA-protein complexes induced during stress. SGs exert cellular pro-survival functions and recent data suggest deregulation of SG response and dynamics as potential pathomechanism in e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration and multisystem proteinopathy. These diseases can be associated with mutations of valosin-containing protein (VCP) and are characterized by impairment of the protein quality control (PQC), as well as aggregation of SG components. Interestingly, SG formation is driven by the reversible self-aggregation of mRNA-binding proteins that contain prion-like domains. Unlike prionogenic fibrillar aggregates, SGs are dynamic structures, which disassemble within few hours after their formation, even if the stress persists. Thus, due to the crowded molecular environment, SGs may, indirectly, require protein quality control (PQC) assistance for proper assembly and disassembly. Here we investigated whether impairment of PQC, autophagy and lysosome-mediated degradation may affect SG response. We demonstrated that inhibition of VCP, autophagy or lysosomes affects SG formation, morphology and composition. In particular, defective ribosomal products (DRIPs) and the large ribosome subunit 60S, which are released from disassembling polysomes, are normally excluded from SGs. Instead, we found that DRIPs and 60S were significantly retained within and/or adjacent to SGs in cells with impaired autophagy, lysosome, or VCP function (Seguin et al., CDD 2014). Next, we observed that depletion of other chaperones and co-chaperones involved in the degradation of ubiquitinated proteins and DRIPs also affects SG dynamics. This further reinforces the hypothesis that PQC and SGs are tightly interconnected. Collectively our data suggest that besides causing a protein homeostasis imbalance, deregulated autophagy, lysosomal or chaperone activities may also alter SG morphology, composition and dynamics. This, in turn, may lead to defective SG disassembly and persistence, thereby ultimately increasing cell vulnerability, especially under challenging/disease conditions.

2015 - Inhibition of autophagy, lysosome and VCP alters stress granule morphology and composition [Relazione in Atti di Convegno]
Carra, Serena
abstract

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many age-related neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation (Buchan et al., Cell 2013). This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here we investigated whether impairment of PQC, autophagy and lysosome-mediated degradation may affect SG assembly. We provide evidence supporting that inhibition of VCP, autophagy or lysosomes affects SG formation, morphology and composition. In particular, defective ribosomal products and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome, or VCP function. Recent data implicate SGs and deregulated proteostasis in amyotrophic lateral sclerosis, frontotemporal lobar degeneration and multisystem proteinopathy, which, intriguingly, are also associated with VCP mutations and where protein aggregates that contain components of SGs accumulate. This suggests that inappropriate SG dynamics may be relevant to pathogenesis. Our data are in line with such a hypothesis and suggest that deregulated autophagy, lysosomal or VCP activities may alter SG morphology and composition. This, in turn, may render the cells particularly vulnerable under challenging/disease conditions, thereby participating to disease progression.

2015 - Investigating the interplay between the protein quality control system, molecular chaperones and stress granules: from cell stress response to disease [Relazione in Atti di Convegno]
Carra, Serena
abstract

Stress granules (SGs) are transient heterogeneous mRNA-protein complexes induced during stress, which exert cellular pro-survival functions. Recent data implicate SGs and deregulated proteostasis in amyotrophic lateral sclerosis, frontotemporal lobar degeneration and multisystem proteinopathy, which are also associated with mutations of valosin-containing protein (VCP) and where protein aggregates that contain components of SGs accumulate. This suggests that inappropriate SG dynamics may be relevant to pathogenesis. Interestingly, SG formation is driven by the reversible self-aggregation of mRNA-binding proteins that contain prion-like domains. Unlike prionogenic fibrillar aggregates, SGs are dynamic structures, which disassemble within few hours after their formation, even if the stress persists. Due to the heterogeneous composition of SGs and to the crowded molecular environment, SGs may, indirectly, require protein quality control (PQC) assistance for proper assembly and disassembly. Previously, the autophagy-lysosome pathway and VCP, key players of the PQC, were shown to regulate SG degradation (Buchan et al., Cell 2013). Here we investigated whether impairment of PQC, autophagy and lysosome-mediated degradation may affect SG response. We provide evidence supporting that inhibition of VCP, autophagy or lysosomes affects SG formation, morphology and composition. In particular, defective ribosomal products (DRIPs) and the large ribosome subunit 60S, which are released from disassembling polysomes, are normally excluded from SGs. Instead, we found that both DRIPs and 60S were significantly retained within and/or adjacent to SGs in cells with impaired autophagy, lysosome, or VCP function (Seguin et al., CDD 2014). Next, we observed that depletion of other chaperones and co-chaperones involved in the degradation of ubiquitinated proteins and DRIPs also affect SG dynamics. Consistently, overexpression of specific chaperone/co-chaperones can modulate the levels of SG markers. These findings further reinforce the hypothesis that PQC and SGs are tightly interconnected. Collectively our data suggest that besides causing a protein homeostasis imbalance, deregulated autophagy, lysosomal or chaperone activities may also alter SG morphology, composition and dynamics. This, in turn, may render the cells particularly vulnerable under challenging/disease conditions, thereby participating to disease progression.

2015 - OVEREXPRESSION OF HSPB8 PROTECTS AGAINST TDP43-MEDIATED TOXICITY IN DROSOPHILA [Poster]
Ganassi, Massimo; Diacci, Chiara; Seguin, SAMUEL JOSEPH ANDRE'; Zelotti, Elena; Morelli, FEDERICA FRANCESCA; Crippa, Valeria; Poletti, Angelo; Gregory, Jenna M; Dobson, Christopher M; Pandey, Udai B; Carra, Serena
abstract

Several neurodegenerative diseases are characterized by the accumulation of aggregates. Boosting aggregate clearance by proteasome and autophagy, with the assistance of chaperones, exerts protective functions in these diseases. We focus on the HSPB8-BAG3-Hsp70 chaperone complex. In cells HSPB8 reduces the aggregation of a truncated form of TDP43 associated with ALS (Crippa 2010). First, we tested whether overexpression of HSP67Bc (Drosophila HSPB8) decreases TDP43-mediated toxicity in vivo, using Drosophila expressing mutated or truncated TDP43. HSP67Bc decreases the eye degeneration caused by NLS-TDP43, which by accumulating in the cytosol causes toxicity (Ritson 2010). This correlates with a reduction of NLS-TDP43 protein levels mediated by HSP67Bc. Inversely, silencing HSP67Bc increases both NLS-TDP43 and ubiquitinated proteins levels, suggesting that HSP67Bc participates to proteostasis. Next, we used flies expressing a truncated form of TDP43 (TDP35), which causes pupae lethality (similarly to truncated TDP25; Gregory 2012). Co-expression of HSP67Bc with TDP35 rescued pupae lethality, further supporting its protective role in vivo. We then asked how mechanistically HSPB8 protects against TDP43-mediated toxicity. Recent studies revealed the presence of RNA-binding proteins component of stress granules (SG) in the proteinaceous inclusions. SGs are mRNA-protein aggregates induced by stress, which serve prosurvival functions. Interestingly, autophagy and specific chaperones indirectly assist SG dynamics (Buchan 2013; Seguin 2014). We investigated whether the HSPB8-BAG3-Hsp70 complex, may indirectly modulate SG dynamics. Intriguingly, upon stress HSPB8 is recruited into SG whereas BAG3 colocalizes with ubiquitinated defective ribosomal products, which are excluded from, but adjacent to SG. Our data open the possibility that HSPB8 and BAG3 may contribute to regulate both protein clearance and SG dynamics, thereby assisting restoration of protein and RNA homeostasis.

2015 - Role of HSPB8 in the Proteostasis Network: From Protein Synthesis to Protein Degradation and Beyond [Capitolo/Saggio]
Poletti, Angelo; Carra, Serena
abstract

Proper protein folding is crucial for protein stability and function; when folding fails, due to stress or genetic mutations, proteins may become toxic. Cells have evolved a complex protein quality control (PQC) system to protect against the toxicity exerted by aberrantly folded proteins, that may aggregate accumulating in various cellular compartments perturbing essential cellular activities, ultimately leading to cell and neuron death. The PQC comprises molecular chaperones, degradative systems (proteasome and autophagy) and components of the unfolded protein response. Prevention of protein aggregation, clearance of misfolded substrates and attenuation of translation, which decreases the amount of misfolding clients to levels manageable by the molecular chaperones, are all key steps for the maintenance of proteostasis and cell survival. In parallel, alterations of proteostasis may also (indirectly) influence RNA homeostasis; in fact, RNA-containing aggregates, known as stress granules, accumulate in cells with impaired PQC and autophagy colocalizing with proteinaceous aggregates in several neurodegenerative diseases. Among the different molecular chaperones, here we will focus on the small heat shock protein HSPB8, which is expressed in neurons in basal conditions and upregulated in response to misfolded protein accumulation. HSPB8 exerts protective functions in several models of protein conformation neurodegenerative diseases. The putative sites of action of HSPB8 that confer HSPB8 pro-survival and anti-aggregation functions are discussed, as well as its potential role at the crossroad between proteostasis and ribostasis.

