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JONATHAN VINET

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Pubblicazioni

2023 - Characterizing microglial gene expression in a model of secondary progressive multiple sclerosis [Articolo su rivista]
Vainchtein, Ilia D.; Alsema, Astrid M.; Dubbelaar, Marissa L.; Grit, Corien; Vinet, Jonathan; van Weering, Hilmar R. J.; Al‐izki, Sarah; Biagini, Giuseppe; Brouwer, Nieske; Amor, Sandra; Baker, David; Eggen, Bart J. L.; Boddeke, Erik W. G. M.; Kooistra, Susanne M.
abstract

Multiple sclerosis (MS) is the most common inflammatory, demyelinating and neurodegenerative disease of the central nervous system in young adults. Chronic-relapsing experimental autoimmune encephalomyelitis (crEAE) in Biozzi ABH mice is an experimental model of MS. This crEAE model is characterized by an acute phase with severe neurological disability, followed by remission of disease, relapse of neurological disease and remission that eventually results in a chronic progressive phase that mimics the secondary progressive phase (SPEAE) of MS. In both MS and SPEAE, the role of microglia is poorly defined. We used a crEAE model to characterize microglia in the different phases of crEAE phases using morphometric and RNA sequencing analyses. At the initial, acute inflammation phase, microglia acquired a pro-inflammatory phenotype. At the remission phase, expression of standard immune activation genes was decreased while expression of genes associated with lipid metabolism and tissue remodeling were increased. Chronic phase microglia partially regain inflammatory gene sets and increase expression of genes associated with proliferation. Together, the data presented here indicate that microglia obtain different features at different stages of crEAE and a particularly mixed phenotype in the chronic stage. Understanding the properties of microglia that are present at the chronic phase of EAE will help to understand the role of microglia in secondary progressive MS, to better aid the development of therapies for this phase of the disease.

2023 - Loss of PML nuclear bodies in familial amyotrophic lateral sclerosis-frontotemporal dementia [Articolo su rivista]
Antoniani, F.; Cimino, M.; Mediani, L.; Vinet, J.; Verde, E. M.; Secco, V.; Yamoah, A.; Tripathi, P.; Aronica, E.; Cicardi, M. E.; Trotti, D.; Sterneckert, J.; Goswami, A.; Carra, S.
abstract

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two neurodegenerative disorders that share genetic causes and pathogenic mechanisms. The critical genetic players of ALS and FTD are the TARDBP, FUS and C9orf72 genes, whose protein products, TDP-43, FUS and the C9orf72-dipeptide repeat proteins, accumulate in form of cytoplasmic inclusions. The majority of the studies focus on the understanding of how cells control TDP-43 and FUS aggregation in the cytoplasm, overlooking how dysfunctions occurring at the nuclear level may influence the maintenance of protein solubility outside of the nucleus. However, protein quality control (PQC) systems that maintain protein homeostasis comprise a cytoplasmic and a nuclear arm that are interconnected and share key players. It is thus conceivable that impairment of the nuclear arm of the PQC may have a negative impact on the cytoplasmic arm of the PQC, contributing to the formation of the cytoplasmic pathological inclusions. Here we focused on two stress-inducible condensates that act as transient deposition sites for misfolding-prone proteins: Promyelocytic leukemia protein (PML) nuclear bodies (PML-NBs) and cytoplasmic stress granules (SGs). Upon stress, PML-NBs compartmentalize misfolded proteins, including defective ribosomal products (DRiPs), and recruit chaperones and proteasomes to promote their nuclear clearance. SGs transiently sequester aggregation-prone RNA-binding proteins linked to ALS-FTD and mRNAs to attenuate their translation. We report that PML assembly is impaired in the human brain and spinal cord of familial C9orf72 and FUS ALS-FTD cases. We also show that defective PML-NB assembly impairs the compartmentalization of DRiPs in the nucleus, leading to their accumulation inside cytoplasmic SGs, negatively influencing SG dynamics. Although it is currently unclear what causes the decrease of PML-NBs in ALS-FTD, our data highlight the existence of a cross-talk between the cytoplasmic and nuclear PQC systems, whose alteration can contribute to SG accumulation and cytoplasmic protein aggregation in ALS-FTD.

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 - 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.

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.

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.

