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Valeria MARIGO

Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche


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Pubblicazioni

2024 - Molecular mechanisms underlying inherited photoreceptor degeneration as targets for therapeutic intervention [Articolo su rivista]
Bighinati, A.; Adani, E.; Stanzani, A.; D'Alessandro, S.; Marigo, V.
abstract

Retinitis pigmentosa (RP) is a form of retinal degeneration characterized by primary degeneration of rod photoreceptors followed by a secondary cone loss that leads to vision impairment and finally blindness. This is a rare disease with mutations in several genes and high genetic heterogeneity. A challenging effort has been the characterization of the molecular mechanisms underlying photoreceptor cell death during the progression of the disease. Some of the cell death pathways have been identified and comprise stress events found in several neurodegenerative diseases such as oxidative stress, inflammation, calcium imbalance and endoplasmic reticulum stress. Other cell death mechanisms appear more relevant to photoreceptor cells, such as high levels of cGMP and metabolic changes. Here we review some of the cell death pathways characterized in the RP mutant retina and discuss preclinical studies of therapeutic approaches targeting the molecular outcomes that lead to photoreceptor cell demise.


2024 - New Improved cGMP Analogues to Target Rod Photoreceptor Degeneration [Articolo su rivista]
Pérez, O.; Stanzani, A.; Huang, L.; Schipper, N.; Loftsson, T.; Bollmark, M.; Marigo, V.
abstract

: Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3',5'-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its RP-phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer SP-CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP.


2024 - Strategies for Improved pDNA Loading and Protection Using Cationic and Neutral LNPs with Industrial Scalability Potential Using Microfluidic Technology [Articolo su rivista]
Ottonelli, I.; Adani, E.; Bighinati, A.; Cuoghi, S.; Tosi, G.; Vandelli, M. A.; Ruozi, B.; Marigo, V.; Duskey, J. T.
abstract

Purpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity. Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical “post-formulation” DNA addition vs “pre” addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake. Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the “post” method, while maintaining their characteristics up to 100 µg/mL of pDNA with the “pre” addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes. Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.


2023 - Optimization of an Injectable Hydrogel Depot System for the Controlled Release of Retinal-Targeted Hybrid Nanoparticles [Articolo su rivista]
Ottonelli, I.; Bighinati, A.; Adani, E.; Loll, F.; Caraffi, R.; Vandelli, M. A.; Boury, F.; Tosi, G.; Duskey, J. T.; Marigo, V.; Ruozi, B.
abstract

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.


2022 - Activation of cGMP-Dependent Protein Kinase Restricts Melanoma Growth and Invasion by Interfering with the EGF/EGFR Pathway [Articolo su rivista]
Quadri, M.; Comitato, A.; Palazzo, E.; Tiso, N.; Rentsch, A.; Pellacani, G.; Marconi, A.; Marigo, V.
abstract

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.


2022 - Conformational insights into the C-terminal mutations of human rhodopsin in retinitis pigmentosa [Articolo su rivista]
Picarazzi, F.; Manetti, F.; Marigo, V.; Mori, M.
abstract

Rhodopsin is a light-sensitive transmembrane receptor involved in the visual transduction cascade. Among the several rhodopsin mutations related to retinitis pigmentosa (RP), those affecting the C-terminal VAPA-COOH motif that is implicated in rhodopsin trafficking from the Golgi to the rod outer segment are notably associated with more aggressive RP forms. However, molecular reasons for defective rhodopsin signaling due to VAPA-COOH mutations, which might include steric hindrance, physicochemical features and structural determinants, are yet unknown, thus limiting further drug design approaches. In this work, clinically relevant rhodopsin mutations at the P347 site within the VAPA-COOH motif were investigated by molecular dynamics (MD) simulations and compared to the wild-type (WT) system. In agreement with experimental evidence, conformational fluctuations of the intrinsically disordered C-terminal tail of WT and mutant rhodopsin were found not to affect the overall structure of the transmembrane domain, including binding to the retinal cofactor. The WT VAPA-COOH motif adopts a unique conformation that is not found in pathological mutants, suggesting that structural features could better explain the pathogenicity of P347 rhodopsin mutants than physicochemical or steric determinants. These results were confirmed by MD simulations in both membrane-embedded full-length opsin and membrane-free C-terminal deca-peptides, these latter becoming very useful and small-size model systems for further investigations of rhodopsin C-terminal mutations. Structural details elucidated in this work might facilitate the understanding of the pathological mechanisms of this class of rhodopsin mutants, which will be instrumental to the development of new therapeutic strategies.