2015 - VCP AND AUTOPHAGOLYSOSOMAL PATHWAY: GUARDIANS OF PROTEOSTASIS AND STRESS GRANULE DYNAMICS. UNRAVELING THEIR IMPLICATIONS IN ALS [Poster]
Ganassi, Massimo; Bigi, Ilaria; Seguin, SAMUEL JOSEPH ANDRE'; Morelli, FEDERICA FRANCESCA; Mandrioli, J; Cereda, C; Poletti, A; Carra, Serena
abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease comprising clinically indistinguishable sporadic (s) and familial (f) forms, associated with an aberrant behavior of a number of gene products (SOD1, TDP-43, FUS, UBQLN2, VCP, FIG4, CHMP2B, SQSTM1, C9orf72). Most of these proteins are involved in protein degradation via the ubiquitin proteasome (UPP) or the autophagolysosomal (APLP) pathways, as well as in RNA processing or stress granule (SG) response. In ALS, motoneurons accumulate protein aggregates that contain RNA-binding proteins markers of SGs. Thus, proteostasis (mediated by the protein quality control, PQC) and ribostasis (involving SGs) may be interconnected and their imbalance may participate to ALS. At present, it is largely unknown whether interplay between PQC (chaperones, UPP and APLP) and SG dynamics exists and to what extent deregulated PQC may affect SGs, thereby contributing to ALS. Notably, valosin containing protein (VCP) assists with autophagy SG clearance (1) and we found that inhibition of autophagy, lysosomes and depletion of VCP alter SG size, number and composition, pointing to APLP and chaperone-assisted degradation as interconnected processes. These data imply that imbalances in proteostasis and deregulated APLP, which occur in ALS, will affect SG morphology and composition; thus, defective SG response may also contribute to ALS. Here, we will: 1) dissect how interplay between PQC, VCP and SGs occurs, identifying new players involved in this process and specific clients whose VCP-assisted/APLP-mediated degradation affects SGs; 2) identify the functional consequences of impaired SG response; 3) test whether boosting chaperone-assisted degradation or client targeting may rescue SG morphology and composition. Using motoneuronal ALS cell models we will characterize the impact of ALS mutants of VCP on SG and proteostasis. In parallel, we will characterize and compare SG morphology and composition in fibroblasts and lymphoblasts derived from ALS patients with mutations in VCP, TDP-43, FUS, SOD1 and C9orf72, as well as in motoneurons derived from ALS-iPSCs. Our approach will provide mechanistic insight on the interplay between VCP, PQC and SGs. It will also demonstrate whether/how SG composition and dynamics are affected in ALS and how this correlates with proteostasis alterations, representing a common pathogenic mechanism; finally, our data will provide the molecular basis for the design of new therapeutic strategy.

2014 - Autophagy researchers [Scheda bibliografica]
Carra, Serena; Malagoli, Davide; Ney, Pa; Steffan, Js
abstract

Brief description of the various PI works and interest, especially centered on the autophagy field

2014 - BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch [Articolo su rivista]
Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, FEDERICA FRANCESCA; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena
abstract

Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the "BAG-instructed proteasomal to autophagosomal switch and sorting" (BIPASS).

2014 - Barcoding heat shock proteins to human diseases: looking beyond the heat shock response [Articolo su rivista]
Kakkar, Vaishali; Meister Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H.
abstract

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review.

2014 - Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly [Relazione in Atti di Convegno]
Carra, Serena
abstract

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

2014 - Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly [Articolo su rivista]
Seguin, SAMUEL JOSEPH ANDRE'; Morelli, FEDERICA FRANCESCA; Vinet, Jonathan; Amore, D; DE BIASI, Sara; Poletti, A; Rubinsztein, Dc; Carra, Serena
abstract

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

2014 - Upregulation of HSPB8 as potential therapeutic approach in Amyotrophic Lateral Sclerosis [Relazione in Atti di Convegno]
Carra, Serena
abstract

Several neurodegenerative diseases are characterized by the accumulation of aggregates. Boosting aggregate clearance by proteasome and autophagy, with the assistance of chaperones, exerts protective functions in these diseases. We focus on the HSPB8-BAG3-Hsp70 chaperone complex. In cells HSPB8 reduces the aggregation of a truncated form of TDP43 associated with ALS (Crippa 2010). First, we tested whether overexpression of HSP67Bc (Drosophila HSPB8) decreases TDP43-mediated toxicity in vivo, using Drosophila expressing mutated or truncated TDP43. HSP67Bc decreases the eye degeneration caused by NLS-TDP43, which by accumulating in the cytosol causes toxicity (Ritson 2010). This correlates with a reduction of NLS-TDP43 protein levels mediated by HSP67Bc. Inversely, silencing HSP67Bc increases both NLS-TDP43 and ubiquitinated proteins levels, suggesting that HSP67Bc participates to proteostasis. Next, we used flies expressing a truncated form of TDP43 (TDP35), which causes pupae lethality (similarly to truncated TDP25; Gregory 2012). Co-expression of HSP67Bc with TDP35 rescued pupae lethality, further supporting its protective role in vivo.

2013 - BAG3 induces the sequestration of ubiquitinated proteins into cytoplasmic puncta and re-routes them to autophagy upon proteasomal impairment [Poster]
Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena
abstract

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). As BAG3, a partner of the heat shock proteins HSPB8 and Hsp70, stimulates autophagy and its levels increase with aging, a condition characterized by decreased proteasome function and autophagy activation, it is tempting to speculate that BAG3 is required to re-route ubiquitinated clients to autophagy. Here, we show that BAG3 interacts via its BAG domain with ubiquitinated proteins and induces their sequestration into cytoplasmic puncta. Similarly, BAG3 drives, in an Hsp70-dependent manner, the recruitment of the proteasome client Ub-R-GFP into similar cytoplasmic puncta. These cytoplasmic puncta are co-labelled with canonical autophagy markers and linkers, suggesting that proteasomal client are re-routed to autophagy by BAG3. Indeed, upon proteasome inhibition ubiquitinated (insoluble) proteins accumulate in control cells, whilst in cells overexpressing BAG3 they are efficiently re-routed to autophagy for clearance. This action might be independent of HSPB8, which we find to dissociate from BAG3 early after proteasomal inhibition. Rather, HSPB8 becomes associated with RNA-containing stress granules, likely participating in translational arrest under proteasomal stress. Upon prolonged proteasomal inhibition, HSPB8 is then also massively recruited to the BAG3-positive puncta, tentatively to contribute to autophagy-mediated protein degradation of (other) accumulating misfolded substrates.

2013 - BAG3-mediated re-routing of protein degradation towards autophagy upon proteasomal impairment [Relazione in Atti di Convegno]
Carra, Serena
abstract

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). As BAG3, a partner of the heat shock proteins HSPB8 and Hsp70, stimulates autophagy and its levels increase with aging, a condition characterized by decreased proteasome function and autophagy activation, it is tempting to speculate that BAG3 is required to re-route ubiquitinated clients to autophagy. Here, we show that BAG3 interacts via its BAG domain with ubiquitinated proteins and induces their sequestration into cytoplasmic puncta. Similarly, BAG3 drives, in an Hsp70-dependent manner, the recruitment of the proteasome client Ub-R-GFP into similar cytoplasmic puncta. These cytoplasmic puncta are co-labelled with canonical autophagy markers and linkers, suggesting that proteasomal client are re-routed to autophagy by BAG3. Indeed, upon proteasome inhibition ubiquitinated (insoluble) proteins accumulate in control cells, whilst in cells overexpressing BAG3 they are efficiently re-routed to autophagy for clearance. This action might be independent of HSPB8, which we find to dissociate from BAG3 early after proteasomal inhibition. Rather, HSPB8 becomes associated with RNA-containing stress granules, likely participating in translational arrest under proteasomal stress. Upon prolonged proteasomal inhibition, HSPB8 is then also massively recruited to the BAG3-positive puncta, tentatively to contribute to autophagy-mediated protein degradation of (other) accumulating misfolded substrates.

2013 - BAG3-mediated re-routing of protein degradation towards autophagy upon proteasomal impairment [Poster]
Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena
abstract

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). How the proteasomal clients are re-routed to the autophagosomal pathway has remained unclear. We found that several stress pathways that are induced upon disturbances in protein homeostasis as well as by direct chemically-induced proteasomal inhibition increase the protein levels of BAG3, a partner of the heat shock proteins HSPB8 and Hsp70. BAG3 induces the sequestration of Hsp70-bound proteasomal clients into cytoplasmic puncta which leads to their re-routing to autophagosomes for degradation. This occurs through competitive inhibition with its family member BAG1, which normally directs Hsp70-bound clients to the proteasome.

2013 - Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease. [Poster]
Heldens, Lonneke; Morelli, FEDERICA FRANCESCA; Verbeek, Dineke; Vinet, Jonathan; Angelini, Corrado; Boelens, Wilbert; Tupler, Rossella; Carra, Serena
abstract

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2 with a defined 1:3 ratio. The HSPB2/HSPB3 complex is induced during muscle differentiation and plays a role in muscle maintenance. Recently the R7S mutation in HSPB3 has been associated with distal hereditary motor neuropathy type 2C (dHMN 2C). Here we report the identification in myopathic patients of two novel mutations in HSPB3: 1) one mutation affects the R116 residue, which corresponds to a key amino acid in the alpha-crystallin domain, whose mutation in other members of the HSPB family also causes disease (it is equivalent to e.g. R120 in HSPB5, whose mutation into G causes MFM and to K141 in HSPB8, whose mutation into E or N causes dHMN); 2) the other mutation disrupts the reading frame leading to a premature stop codon at amino acid 50. Both mutations were not found in more than 400 normal alleles. Expression studies allowed us to confirm that the mutation causing a premature stop codon leads to the generation of an unstable protein that is likely immediately degraded after synthesis and cannot be detected. Also, while both expressed, the R7S mutant was more stable than the R116 one. We next characterized in cells and in vitro the ability of these HSPB3 mutants to interact with HSPB2 and form the HSPB2/HSPB3 complex. We found that while the R7S mutant of HSPB3 was still able to interact with HSPB2, the R116 mutant was not. Future studies will allow us to better characterize how these HSPB3 mutants affect HSPB3 and, indirectly, HSPB2 stability, subcellular localization and function. They will also elucidate on HSPB3 and HSPB2 function in both motor neurons and myoblasts and will shed light on how mechanistically the mutations in HSPB3 affect the function and viability of these cell types, contributing to disease.