2018 - A hydroxypyrone-based inhibitor of metalloproteinase-12 displays neuroprotective properties in both status epilepticus and optic nerve crush animal models [Articolo su rivista]
Vinet, J.; Costa, A. M.; Salinas-Navarro, M.; Leo, G.; Moons, L.; Arkens, L.; Biagini, G.
abstract

Recently, we showed that matrix metalloproteinase-12 (MMP-12) is highly expressed in microglia and myeloid infiltrates, which are presumably involved in blood–brain barrier (BBB) leakage and subsequent neuronal cell death that follows status epilepticus (SE). Here, we assessed the effects of a hydroxypyrone-based inhibitor selective for MMP-12 in the pilocarpine-induced SE rat model to determine hippocampal cell survival. In the hippocampus of rats treated with pilocarpine, intra-hippocampal injections of the MMP-12 inhibitor protected Cornu Ammonis 3 (CA3) and hilus of dentate gyrus neurons against cell death and limited the development of the ischemic-like lesion that typically develops in the CA3 stratum lacunosum-moleculare of the hippocampus. Furthermore, we showed that MMP-12 inhibition limited immunoglobulin G and albumin extravasation after SE, suggesting a reduction in BBB leakage. Finally, to rule out any possible involvement of seizure modulation in the neuroprotective effects of MMP-12 inhibition, neuroprotection was also observed in the retina of treated animals after optic nerve crush. Overall, these results support the hypothesis that MMP-12 inhibition can directly counteract neuronal cell death and that the specific hydroxypyrone-based inhibitor used in this study could be a potential therapeutic agent against neurological diseases/disorders characterized by an important inflammatory response and/or neuronal cell loss.

2018 - Wisp2 overexpression induced by short Teriparatide treatment affects IDG-SW3 osteogenic differentiation. [Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Magaro', MARIA SARA; Poti', Francesco; Tenedini, Elena; Montosi, Giuliana; Magaro', MARIA SARA; Vinet, Jonathan; Maurel, Delphine; Palumbo, Carla
abstract

The study supports the importance of osteocytes in controlling the action of the other bone cells and suggests that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

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 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 - FosB/ΔFosB and p-ERK1/2 expression respectively identified the lateral amygdala and CA1 as critical regions involved in the progressive seizure aggravation observed in mice exposed to repeated 6-Hz corneal stimulation [Abstract in Atti di Convegno]
Costa, Am; Giordano, C; Lucchi, C; Curia, G; Piat, M; Leo, G; Vinet, J; Biagini, G
abstract

FosB/ΔFosB and p-ERK1/2 expression respectively identified the lateral amygdala and CA1 as critical regions involved in the progressive seizure aggravation observed in mice exposed to repeated 6-Hz corneal stimulation

2017 - Involvement of PPARγ in the anticonvulsant activity of EP-80317, a ghrelin receptor antagonist [Articolo su rivista]
Lucchi, Chiara; Costa, ANNA MARIA; Giordano, Carmela; Curia, Giulia; Piat, Marika; Leo, Giuseppina; Vinet, Jonathan; Brunel, Luc; Fehrentz, Jean Alain; Martinez, Jean; Torsello, Antonio; Biagini, Giuseppe
abstract

Ghrelin, des-acyl ghrelin and other related peptides possess anticonvulsant activities. Although ghrelin and cognate peptides were shown to physiologically regulate only the ghrelin receptor, some of them were pharmacologically proved to activate the peroxisome proliferator-activated receptor gamma (PPARγ) through stimulation of the scavenger receptor CD36 in macrophages. In our study, we challenged the hypothesis that PPARγ could be involved in the anticonvulsant effects of EP-80317, a ghrelin receptor antagonist. For this purpose, we used the PPARγ antagonist GW9662 to evaluate the modulation of EP-80317 anticonvulsant properties in two different models. Firstly, the anticonvulsant effects of EP-80317 were studied in rats treated with pilocarpine to induce status epilepticus (SE). Secondly, the anticonvulsant activity of EP-80317 was ascertained in the repeated 6-Hz corneal stimulation model in mice. Behavioral and video electrocorticographic (ECoG) analyses were performed in both models. We also characterized levels of immunoreactivity for PPARγ in the hippocampus of 6-Hz corneally stimulated mice. EP-80317 predictably antagonized seizures in both models. Pre-treatment with GW9662 counteracted almost all EP-80317 effects both in mice and rats. Only the effects of EP-80317 on power spectra of ECoGs recorded during repeated 6-Hz corneal stimulation were practically unaffected by GW9662 administration. Moreover, GW9662 alone produced a decrease in the latency of tonic-clonic seizures and accelerated the onset of SE in rats. Finally, in the hippocampus of mice treated with EP-80317 we found increased levels of PPARγ immunoreactivity. Overall, these results support the hypothesis that PPARγ is able to modulate seizures and mediates the anticonvulsant effects of EP-80317.