2022 - Efficient Delivery of Hydrophilic Small Molecules to Retinal Cell Lines Using Gel Core-Containing Solid Lipid Nanoparticles [Articolo su rivista]
Huang, L.; Himawan, E.; Belhadj, S.; Perez Garcia, R. O.; Paquet Durand, F.; Schipper, N.; Buzgo, M.; Simaite, A.; Marigo, V.
abstract

In this study, we developed a novel solid lipid nanoparticle (SLN) formulation for drug delivery of small hydrophilic cargos to the retina. The new formulation, based on a gel core and composite shell, allowed up to two-fold increase in the encapsulation efficiency. The type of hydrophobic polyester used in the composite shell mixture affected the particle surface charge, colloidal stability, and cell internalization profile. We validated SLNs as a drug delivery system by performing the encapsulation of a hydrophilic neuroprotective cyclic guanosine monophosphate analog, previously demonstrated to hold retinoprotective properties, and the best formulation resulted in particles with a size of ±250 nm, anionic charge > −20 mV, and an encapsulation efficiency of ±60%, criteria that are suitable for retinal delivery. In vitro studies using the ARPE-19 and 661W retinal cell lines revealed the relatively low toxicity of SLNs, even when a high particle concentration was used. More importantly, SLN could be taken up by the cells and the release of the hydrophilic cargo in the cytoplasm was visually demonstrated. These findings suggest that the newly developed SLN with a gel core and composite polymer/lipid shell holds all the characteristics suitable for the drug delivery of small hydrophilic active molecules into retinal cells.


2021 - Identification of novel substrates for cGMP dependent protein kinase (PKG) through kinase activity profiling to understand its putative role in inherited retinal degeneration [Articolo su rivista]
Roy, A.; Groten, J.; Marigo, V.; Tomar, T.; Hilhorst, R.
abstract

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3',5'-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3',5'-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.


2021 - New in vitro cellular model for molecular studies of retinitis pigmentosa [Articolo su rivista]
Huang, L.; Kutluer, M.; Adani, E.; Comitato, A.; Marigo, V.
abstract

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.


2021 - Structural aspects of rod opsin and their implication in genetic diseases [Articolo su rivista]
Fanelli, F.; Felline, A.; Marigo, V.
abstract

Vision in dim-light conditions is triggered by photoactivation of rhodopsin, the visual pigment of rod photoreceptor cells. Rhodopsin is made of a protein, the G protein coupled receptor (GPCR) opsin, and the chromophore 11-cis-retinal. Vertebrate rod opsin is the GPCR best characterized at the atomic level of detail. Since the release of the first crystal structure 20 years ago, a huge number of structures have been released that, in combination with valuable spectroscopic determinations, unveiled most aspects of the photobleaching process. A number of spontaneous mutations of rod opsin have been found linked to vision-impairing diseases like autosomal dominant or autosomal recessive retinitis pigmentosa (adRP or arRP, respectively) and autosomal congenital stationary night blindness (adCSNB). While adCSNB is mainly caused by constitutive activation of rod opsin, RP shows more variegate determinants affecting different aspects of rod opsin function. The vast majority of missense rod opsin mutations affects folding and trafficking and is linked to adRP, an incurable disease that awaits light on its molecular structure determinants. This review article summarizes all major structural information available on vertebrate rod opsin conformational states and the insights gained so far into the structural determinants of adCSNB and adRP linked to rod opsin mutations. Strategies to design small chaperones with therapeutic potential for selected adRP rod opsin mutants will be discussed as well.


2021 - Structure network-based landscape of rhodopsin misfolding by mutations and algorithmic prediction of small chaperone action [Articolo su rivista]
Felline, A.; Schiroli, D.; Comitato, A.; Marigo, V.; Fanelli, F.
abstract

Failure of a protein to achieve its functional structural state and normal cellular location contributes to the etiology and pathology of heritable human conformational diseases. The autosomal dominant form of retinitis pigmentosa (adRP) is an incurable blindness largely linked to mutations of the membrane protein rod opsin. While the mechanisms underlying the noxious effects of the mutated protein are not completely understood, a common feature is the functional protein conformational loss. Here, the wild type and 39 adRP rod opsin mutants were subjected to mechanical unfolding simulations coupled to the graph theory-based protein structure network analysis. A robust computational model was inferred and in vitro validated in its ability to predict endoplasmic reticulum retention of adRP mutants, a feature linked to the mutation-caused misfolding. The structure-based approach could also infer the structural determinants of small chaperone action on misfolded protein mutants with therapeutic implications. The approach is exportable to conformational diseases linked to missense mutations in any membrane protein.