2013 - Characterization of the myopathy associated BAG3 P209L mutation [Poster]
Meister, Melanie; Minoia, Melania; Kanon, Bart; Carra, Serena; Kampinga, Harm H.
abstract

Myofibrillar myopathy is a protein aggregate myopathy characterized by disintegration of the Z-disk and accumulation of protein aggregates. A dominant missense mutation (P209L) in the co-chaperone BAG3 has been associated with a severe early onset form of myofibrillar myopathy. The disease causing P209L mutation in BAG3 is located in it’s binding site for HSPB8 (IPV domain), suggesting it might alter a functional HSPB8-BAG3 interaction. Recently we showed that wildtype BAG3, in association with HSPB8 and HSP70, participates in prevention of misfolded protein aggregation through macro-autophagy. In cultured HEK293T-cells the BAG3 P209L mutant was found to be impaired in its ability to prevent polyQ aggregation. This, however occurs without major effects on binding to the BAG3-partners HSPB8 and HSP70. Just like wildtype BAG3, the BAG3 P209L mutant was able to enhance LC3 lipidation, suggesting that its effect on autophagy stimulation is not impaired. This was confirmed by the absence of direct effects of mutant BAG3 on autophagic flux, instead the mutant strikingly showed impairment in cargo-delivery. Our studies in Drosophila Melanogaster further substantiate the loss of function phenotype of BAG3 P209L as the main mechanism underlying BAG3 P209L-related myopathy.

2013 - Clearance of the mutant androgen receptor in motoneuronal models of spinal and bulbar muscular atrophy. [Articolo su rivista]
Rusmini, P; Crippa, V; Giorgetti, E; Boncoraglio, A; Cristofani, R; Carra, Serena; Poletti, A.
abstract

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.

2013 - D4Z4 reduced allele in myopathic subjects with no FSHD phenotype: why inconsistency between molecular and clinical data should prompt us to further investigations. [Poster]
Daolio, Jessica; Nikolic, Ana; Ricci, Giulia; Govi, Monica; Mele, Fabiano; Carra, Serena; Vercelli, L.; Antonini, G.; Berardinelli, Maria Angela; Mongini, T.; Servida, M.; Ruggiero, L.; Angelini, C.; Cao, M.; D’Angelo, G.; Muzio, A. Di; Moggio, M.; Morandi, L.; Ricci, Eusebio; Rodolico, C.; Santoro, Luisa; Siciliano, G.; Tomelleri, G.; Tupler, Rossella
abstract

Aim of the study. Chacacterization of different FSHD clinical subsets and/or more complex myopathic conditions among carriers of D4Z4 reduced allele (DRA) associated with atypical myopathic phenotypes. Materials and methods. Out of 751 consecutive index cases recruited from the Italian National Registry for FSHD, we identified 298 myopathic patients carrying DRA with 4-8 repats who did not present obvious FSHD phenotype. Re-evaluation of clinical data and depth molecular characterization of 4q35 region were performed. Results. Twenty-three carriers of 4-8 DRA presented myopathic features related to non-FSHD conditions. In this group we observed high clinical heterogeneity. In particular subjects displayed different combinations of clinical signs that were considered atypical for FSHD: 1) involvement of muscles that are not usually affected in FSHD, 2) sparing of muscles that are typically affected in FSHD, 3) additional clinical, genetic and instrumental features that are not included in the FSHD phenotype. In addition, Molecular analysis of the 4q35 subtelomeric haplotype of myopathic DRA patients did not reveal any molecular element enabling us to differentiate these patients from classical FSHD patients. In two patients, heterozygous mutations were found in CAV-3 and HSPB3 genes. Interestingly, these patients were sporadic and no other myopathic subjects were reported in the two families. Discussion and conclusions. Our study shows that not all myopathic patients carrying 4-8 DRA are affected by FSHD. We conclude that in myopathic carriers of 4q short allele who do not display a typical autosomal dominant FSHD additional molecular and clinical investigations should be performed for a more precise characterization of these patients. This approach will favor clinical diagnosis and genetic counseling.

2013 - Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders. [Articolo su rivista]
Carra, Serena; Rusmini, P; Crippa, V; Giorgetti, E; Boncoraglio, A; Cristofani, R; Naujock, M; Meister, M; Minoia, M; Kampinga, Hh; Poletti, A.
abstract

The family of the mammalian small heat-shock proteins consists of 10 members (sHSPs/HSPBs: HSPB1-HSPB10) that all share a highly conserved C-terminal alpha-crystallin domain, important for the modulation of both their structural and functional properties. HSPB proteins are biochemically classified as molecular chaperones and participate in protein quality control, preventing the aggregation of unfolded or misfolded proteins and/or assisting in their degradation. Thus, several members of the HSPB family have been suggested to be protective in a number of neurodegenerative and neuromuscular diseases that are characterized by protein misfolding. However, the pro-refolding, anti-aggregation or pro-degradative properties of the various members of the HSPB family differ largely, thereby influencing their efficacy and protective functions. Such diversity depends on several factors, including biochemical and physical properties of the unfolded/misfolded client, the expression levels and the subcellular localization of both the chaperone and the client proteins. Furthermore, although some HSPB members are inefficient at inhibiting protein aggregation, they can still exert neuroprotective effects by other, as yet unidentified, manners; e.g. by maintaining the proper cellular redox state or/and by preventing the activation of the apoptotic cascade. Here, we will focus our attention on how the differences in the activities of the HSPB proteins can influence neurodegenerative and neuromuscular disorders characterized by accumulation of aggregate-prone proteins. Understanding their mechanism of action may allow us to target a specific member in a specific cell type/disease for therapeutic purposes.

2013 - Differential autophagy power in the spinal cord and muscle of transgenic ALS mice [Articolo su rivista]
Crippa, Valeria; Boncoraglio, Alessandra; Galbiati, Mariarita; Aggarwal, Tanya; Rusmini, Paola; Giorgetti, Elisa; Cristofani, Riccardo; Carra, Serena; Pennuto, Maria; Poletti, Angelo
abstract

Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and to a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analyzed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 mice at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences were also considered. No changes of HSPB8, BAG3, and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analyzed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cord. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded proteins accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.

2013 - Implications of HSPBs and BAG3 in neuro/muscular-protein aggregate diseases [Poster]
Carra, Serena
abstract

The mammalian family of small heat shock proteins (sHSP/HSPB) consists of ten members (HSPB1-HSPB10), which display different expression profiles and different functions. While some members exert mainly refolding activities and have very poor or no anti-aggregation properties (e.g. HSPB1, HSPB4), other members display strong anti-aggregation function (e.g. HSPB7, HSPB8). Moreover, other members seem to exert specialized activities (e.g. HSPB2 and HSPB3 modulate muscle differentiation). Besides these different aspects, the HSPBs also have common properties and alteration of these properties/functions seem to represent a key mechanism leading to disease. In fact, mutations in HSPB1, HSPB3, HSPB4, HSPB5 and HSPB8, as well as the HSPB8 partner BAG3, cause neurological or muscular disorders. These mutations can lead to disease via a gain of function (GOF) mechanism (due to protein instability and tendency to aggregate), or via a loss of function (LOF) mechanism, which can occur as a direct consequence of the GOF or separately (e.g. mutations leading to a truncated non-functional HSPB protein). In parallel, boosting the function of specific HSPBs and their partners (e.g. BAG3) may represent an attractive approach to combat protein aggregate diseases. How alteration (mutation) or modulation (upregulation) of HSPB and BAG3 function may either lead to or combat disease with a special focus on the intracellular pathways crucial for their activity will be addressed here.

2013 - Implications of HSPBs and BAG3 in protein aggregate neuro/muscular diseases [Poster]
Carra, Serena
abstract

The mammalian family of small heat shock proteins (sHSP/HSPB) consists of ten members (HSPB1-HSPB10), which display different expression profiles and different functions. While some members exert mainly refolding activities and have very poor or no anti-aggregation properties (e.g. HSPB1, HSPB4), other members display strong anti-aggregation function (e.g. HSPB7, HSPB8). Moreover, other members seem to exert specialized activities (e.g. HSPB2 and HSPB3 modulate muscle differentiation). Besides these different aspects, the HSPBs also have common properties and alteration of these properties/functions seem to represent a key mechanism leading to disease. In fact, mutations in HSPB1, HSPB3, HSPB4, HSPB5 and HSPB8, as well as the HSPB8 partner BAG3, cause neurological or muscular disorders. These mutations can lead to disease via a gain of function (GOF) mechanism (due to protein instability and tendency to aggregate), or via a loss of function (LOF) mechanism, which can occur as a direct consequence of the GOF or separately (e.g. mutations leading to a truncated non-functional HSPB protein). Here we will focus on mutations of HSPB3 and HSPB8 and their implication in diseases affecting motor neurons and muscle cells. In parallel, boosting the function of specific HSPBs (and their partners, e.g. BAG3) may represent an attractive approach to combat protein aggregate diseases. How modulation of specific HSPBs may help to combat disease will be also addressed here.