2017 - Osteocytes signaling events induced by intermittent vs continuous Teriparatide treatment affect in vitro osteoblast differentiation and mineralization [Abstract in Rivista]
Bertacchini, Jessika; Smargiassi, Alberto; Checchi, Marta; Tenedini, Elena; Montosi, Giuliana; Vinet, Jonathan; Ferretti, Marzia; Palumbo, Carla
abstract

PTH(1-34), also known as Teriparatide, is an active anabolic drug used in the treatment of some forms of osteoporosis and occasionally exploited to speed fracture healing. The effect of such therapies are dependent on the type of administration, in fact it has been largely demonstrated that a short administration of Teriparatide (also called intermittent) increases the bone mass, meanwhile a long administration of the same agent (known as continuous) leads to an increased resorption. The molecular reason why the type of administration is so critical for the fate of the bone remodeling is still largely unknown but it is probably due to the fact that it affects several signaling pathways and alters the biological activity of a cohort of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. In the present work, we firstly focused the attention on molecular events induced by intermittent vs continuous Teriparatide treatment in a well-known osteocytes in vitro model, the MLO-Y4 cells. By the use of a gene array platform, we found many molecules upregulated or downregulated depending on the the temporal administration modes, suggesting that the drug affects in diverse manner the osteocytes related signaling pathways. In particular, we paid attention to Wisp-2, a protein of the Wnt pathway that has been demonstrated to be able to interact and influence the differentiation of osteoblasts into osteocytes and their mineralization. Secondly, through the mineralization assay, we analyzed the functional effects, involving the differentiation of osteoblast IDG-SW3 cell line, upon the conditioning culture with MLO-Y4 medium, that were pre-treated with short and long time administration of Teriparatide. These findings, consistent with the crucial role performed by osteocytes on osteoblast differentiation, clarify the molecular events downstream the short treatment with Teriparatide, suggesting that the perturbation of certain signaling patwhays, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

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 - Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus [Articolo su rivista]
Vinet, Jonathan; Vainchtein, Ilia D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, Domenico; Curia, Giulia; Dominici, Massimo; Boddeke, Hendrikus W. G. M.; Eggen, Bart J. L.; Biagini, Giuseppe
abstract

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile.

2015 - Ischemic-hypoxic mechanisms leading to hippocampal dysfunction as a consequence of status epilepticus [Articolo su rivista]
Lucchi, Chiara; Vinet, Jonathan; Meletti, Stefano; Biagini, Giuseppe
abstract

Status epilepticus (SE) is one of the recognized primary precipitating events that can lead to temporal lobe epilepsy (TLE) associated with hippocampal sclerosis. This type of epilepsy is characterized by poor response to drug treatment, often requiring surgical intervention to remove the mesial temporal regions involved in the seizure onset. However, even neurosurgery may not be completely successful. Thus, the prevention of hippocampal damage and epileptogenesis is currently evaluated as a possible alternative therapeutic approach to prevent the development of pharmacoresistant TLE. Lines of evidence suggest that ischemic-hypoxic lesions might occur in different brain regions, including the hippocampus, during SE. Especially in the hippocampal CA3 region, an ischemic-like lesion develops in the stratum lacunosum-moleculare and is mainly characterized by a loss of astrocytes and neuronal processes and increased immunostaining of pimonidazole which probes areas exposed to hypoxia. Interestingly, these mechanisms can contribute to neuronal cell loss and may be counteracted by drugs that can afford vascular protection, as in the case of ligands of the ghrelin receptor. Notably, some of the ghrelin receptor ligands possess a double edge effect, since they are anticonvulsant and vascular-protective, thus, potentially representing new tools to counteract the consequences of SE. This article is part of a Special Issue entitled Status Epilepticus.