2020 - Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation [Articolo su rivista]
Comitato, A.; Schiroli, D.; Montanari, M.; Marigo, V.
abstract

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2–related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.


2020 - Cellular mechanisms of hereditary photoreceptor degeneration – Focus on cGMP [Articolo su rivista]
Power, M.; Das, S.; Schutze, K.; Marigo, V.; Ekstrom, P.; Paquet-Durand, F.
abstract

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood, a problem that is exacerbated by the enormous genetic heterogeneity of this disease group. However, the last decade has yielded a wealth of new knowledge on degenerative pathways and their diversity. Notably, a central role of cGMP-signalling has surfaced for photoreceptor cell death triggered by a subset of disease-causing mutations. In this review, we examine key aspects relevant for photoreceptor degeneration of hereditary origin. The topics covered include energy metabolism, epigenetics, protein quality control, as well as cGMP- and Ca2+-signalling, and how the related molecular and metabolic processes may trigger photoreceptor demise. We compare and integrate evidence on different cell death mechanisms that have been associated with photoreceptor degeneration, including apoptosis, necrosis, necroptosis, and PARthanatos. A special focus is then put on the mechanisms of cGMP-dependent cell death and how exceedingly high photoreceptor cGMP levels may cause activation of Ca2+-dependent calpain-type proteases, histone deacetylases and poly-ADP-ribose polymerase. An evaluation of the available literature reveals that a large group of patients suffering from hereditary photoreceptor degeneration carry mutations that are likely to trigger cGMP-dependent cell death, making this pathway a prime target for future therapy development. Finally, an outlook is given into technological and methodological developments that will with time likely contribute to a comprehensive overview over the entire metabolic complexity of photoreceptor cell death. Building on such developments, new imaging technology and novel biomarkers may be used to develop clinical test strategies, that fully consider the genetic heterogeneity of hereditary retinal degenerations, in order to facilitate clinical testing of novel treatment approaches.


2020 - Targeting molecular pathways for the treatment of inherited retinal degeneration [Articolo su rivista]
Kutluer, M.; Huang, L.; Marigo, V.
abstract

Inherited retinal degeneration is a major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Inherited retinal degeneration is characterized by high genetic and phenotypic heterogeneity with several genes mutated in patients affected by these genetic diseases. The high genetic heterogeneity of these diseases hampers the development of effective therapeutic interventions for the cure of a large cohort of patients. Common cell demise mechanisms can be envisioned as targets to treat patients regardless the specific mutation. One of these targets is the increase of intracellular calcium ions, that has been detected in several murine models of inherited retinal degeneration. Recently, neurotrophic factors that favor the efflux of calcium ions to concentrations below toxic levels have been identified as promising molecules that should be evaluated as new treatments for retinal degeneration. Here, we discuss therapeutic options for inherited retinal degeneration and we will focus on neuroprotective approaches, such as the neuroprotective activity of the Pigment epithelium-derived factor. The characterization of specific targets for neuroprotection opens new perspectives together with many questions that require deep analyses to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise by neuroprotection may represent a promising treatment strategy for retinal degeneration.


2019 - CHAPTER 3. Modulation of Calcium Overload and Calpain Activity [Capitolo/Saggio]
Paquet-Durand, François; Ekström, Per; Marigo, Valeria
abstract

Fluxes of calcium ions (Ca2+) in rod photoreceptors are major regulators of steady-state and light-evoked intracellular reactions to stimuli. The homeostasis of Ca2+ is regulated by channels and pumps localized at the plasma membrane and in intracellular organelles. Photoreceptor degeneration is frequently associated with Ca2+ homeostasis disruption and stimulation of Ca2+ activated proteases, such as calpains. These events trigger molecular pathways leading to cell death. In this chapter we discuss Ca2+ channels and pumps as well as calpains as potential targets of new therapies for retinal degeneration.