2013 - Implications of HSPBs and BAG3 in protein aggregate neuro/muscular diseases [Relazione in Atti di Convegno]
Carra, Serena
abstract

The mammalian family of small heat shock proteins (sHSP/HSPB) consists of ten members (HSPB1-HSPB10), which display different expression profiles and different functions. While some members exert mainly refolding activities and have very poor or no anti-aggregation properties (e.g. HSPB1, HSPB4), other members display strong anti-aggregation function (e.g. HSPB7, HSPB8). Moreover, other members seem to exert specialized activities (e.g. HSPB2 and HSPB3 modulate muscle differentiation). Besides these different aspects, the HSPBs also have common properties and alteration of these properties/functions seem to represent a key mechanism leading to disease. In fact, mutations in HSPB1, HSPB3, HSPB4, HSPB5 and HSPB8, as well as the HSPB8 partner BAG3, cause neurological or muscular disorders. These mutations can lead to disease via a gain of function (GOF) mechanism (due to protein instability and tendency to aggregate), or via a loss of function (LOF) mechanism, which can occur as a direct consequence of the GOF or separately (e.g. mutations leading to a truncated non-functional HSPB protein). Here we will focus on mutations of HSPB3 and HSPB8 and their implication in diseases affecting motor neurons and muscle cells. In parallel, boosting the function of specific HSPBs (and their partners, e.g. BAG3) may represent an attractive approach to combat protein aggregate diseases. How modulation of specific HSPBs may help to combat disease will be also addressed here.

2013 - Motoneuron and muscle selective removal of ALS-related misfolded proteins. [Poster]
Crippa, Valeria; Galbiati, Mariarita; Boncoraglio, Alessandra; Rusmini, Paola; Onesto, Elisa; Zito, Arianna; Giorgetti, Elisa; Cristofani, Riccardo; Aggarwal, Tanya; Pennuto, Maria; Carra, Serena; Poletti, Angelo
abstract

Amyotrophic lateral sclerosis (ALS) occurs in clinically indistinguishable sporadic (sALS) or familial (fALS) forms. Most of the fALS-related mutant proteins identified so far, like mutant SOD1, TDP-43, FUS, etc., are prone to misfold; the product of the mutant C9ORF72 gene aberrantly codes for small highly hydrophobic dipeptides. Both misfolded proteins and hydrophobic peptides accumulate into insoluble proteinaceous material inside motoneurons. This material must be cleared away from cells with the assistance of the molecular chaperones. Chaperones may act on aberrant proteins either by assisting their refolding, or by directing them to degradation through the proteasome (UPS) or the autophagic system. Motoneurons are very sensitive to misfolded protein toxicity, but other cell types, such as astrocytes, oligodendrocytes, muscle cells could also be affected by their presence. Notably, muscle-restricted expression of mutant SOD1 (mutSOD1), responsible for some fALS, induces muscle atrophy and motoneurons death. We found that several genes are altered in mutSOD1 mice muscles. In fact, we observed up-regulation of typical muscle genes, such as MyoD, myogenin, but also of several components of cell response to proteotoxicity (atrogin-1, HspB8, Bag1, Bag3). Similar changes were found to occur in cultured ALS myoblasts. We then compared the potential mutSOD1 toxicity in motoneuron (NSC34) and muscle (C2C12) cells. Initially, we found that muscle ALS models possess much higher chimotryptic proteasome activity and autophagy power than motoneuron ALS models. The mutSOD1 molecular behaviour was also very different. MutSOD1 clearance was much higher in muscle than in motoneurons and the misfolded protein formed aggregates and impaired proteasome only in motoneurons. The motoneuronal cells were also more sensitive to superoxide-induced oxidative stress. In muscle cells, mutSOD1 remained soluble even after proteasome inhibition, possibly because of high mutSOD1 autophagic clearance. Finally, a N-terminal TDP-43 fragment accumulated in NSC34, but not in C2C12 cells. In the case of TDP-43, proteasome inhibition resulted in a large accumulation of both wt and N-terminal fragment of TDP-43. Therefore, our results suggest that muscle cells differentially manage misfolded mutSOD1 and TDP-43 and their toxicity in muscle may not directly depend on aggregation.

2013 - The regulation of the autophagic network and its implications for human disease [Articolo su rivista]
Yang, Jing; Carra, Serena; Zhu, Wei Guo; Kampinga, Harm H.
abstract

Autophagy has attracted a lot of attention in recent years. More and more proteins and signaling pathways have been discovered that somehow feed into the autophagy regulatory pathways. Regulation of autophagy is complex and condition-specific, and in several diseases, autophagic fluxes are changed. Here, we review the most well-established concepts in this field as well as the reported signaling pathways or components which steer the autophagy machinery. Furthermore, we will highlight how autophagic fluxes are changed in various diseases either as cause for or as response to deal with an altered cellular homeostasis and how modulation of autophagy might be used as potential therapy for such diseases.

2013 - Upregulation of HSPB8 as potential therapeutic approach in familial and sporadic ALS [Poster]
Carra, Serena; Crippa, V; Boncoraglio, A; Seguin, SAMUEL JOSEPH ANDRE'; Cristofani, R; Rusmini, P; Giorgetti, E; Poletti, A.
abstract

Several data suggest that accumulation of aggregated proteins (mutated SOD1, TDP-43 and FUS/TLS) plays an important role in motor neuronal cell death occurring in Amyotrophic Lateral Sclerosis (ALS). Protein aggregation results from the formation of aberrant conformations (misfolding) of aggregate-prone proteins, some of which have been found mutated in the familial forms of ALS. The removal of misfolded (aggregated) proteins is operated by the cells via two major degradative systems the ubiquitin-proteasome pathway (UPP) and the autophagy. Both systems require the assistance of intracellular chaperons. The molecular chaperones recognize and bind misfolded proteins, preventing their aggregation and facilitating their degradation, thus exerting neuroprotective functions. In this project, we will focus on the chaperone HSPB8, which forms a stable complex with the co-chaperone Bag3. Overexpression of HSPB8 (and Bag3) prevents aggregation of mutated SOD1 and TDP-43, associated with familial and sporadic ALS, by increasing their degradation via autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. Thus, HSPB8 (and Bag3) may help motor neurons to cope with misfolded TDP-43 and SOD1 by either directly targeting them to the autophagic vacuoles for degradation and/or restoring/boosting the autophagy flux. Interestingly, deregulated autophagy is amongst the causes for motor neuron diseases (MNDs), further pointing to a link between protein aggregation, protein quality control, HSPB8-Bag3 and autophagy. Besides, mislocalization/aggregation of TDP-43 and FUS/TLS to the mRNA containing cytoplasmic stress granules (SGs) alters RNA metabolism and has been suggested as pathomechanism contributing to ALS. Our preliminary data indicate that HSPB8 is recruited to SG, where it colocalizes with TDP-43. Therefore, HSPB8, by either preventing the mislocalization/aggregation of mutated TDP-43 and FUS to SG and/or by targeting them for degradation may also contribute to disease amelioration by avoiding impairment of specific RNA translation/processing. The hypothesis that HSPB8 may exert essential functions for motor neuron viability is further supported by the observation that HSPB8 is upregulated in surviving motor neurons in both an ALS mouse model and in human ALS tissues, as well as by the finding that mutated forms of HSPB8 (which are found in aggregates) cause dominant hereditary motor neuropathy. In this project, we will investigate the hypothesis that upregulation of HSPB8 (and Bag3) may protect against ALS, using both motor neuronal cells and the mutated SOD1, TDP-43 and FUS/TLS based Drosophila models of ALS. We will also perform a drug screening to find compounds able to induce HSPB8 expression specifically in motor neurons. This work will provide insights in the role of HSPB8 as modulator of ALS and will identify specific sites of action of HSPB8 whose modulation may also represent new therapeutic targets for ALS.

2012 - Alteration of protein folding and degradation in motor neuron diseases: Implications and protective functions of small heat shock proteins. [Articolo su rivista]
Carra, Serena; Crippa, V; Rusmini, P; Boncoraglio, A; Minoia, M; Giorgetti, E; Kampinga, Hh; Poletti, A.
abstract

Motor neuron diseases (MNDs) are neurodegenerative disorders that specifically affect the survival and function of upper and/or lower motor neurons. Since motor neurons are responsible for the control of voluntary muscular movement, MNDs are characterized by muscle spasticity, weakness and atrophy. Different susceptibility genes associated with an increased risk to develop MNDs have been reported and several mutated genes have been linked to hereditary forms of MNDs. However, most cases of MNDs occur in sporadic forms and very little is known on their causes. Interestingly, several molecular mechanisms seem to participate in the progression of both the inherited and sporadic forms of MNDs. These include cytoskeleton organization, mitochondrial functions, DNA repair and RNA synthesis/processing, vesicle trafficking, endolysosomal trafficking and fusion, as well as protein folding and protein degradation. In particular, accumulation of aggregate-prone proteins is a hallmark of MNDs, suggesting that the protein quality control system (molecular chaperones and the degradative systems: ubiquitin-proteasome-system and autophagy) are saturated or not sufficient to allow the clearance of these altered proteins. In this review we mainly focus on the MNDs associated with disturbances in protein folding and protein degradation and on the potential implication of a specific class of molecular chaperones, the small heat shock proteins (sHSPs/HSPBs), in motor neuron function and survival. How boosting of specific HSPBs may be a potential useful therapeutic approach in MNDs and how mutations in specific HSPBs can directly cause motor neuron degeneration is discussed.