2015 - Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus [Articolo su rivista]
Giordano, Carmela; Vinet, Jonathan; Curia, Giulia; Biagini, Giuseppe
abstract

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.

2015 - Repeated 6-Hz corneal stimulation progressively increases FosB/∆FosB levels in the lateral amygdala and induces seizure generalization to the hippocampus. [Abstract in Atti di Convegno]
Giordano, C; Vinet, Jonathan; Curia, Giulia; Biagini, Giuseppe
abstract

Exposure to repetitive seizures is known to promote convulsions which depends on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by the modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and an inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures appeared always in frontal cortex, but only at the fourth stimulation they involved the hippocampus. Duration of non-motor seizures progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session most likely due to activity of principal cells. These findings suggest a predominant role of amygdala in promoting progressively more severe convulsions.

2015 - The phenotypes of microglia and macrophages during epileptogenesis [Abstract in Rivista]
Vinet, Jonathan; Vainchtein, I. D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, D.; Dominici, Massimo; Eggen, B. J. L.; Biagini, Giuseppe
abstract

Question: A growing body of evidence is now supporting a relationship between inflammation and epilepsy. Indeed, activated microglia, reactive astrocytes, local expression of pro-inflammatory cytokines, blood brain barrier leakage and peripheral immune cell infiltration have all been observed in temporal lobe epilepsy (TLE) animal models as well as in humans. Accordingly, inflammatory mechanisms are thought to play a central role in the initiation and maintenance of seizures, starting in the acute phase represented by status epilepticus (SE) induction. Microglia activation has been correlated with the expression of several pro-inflammatory cytokines which are thought to contribute to the neuronal cell death occurring after SE. Data point towards a pro-inflammatory phenotype of microglia that precedes neuronal injury and cell death. Because of this, microglia are generally considered to play a pro-epileptogenic role. However, infiltration of peripheral immune cells during epileptogenesis such as leukocytes, granulocytes and monocytes/macrophages might also contribute to the development of chronic epilepsy and recurrent seizures. Uncertainty on the role of these POSTER ABSTRACTS E355 GLIA different inflammatory cells depended on technical limitations in the discrimination of microglia from macrophages. For this reason, it is possible that the detrimental function that is currently attributed to microglia might be incorrect and should be ascribed to infiltrating macrophages. Methods: Both microglia and macrophages were acutely isolated from the hippocampi of control and pilocarpine-treated CD1 mice (24h and 96h after SE) and FACS sorted. Microglia were defined as CD11b+ CD45int Ly-6Cneg and infiltrated macrophages as CD11b+ CD45hi Ly-6Cpos. After sorting, qPCR and flow cytometry analysis were performed. Results: During epileptogenesis, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecule CD40 and pro-inflammatory gene IL-1. In contrast, infiltrated macrophages were strongly immune activated. Both cell types expressed high levels of the phagocytosis marker AXL. Conclusions: These data suggest that macrophages might be more detrimental than microglia during epileptogenesis.

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 - In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed. [Articolo su rivista]
Vainchtein, Id; Vinet, Jonathan; Brouwer, N; Brendecke, S; Biagini, Giuseppe; Biber, K; Boddeke, Hw; Eggen, Bj
abstract

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. Therefore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11bpos CD45int Ly-6Cneg, and infiltrated macrophages as CD11bpos CD45high Ly-6Cpos. During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a)]. In contrast, CD11bpos CD45high Ly-6Cpos infiltrated macrophages were strongly activated and could be divided into two populations Ly-6Cint and Ly-6Chigh, respectively. Ly-6Chigh macrophages contained less myelin than Ly-6Cint macrophages and expression levels of the proinflammatory cytokines IL-1b and TNF-a were higher in Ly-6Cint macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive.

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 - Pathophysiogenesis of Mesial Temporal Lobe Epilepsy: Is Prevention of Damage Antiepileptogenic? [Articolo su rivista]
Curia, Giulia; Lucchi, Chiara; Vinet, Jonathan; Gualtieri, F; Marinelli, C; Torsello, A; Costantino, Luca; Biagini, Giuseppe
abstract

Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies.