2019 - CHAPTER 6. Modulation of cGMP-signalling to Prevent Retinal Degeneration [Capitolo/Saggio]
Marigo, Valeria; Ekström, Per; Schwede, Frank; Rentsch, Andreas; Paquet-Durand, François
abstract

In the photoreceptors of the retina, the second-messenger molecule cyclic guanosine monophosphate (cGMP) occupies centre stage in the phototransduction cascade. Remarkably, cGMP is also involved in hereditary photoreceptor degeneration caused by a variety of different genetic insults. This provides an entry point for the development of inhibitory cGMP analogues for a mutation-independent treatment. Here, we outline how cGMP signalling can be targeted for the treatment of retinal degeneration, how inhibitory cGMP analogues may be designed and formulated, and how test systems of rising complexity can be used to identify new compounds with photoreceptor neuroprotective properties. In this context, we cite the European Union-funded DRUGSFORD project and provide an example for the efficacy of a specific cGMP analogue to prevent photoreceptor loss and preserve retinal function.


2019 - CRISPR/Cas9 gene editing in vitro and in retinal cells in vivo [Capitolo/Saggio]
Benati, D.; Marigo, V.; Recchia, A.
abstract

CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knock-down of human Rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids.


2019 - Differential Contribution of Calcium-Activated Proteases and ER-Stress in Three Mouse Models of Retinitis Pigmentosa Expressing P23H Mutant RHO [Capitolo/Saggio]
Comitato, A.; Schiroli, D.; La Marca, C.; Marigo, V.
abstract

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.


2019 - Drug delivery to retinal photoreceptors [Articolo su rivista]
Himawan, Erico; Ekström, Per; Buzgo, Matej; Gaillard, Pieter; Stefánsson, Einar; Marigo, Valeria; Loftsson, Thorsteinn; Paquet-Durand, François
abstract

The photoreceptors of the retina are afflicted by diseases that still often lack satisfactory treatment options. Although suitable drugs might be available in some cases, the delivery of these compounds into the eye and across the blood–retinal barrier remains a significant challenge for therapy development. Here, we review the routes of drug administration to the retina and highlight different options for drug delivery to the photoreceptor cells.


2019 - RD Genes Associated with High Photoreceptor cGMP-Levels (Mini-Review) [Capitolo/Saggio]
Paquet-Durand, F.; Marigo, V.; Ekstrom, P.
abstract

Many RD-causing mutations lead to a dysregulation of cyclic guanosine monophosphate (cGMP), making cGMP signalling a prime target for the development of new treatment approaches. We showed previously that an analogue of cGMP, which inhibited cGMP signalling targets, increased photoreceptor viability in three rodent RD models carrying different genetic defects, in different RD genes. This raises the question of the possible generality of this approach as a treatment for RD. Here, we review RD genes that can be associated with high cGMP and discuss which RD genes might be amenable to a treatment aimed at inhibiting excessive cGMP signalling.


2018 - A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness [Articolo su rivista]
Behnen, Petra; Felline, Angelo; Comitato, Antonella; Di Salvo, Maria Teresa; Raimondi, Francesco; Gulati, Sahil; Kahremany, Shirin; Palczewski, Krzysztof; Marigo, Valeria; Fanelli, Francesca
abstract

The autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.


2018 - Combination of cGMP analogue and drug delivery system provides functional protection in hereditary retinal degeneration [Articolo su rivista]
Vighi, E.; Trifunović, D.; Veiga-Crespo, P.; Rentsch, A.; Hoffmann, D.; Sahaboglu, A.; Strasser, T.; Kulkarni, M.; Bertolotti, E.; van den Heuvel, A.; Peters, T.; Reijerkerk, A.; Euler, T.; Ueffing, M.; Schwede, F.; Genieser, H. -G.; Gaillard, P.; Marigo, V.; Ekström, P.; Paquet-Durand, F.
abstract

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.