2012 - Guidelines for the use and interpretation of assays for monitoring autophagy. [Articolo su rivista]
Klionsky, Dj; Abdalla, Fc; Abeliovich, H; Abraham, Rt; Acevedo Arozena, A; Adeli, K; Agholme, L; Agnello, M; Agostinis, P; Aguirre Ghiso, Ja; Ahn, Hj; Ait Mohamed, O; Ait Si Ali, S; Akematsu, T; Akira, S; Al Younes, Hm; Al Zeer, Ma; Albert, Ml; Albin, Rl; Alegre Abarrategui, J; Aleo, Mf; Alirezaei, M; Almasan, A; Almonte Becerril, M; Amano, A; Amaravadi, R; Amarnath, S; Amer, Ao; Andrieu Abadie, N; Anantharam, V; Ann, Dk; Anoopkumar Dukie, S; Aoki, H; Apostolova, N; Auberger, P; Baba, M; Backues, Sk; Baehrecke, Eh; Bahr, Ba; Bai, Xy; Bailly, Y; Baiocchi, R; Baldini, G; Balduini, W; Ballabio, A; Bamber, Ba; Bampton, Et; Bánhegyi, G; Bartholomew, Cr; Bassham, Dc; Bast RC, Jr; Batoko, H; Bay, Bh; Beau, I; Béchet, Dm; Begley, Tj; Behl, C; Behrends, C; Bekri, S; Bellaire, B; Bendall, Lj; Benetti, L; Berliocchi, L; Bernardi, H; Bernassola, F; Besteiro, S; Bhatia Kissova, I; Bi, X; Biard Piechaczyk, M; Blum, Js; Boise, Lh; Bonaldo, P; Boone, Dl; Bornhauser, Bc; Bortoluci, Kr; Bossis, I; Bost, F; Bourquin, Jp; Boya, P; Boyer Guittaut, M; Bozhkov, Pv; Brady, Nr; Brancolini, C; Brech, A; Brenman, Je; Brennand, A; Bresnick, Eh; Brest, P; Bridges, D; Bristol, Ml; Brookes, Ps; Brown, Ej; Brumell, Jh; Brunetti Pierri, N; Brunk, Ut; Bulman, De; Bultman, Sj; Bultynck, G; Burbulla, Lf; Bursch, W; Butchar, Jp; Buzgariu, W; Bydlowski, Sp; Cadwell, K; Cahová, M; Cai, D; Cai, J; Cai, Q; Calabretta, Bruno; Calvo Garrido, J; Camougrand, N; Campanella, M; Campos Salinas, J; Candi, E; Cao, L; Caplan, Ab; Carding, Sr; Cardoso, Sm; Carew, Js; Carlin, Cr; Carmignac, V; Carneiro, La; Carra, Serena; Caruso, Ra; Casari, G; Casas, C; Castino, R; Cebollero, E; Cecconi, F; Celli, J; Chaachouay, H; Chae, Hj; Chai, Cy; Chan, Dc; Chan, Ey; Chang, Rc; Che, Cm; Chen, Cc; Chen, Gc; Chen, Gq; Chen, M; Chen, Q; Chen, Ss; Chen, W; Chen, X; Chen, X; Chen, X; Chen, Yg; Chen, Y; Chen, Y; Chen, Yj; Chen, Z; Cheng, A; Cheng, Ch; Cheng, Y; Cheong, H; Cheong, Jh; Cherry, S; Chess Williams, R; Cheung, Zh; Chevet, E; Chiang, Hl; Chiarelli, R; Chiba, T; Chin, Ls; Chiou, Sh; Chisari, Fv; Cho, Ch; Cho, Dh; Choi, Am; Choi, D; Choi, Ks; Choi, Me; Chouaib, S; Choubey, D; Choubey, V; Chu, Ct; Chuang, Th; Chueh, Sh; Chun, T; Chwae, Yj; Chye, Ml; Ciarcia, R; Ciriolo, Mr; Clague, Mj; Clark, Rs; Clarke, Pg; Clarke, R; Codogno, P; Coller, Ha; Colombo, Mi; Comincini, S; Condello, M; Condorelli, F; Cookson, Mr; Coombs, Gh; Coppens, I; Corbalan, R; Cossart, P; Costelli, P; Costes, S; Coto Montes, A; Couve, E; Coxon, Fp; Cregg, Jm; Crespo, Jl; Cronjé, Mj; Cuervo, Am; Cullen, Jj; Czaja, Mj; D'Amelio, M; Darfeuille Michaud, A; Davids, Lm; Davies, Fe; De Felici, M; de Groot, Jf; de Haan, Ca; De Martino, L; De Milito, A; De Tata, V; Debnath, J; Degterev, A; Dehay, B; Delbridge, Lm; Demarchi, F; Deng, Yz; Dengjel, J; Dent, P; Denton, D; Deretic, V; Desai, Sd; Devenish, Rj; Di Gioacchino, M; Di Paolo, G; Di Pietro, C; Díaz Araya, G; Díaz Laviada, I; Diaz Meco, Mt; Diaz Nido, J; Dikic, I; Dinesh Kumar, Sp; Ding, Wx; Distelhorst, Cw; Diwan, A; Djavaheri Mergny, M; Dokudovskaya, S; Dong, Z; Dorsey, Fc; Dosenko, V; Dowling, Jj; Doxsey, S; Dreux, M; Drew, Me; Duan, Q; Duchosal, Ma; Duff, K; Dugail, I; Durbeej, M; Duszenko, M; Edelstein, Cl; Edinger, Al; Egea, G; Eichinger, L; Eissa, Nt; Ekmekcioglu, S; El Deiry, Ws; Elazar, Z; Elgendy, M; Ellerby, Lm; Eng, Ke; Engelbrecht, Am; Engelender, S; Erenpreisa, J; Escalante, R; Esclatine, A; Eskelinen, El; Espert, L; Espina, V; Fan, H; Fan, J; Fan, Qw; Fan, Z; Fang, S; Fang, Y; Fanto, M; Fanzani, A; Farkas, T; Farré, Jc; Faure, M; Fechheimer, M; Feng, Cg; Feng, J; Feng, Q; Feng, Y; Fésüs, L; Feuer, R; Figueiredo Pereira, Me; Fimia, Gm; Fingar, Dc; Finkbeiner, S; Finkel, T; Finley, Kd; Fiorito, F; Fisher, Ea; Fisher, Pb; Flajolet, M; Florez McClure, Ml; Florio, S; Fon, Ea; Fornai, F; Fortunato, F; Fotedar, R; Fowler, Dh; Fox, Hs; Franco, R; Frankel, Lb; Fransen, M; Fuentes, Jm; Fueyo, J; Fujii, J; Fujisaki, K; Fuj
abstract

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

2012 - SENSING AND REROUTING OF PROTEIN DEGRADATION TOWARDS AUTOPHAGY UPON PROTEASOMAL IMPAIRMENT [Poster]
Boncoraglio, Alessandra; Minoia, Melania; Brunsting, Jeanette F.; Reits, Eric; Kampinga, Harm H.; Carra, Serena
abstract

The accumulation of misfolded, mutant proteins is a common basis for many adult onset neurodegenerative diseases. Cells have evolved an elaborate protein quality control system, which acts to facilitate the folding or refolding of misfolded protein species by molecular chaperones or, if folding is unsuccessful, these same chaperones often target the misfolded proteins for degradation, thereby preventing protein aggregation. Intracellular degradation is primarily mediated by two proteolytic systems: the autophagy and the ubiquitin proteasomal systems. Proteotoxic stress can lead to proteasomal impairment and augmented authophagosomal capacity in order to ensure proper clearance of clients (proteasome-autophagy switch). However, neither the mechanism of sensing nor that of switching is understood. Here, we show that the ER is main sensor for proteasomal inhibition through the IRE-1alpha-Xbp-1 signalling cascade. After proteasome inhibition, BAG-3 is upregulated in a HSF-1 independent manner, but in a Xbp-1 dependent manner and is a major executor of the proteasome-autophagy switch. BAG-3 both boosts autophagy and redirects HSP70-bound proteasomal clients to autophagosomes through competitive inhibition with its family member BAG-1, that normally directs HSP70-bound clients to the proteasome, thus playing a key role in the maintenance of protein homeostasis under proteotoxic stress conditions.

2012 - The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases. [Articolo su rivista]
Seidel, K; Vinet, Jonathan; den Dunnen, Wfa; Brunt, Er; Meister, M; Boncoraglio, A; Zijlstra, Mp; Boddeke, Hwgm; Rüb, U; Kampinga, Hh; Carra, Serena
abstract

AIMS:HSPB8 is a small heat shock protein that forms a complex with the co-chaperone BAG3. Overexpression of the HSPB8-BAG3 complex in cells stimulates autophagy and facilitates the clearance of mutated aggregation-prone proteins, whose accumulation is a hallmark of many neurodegenerative disorders. HSPB8-BAG3 could thus play a protective role in protein aggregation diseases and might be specifically upregulated in response to aggregate-prone protein-mediated toxicity. Here we analysed HSPB8-BAG3 expression levels in post-mortem human brain tissue from patients suffering of the following protein conformation disorders: Alzheimer's disease, Parkinson's disease, Huntington's disease and spinocerebellar ataxia type 3 (SCA3).METHODS:Western blotting and immunohistochemistry techniques were used to analyse HSPB8 and BAG3 expression levels in fibroblasts from SCA3 patients and post-mortem brain tissues, respectively.RESULTS:In all diseases investigated, we observed a strong upregulation of HSPB8 and a moderate upregulation of BAG3 specifically in astrocytes in the cerebral areas affected by neuronal damage and degeneration. Intriguingly, no significant change in the HSPB8-BAG3 expression levels was observed within neurones, irrespective of their localization or of the presence of proteinaceous aggregates.CONCLUSIONS:We propose that the upregulation of HSPB8 and BAG3 may enhance the ability of astrocytes to clear aggregated proteins released from neurones and cellular debris, maintain the local tissue homeostasis and/or participate in the cytoskeletal remodelling that astrocytes undergo during astrogliosis.