2014 - WONOEP appraisal: new genetic approaches to study epilepsy [Articolo su rivista]
Rossignol, Elsa; Kobow, Katja; Simonato, Michele; Loeb, Jeffrey A; Grisar, Thierry; Gilby, Krista L; Vinet, Jonathan; Kadam, Shilpa D; Becker, Albert J.
abstract

New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type-specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy.

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 [Poster]
Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena
abstract

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 - Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes [Articolo su rivista]
Watts, A. O; van Lipzig, M. M. H; Jaeger, W. C; Seeber, R. M; van Zwam, M; Vinet, Jonathan; van der Lee, M. M. C; Siderius, M; Zaman, G. J. R; Boddeke, H. W. G. M; Smit, M. J; Pfleger, K. D. G; Leurs, R; Vischer, H. F.
abstract

The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

2013 - Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR [Articolo su rivista]
Vinet, Jonathan; van Zwam, M; Dijkstra, I. M; Brouwer, N; van Weering, H. R. J; Watts, A; Meijer, M; Fokkens, M. R; Kannan, V; Verzijl, D; Vischer, H. F; Smit, M. J; Leurs, R; Biber, K; Boddeke, H. W. G. M.
abstract

Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR.

2013 - Neurosteroids And Epileptogenesis [Articolo su rivista]
Biagini, Giuseppe; Rustichelli, Cecilia; Curia, Giulia; Vinet, Jonathan; Lucchi, Chiara; Pugnaghi, Matteo; Meletti, Stefano
abstract

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its upregulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus (SE) induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.

2013 - Protective but Not Anticonvulsant Effects of Ghrelin and JMV-1843 in the Pilocarpine Model of Status epilepticus. [Articolo su rivista]
Lucchi, Chiara; Curia, Giulia; Vinet, Jonathan; Gualtieri, Fabio; Bresciani, E; Locatelli, V; Torsello, A; Biagini, Giuseppe
abstract

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45±0.07 mm2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

2013 - Protective but not anticonvulsive effects of ghrelin and JMV-1843 in the pilocarpine model of status epilepticus. [Abstract in Atti di Convegno]
Lucchi, Chiara; Curia, Giulia; Vinet, Jonathan; Gualtieri, F; Torsello, A; Biagini, Giuseppe
abstract

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

2012 - Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity [Articolo su rivista]
Moidunny, Shamsudheen; Vinet, Jonathan; Wesseling, Evelyn; Bijzet, Johan; Shieh, Chu Hsin; van Ijzendoorn, Sven C. D; Bezzi, Paola; Boddeke, Hendrikus W. G. M; Biber, Knut
abstract

Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling.

2012 - Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3) [Articolo su rivista]
Seidel, K; Meister, M; Dugbartey, G. J; Zijlstra, M. P; Vinet, Jonathan; Brunt, E. R. P; van Leeuwen, F. W; Rüb, U; Kampinga, H. H; den Dunnen, W. F. A.
abstract

A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions.

2012 - Identification of a microglia phenotype supportive of remyelination [Articolo su rivista]
Olah, Marta; Amor, Sandra; Brouwer, Nieske; Vinet, Jonathan; Eggen, Bart; Biber, Knut; Boddeke, Hendrikus W. G. M.
abstract

In multiple sclerosis, endogenous oligodendrocyte precursor cells (OPCs) attempt to remyelinate areas of myelin damage. During disease progression, however, these attempts fail. It has been suggested that modulating the inflammatory environment of the lesion might provide a promising therapeutic approach to promote endogenous remyelination. Microglia are known to play a central role in neuroinflammatory processes. To investigate the microglia phenotype that supports remyelination, we performed genome-wide gene expression analysis of microglia from the corpus callosum during demyelination and remyelination in the mouse cuprizone model, in which remyelination spontaneously occurs after an episode of toxin-induced primary demyelination. We provide evidence for the existence of a microglia phenotype that supports remyelination already at the onset of demyelination and persists throughout the remyelination process. Our data show that microglia are involved in the phagocytosis of myelin debris and apoptotic cells during demyelination. Furthermore, they express a cytokine and chemokine repertoire enabling them to activate and recruit endogenous OPCs to the lesion site and deliver trophic support during remyelination. This study not only provides a detailed transcriptomic analysis of the remyelination-supportive microglia phenotype but also reinforces the notion that the primary function of microglia is the maintenance of tissue homeostasis and the support of regeneration already at the earliest stages in the development of demyelinating lesions.