2018 - New cGMP analogues restrain proliferation and migration of melanoma cells [Articolo su rivista]
Vighi, Eleonora; Rentsch, Andreas; Henning, Philipp; Comitato, Antonella; Hoffmann, Dorit; Bertinetti, Daniela; Bertolotti, Evelina; Schwede, Frank; Herberg, Friedrich W.; Genieser, Hans-Gottfried; Marigo, Valeria
abstract

Melanoma is one of the most aggressive cancers and displays high resistance to conventional chemotherapy underlining the need for new therapeutic strategies. The cGMP/PKG signaling pathway was detected in melanoma cells and shown to reduce migration, proliferation and to increase apoptosis in different cancer types. In this study, we evaluated the effects on cell viability, cell death, proliferation and migration of novel dimeric cGMP analogues in two melanoma cell lines (MNT1 and SkMel28). These new dimeric cGMP analogues, by activating PKG with limited effects on PKA, significantly reduced proliferation, migration and increased cell death. No decrease in cell viability was observed in non-tumor cells suggesting a tumor-specific effect. These effects observed in melanoma are possibly mediated by PKG2 activation based on the decreased toxic effects in tumor cell lines not expressing PKG2. Finally, PKGassociated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP), linked to cell death, proliferation and migration was found increased and with a change of subcellular localization. Increased phosphorylation of RhoA induced by activation of PKG may also contribute to reduced migration ability of the SkMel28 melanoma cell line when treated with cGMP analogues. These findings suggest that the cGMP/PKG pathway can be envisaged as a therapeutic target of novel dimeric cGMP analogues for the treatment of melanoma.


2018 - Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina [Articolo su rivista]
Comitato, Antonella; Subramanian, Preeti; Turchiano, Giandomenico; Montanari, Monica; Becerra, S. Patricia; Marigo, Valeria
abstract

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.


2018 - Primary rod and cone degeneration is prevented by HDAC inhibition [Capitolo/Saggio]
Trifunović, Dragana; Petridou, Eleni; Comitato, Antonella; Marigo, Valeria; Ueffing, Marius; Paquet-Durand, François
abstract

Photoreceptor cell death in inherited retinal degeneration is accompanied by over-activation of histone deacetylases (HDAC). Excessive HDAC activity is found both in primary rod degeneration (such as in the rd10 mouse) and in primary cone death, including the cone photoreceptor function loss 1 (cpfl1) mouse. We evaluated the potential of pharmacological HDAC inhibition to prevent photoreceptor degeneration in primary rod and cone degeneration. We show that a single in vivo treatment of cpfl1 mice with the HDAC inhibitor trichostatin A (TSA) resulted in a significant protection of cpfl1 mutant cones. Similarly, HDAC inhibition with the clinically approved HDAC inhibitor vorinostat (SAHA) resulted in a significant improvement of rod survival in rd10 retinal explant cultures. Altogether, these results highlight the feasibility of targeted neuroprotection in vivo and create hope to maintain vision in patients suffering from both rod and cone dystrophies.


2018 - Specific knock-down of C-terminal dominant mutation in Rhodopsin gene by CRISPR/Cas9 system [Abstract in Atti di Convegno]
Benati, Daniela; Patrizi, Clarissa; Marigo, Valeria; Auricchio, Alberto; Recchia, Alessandra
abstract


2017 - CRISPR/Cas9-mediated specific knock-down of dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Manel, Llado; Patrizi, Clarissa; Schiroli, Davide; Marigo, Valeria; Auricchio, Alberto; Recchia, Alessandra
abstract

Rhodopsin (RHO) mutations represent a common cause of blindness, accounting for 25% of autosomal dominant Retinitis Pigmentosa (RP) and 8-10% of all RP. Although gene therapy has been successfully applied to retinal degeneration caused by recessive mutations, therapeutic intervention for dominant mutations are still lagging behind. In this study, we explored the efficacy of newly described CRISPR/Cas9 variants with altered PAM specificity and nearly completely reduced off-target effects, to specifically inactivate two highly frequent dominant mutations, P23H and P347S, mapped in the N-terminal and the C-terminal region of the RHO gene, respectively. We designed gRNAs on the mutations to compare allele-specific targeting of the high fidelity SpCas9 (SpCas9-HF1), the respective VQR variant (SpCas9-VQR-HF1) or the SaCas9, and we tested gRNAs in vitro on HeLa clones stably expressing P23H, P347S or wild-type RHO. Analysis of insertions or deletions (Indels) in the genomic DNA specifically in the RHO gene, by Cel-I assay and sequencing, identified the most efficient and mutation-specific system able to induce Indels in the P23H or P347S RHO mutated allele, with almost undetectable editing of the wild-type allele. We are going to package the selected CRISPR/Cas9-gRNA in AAV2/8 particles to test this approach in P23H or P347S RHO transgenic mice, to evaluate retina functionality and vision recovery upon CRISPR/Cas9-mediated editing. Our results will provide clear evidences about the employment of CRISPR/Cas9 system to selectively target dominant mutations and the preclinical application of this strategy for patients affected by RP due to mutations in the RHO gene.