2012 - The family of mammalian small heat shock proteins (HSPBs): Implications in protein deposit diseases and motor neuropathies. [Articolo su rivista]
Boncoraglio, A; Minoia, M; Carra, Serena
abstract

A number of neurological and muscular disorders are characterized by the accumulation of aggregate-prone proteins and are referred to as protein deposit or protein conformation diseases. Besides some sporadic forms, most of them are genetically inherited in an autosomal dominant manner, although recessive forms also exist. Although genetically very heterogeneous, some of these diseases are the result of mutations in some members of the mammalian small heat shock protein family (sHSP/HSPB), which are key players of the protein quality control system and participate, together with other molecular chaperones and co-chaperones, in the maintenance of protein homeostasis. Thus, on one hand upregulation of specific members of the HSPB family can exert protective effects in protein deposit diseases, such as the polyglutamine diseases. On the other hand, mutations in the HSPBs lead to neurological and muscular disorders, which may be due to a loss-of-function in protein quality control and/or to a gain-of-toxic function, resulting from the aggregation-proneness of the mutants. In this review we summarize the current knowledge about some of the best characterized functions of the HSPBs (e.g. role in cytoskeleton stabilization, chaperone function, anti-aggregation and anti-apoptotic activities), also highlighting differences in the properties of the various HSPBs and how these may counteract protein aggregation diseases. We also describe the mutations in the various HSPBs associated with neurological and muscular disorders and we discuss how gain-of-toxic function mechanisms (e.g. due to the mutated HSPB protein instability and aggregation) and/or loss-of-function mechanisms can contribute to HSPB-associated pathologies. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

2012 - Upregulation of HSPB8 as potential therapeutic approach in familial and sporadic ALS [Poster]
Carra, Serena; Crippa, V; Boncoraglio, A; Seguin, SAMUEL JOSEPH ANDRE'; Cristofani, R; Rusmini, P; Giorgetti, E; Poletti, A.
abstract

Several data suggest that accumulation of aggregated proteins (mutated SOD1, TDP-43 and FUS/TLS) plays an important role in motor neuronal cell death occurring in Amyotrophic Lateral Sclerosis (ALS). Protein aggregation results from the formation of aberrant conformations (misfolding) of aggregate-prone proteins, some of which have been found mutated in the familial forms of ALS. The removal of misfolded (aggregated) proteins is operated by the cells via two major degradative systems the ubiquitin-proteasome pathway (UPP) and the autophagy. Both systems require the assistance of intracellular chaperons. The molecular chaperones recognize and bind misfolded proteins, preventing their aggregation and facilitating their degradation, thus exerting neuroprotective functions. In this project, we will focus on the chaperone HSPB8, which forms a stable complex with the co-chaperone Bag3. Overexpression of HSPB8 (and Bag3) prevents aggregation of mutated SOD1 and TDP-43, associated with familial and sporadic ALS, by increasing their degradation via autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. Thus, HSPB8 (and Bag3) may help motor neurons to cope with misfolded TDP-43 and SOD1 by either directly targeting them to the autophagic vacuoles for degradation and/or restoring/boosting the autophagy flux. Interestingly, deregulated autophagy is amongst the causes for motor neuron diseases (MNDs), further pointing to a link between protein aggregation, protein quality control, HSPB8-Bag3 and autophagy. Besides, mislocalization/aggregation of TDP-43 and FUS/TLS to the mRNA containing cytoplasmic stress granules (SGs) alters RNA metabolism and has been suggested as pathomechanism contributing to ALS. Our preliminary data indicate that HSPB8 is recruited to SG, where it colocalizes with TDP-43. Therefore, HSPB8, by either preventing the mislocalization/aggregation of mutated TDP-43 and FUS to SG and/or by targeting them for degradation may also contribute to disease amelioration by avoiding impairment of specific RNA translation/processing. The hypothesis that HSPB8 may exert essential functions for motor neuron viability is further supported by the observation that HSPB8 is upregulated in surviving motor neurons in both an ALS mouse model and in human ALS tissues, as well as by the finding that mutated forms of HSPB8 (which are found in aggregates) cause dominant hereditary motor neuropathy. In this project, we will investigate the hypothesis that upregulation of HSPB8 (and Bag3) may protect against ALS, using both motor neuronal cells and the mutated SOD1, TDP-43 and FUS/TLS based Drosophila models of ALS. We will also perform a drug screening to find compounds able to induce HSPB8 expression specifically in motor neurons. This work will provide insights in the role of HSPB8 as modulator of ALS and will identify specific sites of action of HSPB8 whose modulation may also represent new therapeutic targets for ALS.

2011 - BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity. [Articolo su rivista]
Hishiya, A; Salman, Mn; Carra, Serena; Kampinga, Hh; Takayama, S.
abstract

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.

2011 - Emerging roles of molecular chaperones and co-chaperones in selective autophagy: focus on BAG proteins. [Articolo su rivista]
Gamerdinger, M; Carra, Serena; Behl, C.
abstract

Macroautophagy is a catabolic process by which the cell degrades cytoplasmic components through the lysosomal machinery. While initially acknowledged as a rather unspecific bulk degradation process, growing lines of evidence indicate the selectivity of macroautophagy pathways in the removal of misfolded or aggregated proteins. How such substrates are recognized and specifically targeted to the macroautophagy machinery has become a hotspot of investigation, and recent evidence suggests that here molecular chaperones and co-chaperones play a central role. One emerging pathway is mediated by the co-chaperone protein Bcl-2-associated athanogene 3 (BAG 3) which seems to utilize the specificity of molecular chaperones (heat-shock proteins) towards non-native proteins as basis for targeted macroautophagic degradation. In this short review, we focus on the molecular interplay between the macroautophagy system and molecular chaperones and highlight the relevance of the pathway mediated by BAG3 to aging and age-associated protein-misfolding diseases.

2011 - Small heat shock proteins, protein degradation and protein aggregation diseases. [Articolo su rivista]
Vos, Mj; Zijlstra, Mp; Carra, Serena; Sibon, Oc; Kampinga, Hh
abstract

Small heat shock proteins have been characterized in vitro as ATP-independent molecular chaperones that can prevent aggregation of un- or mis-folded proteins and assist in their refolding with the help of ATP-dependent chaperone machines (e.g., the Hsp70 proteins). Comparison of the functionality of the 10 human members of the small HSPB family in cell models now reveals that some members function entirely differently and independently from Hsp70 machines. One member, HSPB7, has strong activities to prevent toxicity of polyglutamine-containing proteins in cells and Drosophila, and seems to act by assisting the loading of misfolded proteins or small protein aggregates into autophagosomes.

2010 - A role of small heat shock protein B8 (HSPB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases. [Articolo su rivista]
Crippa, V; Carra, Serena; Rusmini, P; Sau, D; Bolzoni, E; Bendotti, C; De Biasi, S; Poletti, A.
abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signaling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motor neuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.

2010 - Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3) [Articolo su rivista]
Sun, X; Fontaine, Jm; Hoppe, Ad; Carra, Serena; Deguzman, C; Martin, Jl; Simon, S; Vicart, P; Welsh, Mj; Landry, J; Benndorf, R.
abstract

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

2010 - CYTOPROTECTIVE FUNCTIONS OF SMALL STRESS PROTEINS IN PROTEIN CONFORMATIONAL DISEASES [Capitolo/Saggio]
Carra, Serena; H. H., Kampinga
abstract

The mammalian small heat shock protein family comprises 10 members (HspB1-10) some of which have been implicated indirectly or directly in several neurodegenerative and neuromuscular disorders. Upregulation of some HspB members has been found in brain amyloidosis, where they are often trapped within inclusion bodies and mutations of several HspB proteins have been associated with muscular and neurological disorders. These two findings strongly suggest an important role for the HspB proteins in the maintaining of neuronal and muscular cells viability. How these molecular chaperones can alleviate neurodegeneration and how their mutation can result in toxicity is still largely unknown. This review will summarize the current knowledge about HspB protein function and implication in neurodegenerative diseases. From this, we suggest that, besides the two most characterized biochemical properties of HspB members (chaperone activity and cytosketelal stabilization), also non-canonical pathways seem relevant to the neuroprotective actions of some HspB members.

2010 - HSPB7 is the most potent polyQ aggregation suppressor within the HSPB family of molecular chaperones. [Articolo su rivista]
Vos, Mj; Zijlstra, Mp; Kanon, B; van Waarde Verhagen, Ma; Brunt, Er; Oosterveld Hut, Hm; Carra, Serena; Sibon, Oc; Kampinga, Hh
abstract

A small number of heat-shock proteins have previously been shown to act protectively on aggregation of several proteins containing an extended polyglutamine (polyQ) stretch, which are linked to a variety of neurodegenerative diseases. A specific subfamily of heat-shock proteins is formed by the HSPB family of molecular chaperones, which comprises 10 members (HSPB1-10, also called small HSP). Several of them are known to act as anti-aggregation proteins in vitro. Whether they also act protectively in cells against polyQ aggregation has so far only been studied for few of them (e.g. HSPB1, HSPB5 and HSPB8). Here, we compared the 10 members of the human HSPB family for their ability to prevent aggregation of disease-associated proteins with an expanded polyQ stretch. HSPB7 was identified as the most active member within the HSPB family. It not only suppressed polyQ aggregation but also prevented polyQ-induced toxicity in cells and its expression reduces eye degeneration in a Drosophila polyQ model. Upon overexpression in cells, HSPB7 was not found in larger oligomeric species when expressed in cells and-unlike HSPB1-it did not improve the refolding of heat-denatured luciferase. The action of HSPB7 was also not dependent on the Hsp70 machine or on proteasomal activity, and HSPB7 overexpression alone did not increase autophagy. However, in ATG5-/- cells that are defective in macroautophagy, the anti-aggregation activity of HSPB7 was substantially reduced. Hence, HSPB7 prevents toxicity of polyQ proteins at an early stage of aggregate formation by a non-canonical mechanism that requires an active autophagy machinery.