2012 - Neuroprotective function for ramified microglia in hippocampal excitotoxicity [Articolo su rivista]
Vinet, Jonathan; Weering, Hilmar R. J. van; Heinrich, Annette; Kälin, Roland E; Wegner, Anja; Brouwer, Nieske; Heppner, Frank L; Rooijen, Nico van; Boddeke, Hendrikus W. G. M; Biber, Knut
abstract

Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.

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.

2011 - CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus [Articolo su rivista]
van Weering, Hilmar R. J; Boddeke, Hendrikus W. G. M; Vinet, Jonathan; Brouwer, Nieske; de Haas, Alexander H; van Rooijen, Nico; Thomsen, Allan R; Biber, Knut P. H.
abstract

The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10(-/-) ) and CXCR3-deficient (CXCR3(-/-) ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10(-/-) and CXCR3(-/-) OHSCs in response to a high (50 μM) NMDA-concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3(-/-) and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions.

2011 - Microglia phenotype diversity [Articolo su rivista]
Olah, M; Biber, K; Vinet, Jonathan; Boddeke, H. W. G. M.
abstract

Microglia, the tissue macrophages of the brain, have under healthy conditions a resting phenotype that is characterized by a ramified morphology. With their fine processes microglia are continuously scanning their environment. Upon any homeostatic disturbance microglia rapidly change their phenotype and contribute to processes including inflammation, tissue remodeling, and neurogenesis. In this review, we will address functional phenotypes of microglia in diverse brain regions and phenotypes associated with neuroinflammation, neurogenesis, brain tumor homeostasis, and aging.

2010 - Expression of CXCL10 in cultured cortical neurons [Articolo su rivista]
Vinet, Jonathan; de Jong, Eiko K; Boddeke, Hendrikus W. G. M; Stanulovic, Vesna; Brouwer, Nieske; Granic, Ivica; Eisel, Ulrich L. M; Liem, Robert S. B; Biber, Knut
abstract

Chemokines expressed in neurons are important mediators in neuron-neuron and neuron-glia signaling. One of these chemokines is CCL21 that activates microglia via the chemokine receptor CXCR3. As neurons also express CXCL10, a main ligand for CXCR3, we have thus investigated in detail the expression pattern of CXCL10 in neurons. We show that CXCL10 is constitutively expressed by neurons, is stored in large dense-core vesicles and is not regulated by neuronal injury or stress. Neuronal CXCL10 release occurred constitutively at low level. In vivo CXCL10 expression was found in the developing brain at various embryonic stages and its peak expression correlates with the presence of CD11b- and GFAP-positive cells expressing CXCR3. These results suggest a possible role of neuronal CXCL10 in recruitment and homing of glial cells during embryogenesis.

2010 - Subclasses of oligodendrocytes populate the mouse hippocampus [Articolo su rivista]
Vinet, Jonathan; Lemieux, Philippe; Tamburri, Albert; Tiesinga, Paul; Scafidi, Joseph; Gallo, Vittorio; Sík, Attila
abstract

Oligodendrocytes are the myelin-forming cells of the central nervous system that facilitate transmission of axonal electrical impulses. Using transgenic mice expressing 2',3' cyclic nucleotide 3' phosphodiesterase (CNPase)-enhanced green fluorescent protein, a three-dimensional reconstruction tool and analysis, we illustrate that three morphologically different oligodendrocyte types exist in the hippocampus. Those of the ramified type have the most numerous processes, the largest cell body, occupy the largest area and form beaded-like structures, due to mitochondria aggregates, along the processes. Stellar-shaped oligodendrocytes have smaller cell bodies and their processes cover a significantly smaller area. Those of the smooth subtype have a small cell body with at most two processes. In addition to these types, a large number of oligodendrocytes were found that faintly express CNPase-enhanced green fluorescent protein. More than 50% of the faint type colocalized with NG2 and 91% with oligodendrocyte transcription factor-2, whereas 94% of NG2-immunoreactive and 45% of oligodendrocyte transcription factor-2-immunoreactive cells were faintly CNPase-enhanced green fluorescent protein positive. Based on the complexity of the overall structure, the three types probably represent stages of a maturation process such that one subtype can morph into another. Thus, the least complex 'smooth' cell would represent the youngest oligodendrocyte that matures into the stellar type and eventually progresses to become the most complex ramified oligodendrocyte. Investigation of the distribution pattern revealed that the highest density of oligodendrocytes was found in the stratum lacunosum-moleculare and the hilar region. The distribution analysis of oligodendrocyte subclasses revealed a tendency for different cell types to segregate in large non-overlapping areas. This observation suggests that morphologically, and possible functionally, different oligodendrocytes are topographically segregated.