2017 - Erratum: The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells (Human molecular genetics (2008) 17 22 (3487-3501)) [Articolo su rivista]
Palmisano, I.; Bagnato, P.; Palmigiano, A.; Innamorati, G.; Rotondo, G.; Altimare, D.; Venturi, C.; Sviderskaya, E. V.; Piccirillo, R.; Coppola, M.; Marigo, V.; Incerti, B.; Ballabio, A.; Surace, E. M.; Tacchetti, C.; Bennett, D. C.; Schiaffino, M. V.
abstract


2017 - New dimeric cGMP analogues reduce proliferation in three colon cancer cell lines [Articolo su rivista]
Hoffmann, Dorit; Rentsch, Andreas; Vighi, Eleonora; Bertolotti, Evelina; Comitato, Antonella; Schwede, Frank; Genieser, Hans-Gottfried; Marigo, Valeria
abstract

Activation of the cGMP-dependent protein kinase G (PKG) can inhibit growth and/or induce apoptosis in colon cancer. In this study we evaluated the effects on cell viability, cell death and proliferation of novel dimeric cGMP analogues, compared to a monomeric compound. Three colon cancer cell lines, which only express isoform 2 of PKG, were treated with these novel cGMP analogues and responded with increased PKG activity. cGMP analogues reduced cell viability in the three cell lines and this was due to a cytostatic rather than cytotoxic effect. These findings suggest that activation of PKG2 can be a therapeutic target in the treatment of colon cancer and, most importantly, that dimeric cGMP analogues can further improve the beneficial effects previously observed with monomeric cGMP analogues.


2017 - Targeted liposomal delivery of cGMP analogues [Brevetto]
Per, Ekström; François, PAQUET-DURAND; Pieter Jaap, Gaillard; Marigo, Valeria; Hans-Gottfried, Genieser; Andreas, Rentsch
abstract

The invention relates to means and methods of targeted drug delivery of therapeutic agent to and across the blood-ocular barrier. In particular, the invention relates to cyclic guanosine-3', 5 '-monophosphate analogues as therapeutic agent for treating retinal diseases. The cGMPSs targeted to the blood-ocular barrier by glutathione-based ligands that facilitate the specific binding to and enhanced internalization by glutathione transporters present on the blood-ocular barrier. The glutathione-based ligands are conjugated to nanocontainers such as liposomes encapsulating the cGMPSs.


2016 - Dominant and recessive mutations in rhodopsin activate different cell death pathways [Articolo su rivista]
Comitato, Antonella; DI SALVO, MARIA TERESA; Turchiano, Giandomenico; Montanari, Monica; Sakami, Sanae; Palczewski, Krzysztof; Marigo, Valeria
abstract

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.


2016 - HDAC inhibition in the cpfl1 mouse protects degenerating cone photoreceptors in vivo [Articolo su rivista]
Trifunović, Dragana; Arango Gonzalez, Blanca; Comitato, Antonella; Barth, Melanie; Del Amo, Eva M; Kulkarni, Manoj; Sahaboglu, Ayse; Hauck, Stefanie M; Urtti, Arto; Arsenijevic, Yvan; Ueffing, Marius; Marigo, Valeria; Paquet Durand, François
abstract

Cone photoreceptor cell death as it occurs in certain hereditary retinal diseases is devastating, with the affected patients suffering from a loss of accurate and colour vision. Regrettably, these hereditary cone diseases are still untreatable to date. Thus, the identification of substances able to block or restrain cone cell death is of primary importance. We studied the neuroprotective effects of a histone deacetylase inhibitor, Trichostatin A (TSA), in a mouse model of inherited, primary cone degeneration (cpfl1). We show that HDAC inhibition protects cpfl1 cones in vitro, in retinal explant cultures. More importantly, in vivo, a single intravitreal TSA injection significantly increased cone survival for up to 16 days post-injection. In addition, the abnormal, incomplete cone migration pattern in the cpfl1 retina was significantly improved by HDAC inhibition. These findings suggest a crucial role for HDAC activity in primary cone degeneration and highlight a new avenue for future therapy developments for cone dystrophies and retinal diseases associated with impaired cone migration.