2010 - Identification of the Drosophila ortholog of HSPB8: implication of HSPB8 loss of function in protein folding diseases. [Articolo su rivista]
Carra, Serena; Boncoraglio, A; Kanon, B; Brunsting, Jf; Minoia, M; Rana, A; Vos, Mj; Seidel, K; Sibon, Oc; Kampinga, Hh
abstract

Protein aggregation is a hallmark of many neuronal disorders, including the polyglutamine disorder spinocerebellar ataxia 3 and peripheral neuropathies associated with the K141E and K141N mutations in the small heat shock protein HSPB8. In cells, HSPB8 cooperates with BAG3 to stimulate autophagy in an eIF2α-dependent manner and facilitates the clearance of aggregate-prone proteins (Carra, S., Seguin, S. J., Lambert, H., and Landry, J. (2008) J. Biol. Chem. 283, 1437-1444; Carra, S., Brunsting, J. F., Lambert, H., Landry, J., and Kampinga, H. H. (2009) J. Biol. Chem. 284, 5523-5532). Here, we first identified Drosophila melanogaster HSP67Bc (Dm-HSP67Bc) as the closest functional ortholog of human HSPB8 and demonstrated that, like human HSPB8, Dm-HSP67Bc induces autophagy via the eIF2α pathway. In vitro, both Dm-HSP67Bc and human HSPB8 protected against mutated ataxin-3-mediated toxicity and decreased the aggregation of a mutated form of HSPB1 (P182L-HSPB1) associated with peripheral neuropathy. Up-regulation of both Dm-HSP67Bc and human HSPB8 protected and down-regulation of endogenous Dm-HSP67Bc significantly worsened SCA3-mediated eye degeneration in flies. The K141E and K141N mutated forms of human HSPB8 that are associated with peripheral neuropathy were significantly less efficient than wild-type HSPB8 in decreasing the aggregation of both mutated ataxin 3 and P182L-HSPB1. Our current data further support the link between the HSPB8-BAG3 complex, autophagy, and folding diseases and demonstrate that impairment or loss of function of HSPB8 might accelerate the progression and/or severity of folding diseases.

2010 - The small heat shock protein B8 (HSPB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). [Articolo su rivista]
Crippa, V; Sau, D; Rusmini, P; Boncoraglio, A; Onesto, E; Bolzoni, E; Galbiati, M; Fontana, E; Marino, M; Carra, Serena; Bendotti, C; De Biasi, S; Poletti, A.
abstract

Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are characterized by the presence of misfolded proteins, thought to trigger neurotoxicity. Some familial forms of ALS (fALS), clinically indistinguishable from sporadic ALS (sALS), are linked to superoxide dismutase 1 (SOD1) gene mutations. It has been shown that the mutant SOD1 misfolds, forms insoluble aggregates and impairs the proteasome. Using transgenic G93A-SOD1 mice, we found that spinal cord motor neurons, accumulating mutant SOD1 also over-express the small heat shock protein HspB8. Using motor neuronal fALS models, we demonstrated that HspB8 decreases aggregation and increases mutant SOD1 solubility and clearance, without affecting wild-type SOD1 turnover. Notably, HspB8 acts on mutant SOD1 even when the proteasome activity is specifically blocked. The pharmacological blockage of autophagy resulted in a dramatic increase of mutant SOD1 aggregates. Immunoprecipitation studies, performed during autophagic flux blockage, demonstrated that mutant SOD1 interacts with the HspB8/Bag3/Hsc70/CHIP multiheteromeric complex, known to selectively activate autophagic removal of misfolded proteins. Thus, HspB8 increases mutant SOD1 clearance via autophagy. Autophagy activation was also observed in lumbar spinal cord of transgenic G93A-SOD1 mice since several autophago-lysosomal structures were present in affected surviving motor neurons. Finally, we extended our observation to a different ALS model and demonstrated that HspB8 exerts similar effects on a truncated version of TDP-43, another protein involved both in fALS and in sALS. Overall, these results indicate that the pharmacological modulation of HspB8 expression in motor neurons may have important implications to unravel the molecular mechanisms involved both in fALS and in sALS.

2009 - HspB8 participates in protein quality control by a non chaperone-like mechanism that requires eIF2alpha phosphorylation. [Articolo su rivista]
Carra, Serena; Brunsting, Jf; Lambert, H; Landry, J; Kampinga, Hh
abstract

Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8.Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as ATP-independent molecular chaperones that can bind unfolded proteins and facilitate their processing via the help of ATP-dependent chaperones such as the Hsp70 machine, in which Bag3 may act as a molecular link between HspB, Hsp70, and the ubiquitin ligases. However, here we show that HspB8 and Bag3 act in a non-canonical manner unrelated to the classical chaperone model. Rather, HspB8 and Bag3 induce the phosphorylation of the alpha-subunit of the translation initiator factor eIF2, which in turn causes a translational shut-down and stimulates autophagy. This function of HspB8.Bag3 does not require Hsp70 and also targets fully folded substrates. HspB8.Bag3 activity was independent of the endoplasmic reticulum (ER) stress kinase PERK, demonstrating that its action is unrelated to ER stress and suggesting that it activates stress-mediated translational arrest and autophagy through a novel pathway.

2009 - Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction. [Articolo su rivista]
Fuchs, M; Poirier, Dj; Seguin, Sj; Lambert, H; Carra, Serena; Charette, Sj; Landry, J.
abstract

The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated athanogene 3). In the present study we identify the binding regions in HspB8 and Bag3 crucial for their interaction. We present evidence that HspB8 binds to Bag3 through the hydrophobic groove formed by its strands beta4 and beta8, a region previously known to be responsible for the formation and stability of higher-order oligomers of many sHsps (small Hsps). Moreover, we demonstrate that two conserved IPV (Ile-Pro-Val) motifs in Bag3 mediate its binding to HspB8 and that deletion of these motifs suppresses HspB8 chaperone activity towards mutant Htt43Q (huntingtin exon 1 fragment with 43 CAG repeats). In addition, we show that Bag3 can bind to the molecular chaperone HspB6. The interaction between HspB6 and Bag3 requires the same regions that are involved in the HspB8-Bag3 association and HspB6-Bag3 promotes clearance of aggregated Htt43Q. Our findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their substrate proteins for degradation. Interestingly, a mutation in one of Bag3 IPV motifs has recently been associated with the development of severe dominant childhood muscular dystrophy, suggesting a possible important physiological role for HspB-Bag3 complexes in this disease.

2009 - The stress-inducible HspB8-Bag3 complex induces the eIF2alpha kinase pathway: Implications for protein quality control and viral factory degradation? [Articolo su rivista]
Carra, Serena
abstract

The recently discovered HspB8-Bag3 complex participates in protein quality control through a mechanism that requires the activation of the eIF2alpha signaling pathway and that leads to protein synthesis inhibition and autophagy stimulation. Both processes help to protect the cells against the accumulation of aggregate-prone proteins, which may be relevant in many protein-conformational neurodegenerative disorders. Besides, this activity of HspB8-Bag3 may have important implications during viral infection.

2008 - HspB8 and Bag3: A new chaperone complex targeting misfolded proteins to macroautophagy [Articolo su rivista]
Carra, Serena; Seguin, Sj; Landry, J.
abstract

Protein quality control involves molecular chaperones that recognize misfolded proteins thereby preventing their aggregation, and associated co-chaperones that modulate substrate sorting between renaturation and proteasomal degradation. We recently described a new chaperone complex that stimulates degradation of protein substrates by macroautophagy. The complex is formed of HspB8, a member of the HspB family of molecular chaperones, which is found mutated in neuromuscular diseases, and Bag3, a member of the co-chaperone family of Bag domain-containing proteins. In this complex, Bag3 was shown to be responsible for macroautophagy stimulation. Here we analyzed the role of the three Bag3 canonical protein interaction domains. We show that the proline-rich region is essential for the Bag3-mediated stimulation of mutated huntingtin clearance. Surprisingly, deletion of the BAG domain that mediates Bag3 interaction with Hsp70 and Blc-2, did not affect its activity. We propose that in the HspB8- Bag3 complex, HspB8 is responsible for recognizing the misfolded proteins whereas Bag3, at least in part through its proline-rich domain, might recruit and activate the macroautophagy machinery in close proximity to the chaperone-loaded substrates.

2008 - HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy. [Articolo su rivista]
Carra, Serena; Seguin, Sj; Lambert, H; Landry, J.
abstract

Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy.

2008 - Role of HspB1 and HspB8 in hereditary peripheral neuropathies: beyond the chaperone function [Capitolo/Saggio]
Carra, Serena; Landry, J.
abstract

Within the last ten years, mutations in genes encoding the small heat shock proteins (also called HspB) HspB1, HspB4, HspB5 and HspB8 have been associated with neurological and muscular disorders. In particular, HspB1 and HspB8 mutations result in hereditary peripheral neuropathies, which primarily affect motor and/or sensory peripheral neurons. Due to their extremely long axons, peripheral neurons are particularly dependent on an efficient vesicular trafficking and axonal transport, whose defects have been directly linked to the development of hereditary peripheral neuropathies. Genetic analyses generated new insights into the molecular pathways involved in hereditary peripheral neuropathies, which include not only vesicular trafficking but also protein quality control, protein degradation and RNA processing. This review summarizes the current understanding of HspB implication in motor diseases, starting with a general picture of HspB functions followed by a description of new emerging roles for HspB1 and HspB8 in axonal transport, protein sorting and degradation.