2008 - Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles [Articolo su rivista]
de Jong, Eiko K; Vinet, Jonathan; Stanulovic, Vesna S; Meijer, Michel; Wesseling, Evelyn; Sjollema, Klaas; Boddeke, Hendrikus W. G. M; Biber, Knut
abstract

Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuron-microglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.

2008 - Neuron-microglia signaling: chemokines as versatile messengers [Articolo su rivista]
Biber, K; Vinet, Jonathan; Boddeke, H. W. G. M.
abstract

Our understanding of microglia biology has significantly changed in the last couple of years. Instead of being predominantly detrimental cells showing a stereotypic activation pattern, microglia today are considered highly adaptive elements with many distinct phenotypes. Microglia activity is aimed to protect and to restore and only in case of uncontrolled or impaired microglia function these cells may have detrimental effects. The control of microglia activity is thus an important issue to understand. The family of chemokines are versatile signals specialized to control cell-cell interactions. Neurons express chemokines in a temporarily and spatially regulated manner and microglia respond to these messengers via the appropriate receptors. Due to these features are chemokines ideal messengers for the communication between neurons and microglia.

2006 - Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice [Articolo su rivista]
Vinet, Jonathan; Sík, A.
abstract

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.

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 - Bcl-2 expression in thalamus, brainstem, cerebellum and visual cortex of adult primate [Articolo su rivista]
Vinet, Jonathan; Bernier, Patrick J; Parent, André
abstract

Due to the functional importance of Bcl-2, which acts as an anti-apoptotic protein that also affects neural differentiation and adult neurogenesis, we undertook a detailed immunohistochemical study of the distribution of this protein in the brain of squirrel monkeys. The present study describes findings obtained at thalamic, brainstem, cerebellum and visual cortex levels, and the data are compared with our previous results gathered in the same species. At thalamic level, Bcl-2-positive neurons occur in anterior, rostral intralaminar, midline and lateral habenular nuclei. The protein is also expressed in several structures associated with the ventricular system, including the subventricular zone (SVZ), the subcommissural organ, and the periventricular grey at rostral and caudal tips of the fourth ventricle. At brainstem and cerebellar levels, Bcl-2-positive neurons occur in the dorsal raphe nucleus, inferior olivary complex, and in molecular and granular layers of the cerebellum. Finally, neurons of layer IV of the striate cortex display a very strong Bcl-2 immunoreactivity that contrasts with the poor labeling of neurons in adjacent parastriate and peristriate cortices. These finding suggests that Bcl-2 plays a role in the plasticity and structural maintenance of various structures in the primate brain and indicate that the mitotically active SVZ might be more extended along the rostrocaudal axis in primates than in rodents.

2002 - Newly generated neurons in the amygdala and adjoining cortex of adult primates [Articolo su rivista]
Bernier, Patrick J; Bedard, Andreanne; Vinet, Jonathan; Levesque, Martin; Parent, Andre
abstract