2016 - In vitro and in vivo CRISPR/Cas9-mediated genome editing to downregulate dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Cocchiarella, Fabienne; Marigo, Valeria; Recchia, Alessandra
abstract

Many progresses have been made in understanding the genetic basis for Retinitis Pigmentosa (RP), however therapeutic interventions are still lagging behind. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of autosomal dominant RP and 8-10% of all RP (Hartong et al., 2006) with more than 100 different mutations identified so far. Here we show the application of CRISPR-Cas9 technology to knock out the RHO defective alleles by introducing double strand breaks into the target gene. We designed single or double gRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. The two gRNAs were tested singularly or together in vitro in HeLa clones stably expressing P23H RHO. Cel I assay, TIDE and sequencing analyses demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene, which caused strong reduction of RHO expression up to 90%. The higher effect was obtained with two gRNAs together. The CRISPR/Cas9 plasmid expressing two gRNAs were then in vivo tested in P23H RHO transgenic mice by sub-retinal electroporation, together with EGFP expressing plasmids. Analysis of indels in FACS-sorted EGFP+ cells demonstrated up to 30% of in vivo genome editing of the human P23H RHO gene, without targeting of the murine Rho allele. We also detected reduction of RHO at mRNA and protein levels. Thus, successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool and its potential use in gene therapy.


2016 - In vitro and in vivo genome editing of the RHO gene to downregulate dominant mutations [Abstract in Rivista]
Marigo, Valeria; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Recchia, Alessandra
abstract

Purpose: Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand break (DSB) into the target gene. Methods: The CRISPR/Cas9 system was developed to target the first exon of the human RHO gene by designing 2 gRNAs. The system was tested in vitro in HeLa clones expressing RHO. The effects on the RHO gene were evaluated by sequencing and by analyses at mRNA and protein levels. We expressed by electroporation these 2 gRNAs and Cas9 in vivo in transgenic mice expressing human RHO with P23H mutation. Genome editing was evaluated by sequencing genomic DNA from targeted cells and by analyzing RHO mRNA and protein. Results: Two gRNAs were designed in the first exon of the RHO gene and one of them targeted the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice by electroporation. Targeted cells were tracked by co-expression with EGFP. EGFP+ cells were FACS sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. We also detected reduction of human RHO mRNA expression as well as RHO protein. Conclusions: We developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.


2016 - In vitro and in vivo genome editing of the RHO gene to downregulate dominant mutations [Abstract in Atti di Convegno]
Latella, Maria C.; Di Salvo, Maria T.; Cocchiarella, Fabienne; Benati, Daniela; Marigo, Valeria; Recchia, Alessandra
abstract


2016 - In vivo editing of the human mutant Rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina [Articolo su rivista]
Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra
abstract

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/ Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.


2016 - Unravelling the Complexity of Inherited Retinal Dystrophies Molecular Testing: Added Value of Targeted Next-Generation Sequencing [Articolo su rivista]
Bernardis, Isabella; Chiesi, Laura; Tenedini, Elena; Artuso, Lucia; Percesepe, Antonio; Artusi, Valentina; Simone, Maria Luisa; Manfredini, Rossella; Camparini, Monica; Rinaldi, Chiara; Ciardella, Antonio; Graziano, Claudio; Balducci, Nicole; Tranchina, Antonia; Cavallini, Gian Maria; Pietrangelo, Antonello; Marigo, Valeria; Tagliafico, Enrico
abstract

To assess the clinical utility of targeted Next-Generation Sequencing (NGS) for the diagnosis of Inherited Retinal Dystrophies (IRDs), a total of 109 subjects were enrolled in the study, including 88 IRD affected probands and 21 healthy relatives. Clinical diagnoses included Retinitis Pigmentosa (RP), Leber Congenital Amaurosis (LCA), Stargardt Disease (STGD), Best Macular Dystrophy (BMD), Usher Syndrome (USH), and other IRDs with undefined clinical diagnosis. Participants underwent a complete ophthalmologic examination followed by genetic counseling. A custom AmpliSeq� panel of 72 IRD-related genes was designed for the analysis and tested using Ion semiconductor Next-Generation Sequencing (NGS). Potential disease-causing mutations were identified in 59.1% of probands, comprising mutations in 16 genes. The highest diagnostic yields were achieved for BMD, LCA, USH, and STGD patients, whereas RP confirmed its high genetic heterogeneity. Causative mutations were identified in 17.6% of probands with undefined diagnosis. Revision of the initial diagnosis was performed for 9.6% of genetically diagnosed patients. This study demonstrates that NGS represents a comprehensive cost-effective approach for IRDs molecular diagnosis. The identification of the genetic alterations underlying the phenotype enabled the clinicians to achieve a more accurate diagnosis. The results emphasize the importance of molecular diagnosis coupled with clinic information to unravel the extensive phenotypic heterogeneity of these diseases.