2008 - Structural and functional diversities between members of the human HspB, HspH, HspA, and DnaJ chaperones families. [Articolo su rivista]
Vos, Mj; Hageman, J; Carra, Serena; Kampinga, Hh
abstract

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins required to deal with proteotoxic stresses. Besides being stress-protective and possible targets for delaying progression of protein folding diseases, mutations in chaperones also have been shown to cause disease (chaperonopathies). The mechanism of action of the "classical", stress-inducible HSPs in serving as molecular chaperones preventing the irreversible aggregation of stress-unfolded or disease-related misfolded proteins is beginning to emerge. However, the human genome encodes several members for each of the various HSP families that are not stress-related but contain conserved domains. Here, we have reviewed the existing literature on the various members of the human HSPB (HSP27), HSPH (HSP110), HSPA (HSP70), and DNAJ (HSP40) families. Apart from structural and functional homologies, several diversities between members and families can be found that not only point to differences in client specificity but also seem to serve differential client handling and processing. How substrate specificity and client processing is determined is far from being understood.

2006 - Small Heat Shock Proteins in Neurodegenerative Diseases. [Capitolo/Saggio]
Carra, Serena; Landry, J.
abstract

The small heat shock proteins (sHSP) comprise 10 members in mammals where they are called the HspB proteins (HspB1-10). Like other HSP, the HspB proteins have chaperone activity in vitro. This activity is poorly characterized in vivo, however, many of these proteins protect cells against diverse stress and have been associated, in numerous studies, with protein conformation diseases. HspB proteins are upregulated in neurodegenerative disorders, including brain amyloidosis, and immunohistochemicalstudies showed that they are often trapped within proteininclusions that are formed in these diseases. Moreover, HspB proteins show a protective effect against protein aggregation and toxicity in cellular model of conformational diseases. Furthermore, mutations in four HspB proteins (HspB1, HspB4, HspB5 and HspB8) have been associated with neuromuscular and other protein conformation disorders. These observations constitute strong support for an important role of the HspB proteins in neurodegenerative disorders.

2005 - HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells. [Articolo su rivista]
Carra, Serena; Sivilotti, M; Chavez Zobel, At; Lambert, H; Landry, J.
abstract

The family of small heat shock proteins (sHsp) is composed of 10 members in mammals, four of which are found mutated in diseases associated with the accumulation of protein aggregates. Though many sHsp have demonstrated molecular chaperone activity in vitro in cell-free conditions, their activity in vivo in the normal cellular context remains unclear. In the present study, we investigated the capacity of the sHsp, HspB8/Hsp22, to prevent protein aggregation in the cells using the polyglutamine protein Htt43Q as a model. In control conditions, Htt43Q accumulated in perinuclear inclusions composed of SDS-insoluble aggregates. Co-transfected with Htt43Q, HspB8 became occasionally trapped within the inclusions; however, in most cells, HspB8 blocked inclusion formation. Biochemical analyses indicated that HspB8 inhibited the accumulation of SDS-insoluble Htt43Q as efficiently as Hsp40 which was taken as a positive control. Htt43Q then accumulated in the SDS-soluble fraction, provided that protein degradation was blocked by proteasome and autophagy inhibitors. In contrast, the other sHsp Hsp27/HspB1 and alphaB-crystallin/HspB5 had no effect. This suggested that HspB8 functions as a molecular chaperone, maintaining Htt43Q in a soluble state competent for rapid degradation. Analyses of Hsp27-HspB8 chimeric proteins indicated that the C-terminal domain of HspB8 contains the specific sequence necessary for chaperone activity. Missense mutations in this domain at lysine 141, which are found in human motor neuropathies, significantly reduced the chaperone activity of the protein. A decrease in the HspB8 chaperone activity may therefore contribute to the development of these diseases.

2004 - Chronic treatment with desipramine and fluoxetine modulate BDNF, CaMKK alpha and CaMKK beta mRNA levels in the hippocampus of transgenic mice expressing antisense RNA against the glucocorticoid receptor [Articolo su rivista]
J., Vinet; Carra, Serena; Blom, Johanna Maria Catharina; Brunello, Nicoletta; N., Barden; Tascedda, Fabio
abstract

Antidepressants up-regulate the cAMP response element binding protein (CREB) and the brain-derived neurotrophic factor (BDNF) in hippocampus and these effects contribute to the protection of hippocampal neurons from stressful stimuli such as high glucocorticoid levels. CREB can be activated by both protein kinase A and by Ca2+-calmodulin-dependent protein kinases (CaMKs), which are in turn phosphorylated by their upstream activators CaMKKalpha and CaMMKKbeta. Using in situ hybridization, we examined the effects of chronic treatment with fluoxetine (FLU) or desipramine (DMI) on BDNF, CaMKKalpha and CaMKKbeta mRNAs in the hippocampus of wild-type (Wt) and transgenic (TG) mice characterized by glucocorticoid receptor (GR) dysfunction. Basal levels of CaMKKbeta were down regulated in the CA3 region of TG mice. DMI decreased the expression of both CaMKKalpha and CaMMKKbeta in the CA3 region of Wt mice. FLU up-regulated BDNF mRNA levels in the CA3 of TG animals while both FLU and DMI increased BDNF gene expression in the dentate gyrus (DG) of TG animals. Our results demonstrate a different regulation of BDNF expression by antidepressant drugs in the hippocampus of Wt and TG animals. Moreover, for the first time, a role for CaMKKs in the mechanism of action of antidepressant agents, at least in the hippocampus, is reported. These data are discussed in view of interactions existing between CaMK pathway and GR-mediated gene transcription.

2003 - Cloning of mouse Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta) and characterization of CaMKKbeta and CaMKKalpha distribution in the adult mouse brain. [Articolo su rivista]
Vinet, Jonathan; Carra, Serena; Blom, Johanna Maria Catharina; Harvey, M; Brunello, Nicoletta; Barden, N; Tascedda, Fabio
abstract

The Ca(2+)/calmodulin-dependent protein kinase kinases alpha and beta (CaMKKs alpha and beta) are novel members of the CaM kinase family. The CaMKKbeta was cloned from mouse brain. The deduced amino acid sequence shared 96.43% homology with the rat CaMKKbeta. Both the alpha and beta isoforms were widely distributed throughout the adult mouse brain. Additionally, all peripheral tissues examined displayed CaMKK alpha and beta expression.

2002 - Altered regulation of CREB by chronic antidepressant administration in the brain of transgenic mice with impaired glucocorticoid receptor function. [Articolo su rivista]
Blom, Johanna Maria Catharina; Tascedda, Fabio; Carra, Serena; Ferraguti, Chiara; Barden, N; Brunello, Nicoletta
abstract

Various effects of antidepressant drugs on gene transcription have been described and altered gene expression has been proposed as being a common biological basis underlying depressive illness. One target for the common action of antidepressants is a modifying effect on the regulation of postreceptor pathways and genes related to the cAMP cascade. Recent studies have demonstrated that long-term antidepressant treatment resulted in sustained activation of the cyclic adenosine 3',5'-monophosphate system and in increased expression of the transcription factor cAMP response element binding protein (CREB). A transgenic animal model of depression with impaired glucocorticoid receptor function was used to investigate the effect of chronic antidepressant treatments on CREB expression in different brain areas. Wild-type and transgenic mice received one administration of saline, desipramine, or fluoxetine, daily for 21 days. The effects of antidepressants on CREB mRNA were analyzed using a sensitive RNase protection assay. Antidepressant treatment resulted in a neuroanatomically and animal specific expression pattern of CREB. Our findings suggest that life-long central glucocorticoid receptor dysfunction results in an altered sensitivity with respect to the effects of antidepressants on the expression of CREB.

2001 - Modulation of glutamate receptors in response to the novel antipsychotic olanzapine in rats. [Articolo su rivista]
Tascedda, Fabio; Blom, Johanna Maria Catharina; Brunello, Nicoletta; Zolin, K; Gennarelli, Massimo; Colzi, A; Bravi, D; Carra, Serena; Racagni, G; Riva, M. A.
abstract

BACKGROUND:A disturbance in glutamate neurotransmission has been hypothesized in schizophrenia. Hence, the beneficial effects of pharmacological treatment may be related to adaptive changes taking place in this neurotransmitter system.METHODS:In this study, we investigated the modulation of ionotropic and metabotropic glutamate receptors in the rat brain following acute or chronic exposure to the novel antipsychotic olanzapine.RESULTS:In accordance with the clear distinction between classical and atypical drugs, olanzapine did not alter glutamate receptor expression in striatum. Chronic, not acute, exposure to olanzapine was capable of up-regulating hippocampal mRNA levels for GluR-B and GluR-C, two alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-forming subunits. This effect could be relevant for the improvement of schizophrenic alterations, which are thought to depend on dysfunction of the glutamatergic transmission within the hippocampal formation. We also found that the expression of group II glutamate metabotropic receptors was up-regulated in the frontal cortex after chronic exposure to clozapine, and to a lesser extent olanzapine, but not with haloperidol.CONCLUSIONS:The adaptive mechanisms taking place in glutamatergic transmission might prove useful in ameliorating some of the dysfunction observed in the brain of schizophrenic patients.