The subventricular zone remains mitotically active throughout life in rodents. Studies with tritiated thymidine, which is incorporated into the DNA of mitotic cells, have revealed that the rodent subventricular zone produces neuroblasts that migrate toward the olfactory bulb along the rostral migratory stream. A similar migratory stream has been documented in monkeys by using the thymidine analogue BrdUrd. The same approach showed that neurogenesis occurred in the dentate gyrus of adult primates, including humans. In the present study, experiments combining injections of BrdUrd and the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine, with the immunostaining for molecular markers of neurogenesis (polysialylated neural cell adhesion molecule, beta-tubulin-III, collapsin response mediator protein-4, neuronal nuclear protein) in New World (Saimiri sciureus) and Old World (Macaca fascicularis) monkeys have revealed that new neurons are produced in the amygdala, piriform cortex, and adjoining inferior temporal cortex in adult primates. These newborn neurons expressed the antiapoptotic protein Bcl-2 and formed a more-or-less continuous pathway that extended from the tip of the temporal ventricular horn to the deep portion of the temporal lobe. The production of newborn neurons in the amygdala, piriform cortex, and inferior temporal cortex seems to parallel the continuing addition of neurons in the olfactory bulb. These two concomitant phenomena may ensure structural stability and functional plasticity to the primate olfactory system and temporal lobe.

2000 - Characterization of the subventricular zone of the adult human brain: evidence for the involvement of Bcl-2 [Articolo su rivista]
Bernier, P. J; Vinet, Jonathan; Cossette, M; Parent, A.
abstract

The subventricular zone (SVZ) is an embryonic remnant that persists and remains mitotically active throughout adulthood. The rodent SVZ harbors neuronal precursors, principally in its anterior part, and generates neuroblasts that migrate tangentially into the olfactory bulb, thus forming the so-called rostral migratory stream. This study aimed at characterizing the SVZ in the human brain. Antibodies raised against the widely used SVZ molecular markers nestin, glial fibrillary acidic protein, beta-tubulin-III and polysialylated neural cell adhesion molecule, have allowed us to characterize in detail a zone similar to the rodent SVZ in humans. Virtually all portions of the lateral ventricle, as well as the ventral (hypothalamic) sector of the third ventricle, displayed immunoreactivity for most of the molecular markers. The midline region of the septum (septal recess) and the ventral portion of the SVZ displayed a particularly intense immunostaining for all SVZ markers. These two regions may represent zones of adult neurogenesis that are unique to primates. Furthermore, the anti-apoptotic protein Bcl-2 was found to be actively synthesized and co-expressed with all the other markers throughout the entire SVZ. This study reveals that a well-developed SVZ exists in the adult human brain and suggests that Bcl-2 might play an important role in the functional organization of such a system.

1999 - Differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits by calretinin-immunoreactive neurons in the human striatum [Articolo su rivista]
Cicchetti, F; Vinet, Jonathan; Beach, T. G; Parent, A.
abstract

We recently reported the existence of medium and large intemeurons immunoreactive for the calcium-binding protein calretinin in the human striatum. We also showed a selective sparing of all medium, but not all large, calretinin-immunoreactive striatal neurons in Huntington's disease striatum. Because glutamate receptor-mediated excitotoxicity has been implicated in the massive loss of striatal projection neurons that characterizes Huntington's disease, we have applied a double-antigen localization procedure to post mortem tissue from eight normal human subjects to determine the expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits 1/2/4 by the calretinin-immunoreactive interneurons. The two types of calretinin-immunoreactive neurons were found to display various patterns of glutamate receptor subunit expression and a specific regionalization was also noted in the expression of these glutamate receptor subunits. Approximately half of the large calretinin-immunoreactive neurons displayed immunoreactivity for glutamate receptor subunits 1 and 2, and about the same proportion of medium calretinin-immunoreactive neurons expressed glutamate receptor subunits 1 and 4. These double-labeled neurons were rather uniformly distributed in the caudate nucleus and putamen. In contrast, as much as 70.1% of the large calretinin-immunoreactive neurons displayed glutamate receptor subunit 4 immunoreactivity in the postcommissural portion of the putamen, an area that corresponds to the sensorimotor striatal territory. For their part, the medium calretinin-immunoreactive neurons were markedly enriched with glutamate receptor subunit 2, 76% of them being double labeled in the caudate nucleus, which corresponds to the striatal associative territory, compared with 85.5% in the postcommissural putamen. Receptor subunit composition plays a key role in determining the functional properties of glutamate receptors, including their permeability to calcium and susceptibility to excitotoxic insults. Thus, the differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits reported here may help to explain the selective sparing of certain types of calretinin-immunoreactive striatal interneurons in Huntington's disease, although other factors, such as post-transcriptional editing, are also likely to be involved.

Università degli studi di Modena e Reggio Emilia

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