2015 - A Next Generation Sequencing amplicon-based strategy to explore Inherited Retinal Degeneration complexity [Abstract in Atti di Convegno]
Artusi, Valentina; Chiesi, Laura; Bernardis, Isabella; Tenedini, Elena; Artuso, Lucia; Cavallini, Gian Maria; Percesepe, Antonio; Marigo, Valeria; Tagliafico, Enrico
abstract

Inherited Retinal Degeneration (IRD) are a group of eye disorders, characterized by photoreceptors degeneration which include: Retinitis Pigmentosa, Stargardt disease, Usher Syndrome and Leber congenital amaurosis. The high genetic heterogeneity, the incompleteness of disease specific databases and the elevated number of genes involved in IRD, often hamper the correct molecular diagnosis and patients stratification. To clarify IRD molecular profile, we used a next Generation Sequencing (NGS) strategy and designed a custom AmpliSeq panel (Life Technologies), containing the coding sequences of 72 disease related genes, for a total of 1649 amplicons. An in-house bioinformatic pipeline was optimized to filter synonymous variants and polymorphism and to annotate variants with prediction algorithm (dbNSFP) and disease specific databases (LOVD eye diseases, Retina International, RPGR database). A cohort of 40 samples was selected (29 patients, 11 healthy relatives). They underwent a complete ophthalmologic examination (visual acuity, anterior segment and fundus examination, ERG and/or EOG, OCT), as well as a genetic counselling. Possibly causative mutations were detected in 62% patients (n=18). We found mutations in 8 genes. The most recurrent gene was mutated in 38% (n=7) of patients. The remaining seven genes harboured lower frequencies with just one or two patients mutated. Overall, seven genes were inherited with an autosomal recessive pattern and one gene was X-linked. Of note, less than 21% of variants have been already described in specific databases. These preliminary results highlight the need to further explore the molecular complexity and heterogeneity of RD in order to translate these analyses into clinical practice.


2015 - Classification of Rhodopsin mutations by integrated in silico and in vitro analyses for screening of chaperon molecules to rescue misfolding. [Abstract in Rivista]
Marigo, Valeria; Behnen, PETRA JOHANNA; Felline, Angelo Nicola; Fanelli, Francesca
abstract

Purpose: About 140 point mutations were identified in the rhodopsin gene (RHO) as cause of Autosomal Dominant Retinitis Pigmentosa (ADRP), a genetic degenerative disease causing blindness in later life. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Characterization of the structural and molecular features of such mutants will support the development of rational drug design. Methods: Wild type RHO and 33 different RHO mutations were analyzed in silico either in the rhodopsin form bound to retinal or in the opsin form by thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells either in the absence or presence of 9-cis retinal in the culture medium. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. Retention in the endoplasmic reticulum (ER) was assessed by analysis of co-localization with calnexin and calculation of the Pearson Correlation Coefficient (PCC) of co-localization. Results: In silico studies revealed that the selected ADRP RHO mutations share marked abilities to impair highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates to the stability of the protein. We defined computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed increased ER retention and reduction of plasma membrane localization of most of the mutants compared to wild type RHO. We found a strong correlation of the perturbation indexes calculated by in silico analyses with PCC calculated by in vitro analyses. We could also characterize different abilities of the mutated proteins to be affected by treatment with 9-cis retinal. Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also developed a PCC index to evaluate the effect of retinal on protein folding and protein localization at the plasma membrane. This knowledge will be our starting point for in silico screening of compounds able to bind the retinal site and act as chaperones. The in vitro studies have developed a quantitative analysis to assess therapeutic effects of chaperone molecules.


2015 - Directed differentiation of Photoreceptors and Retinal Pigment Epithelium from adult Mouse Retinal Stem Cells [Capitolo/Saggio]
Ballios, Brian G.; Marigo, Valeria; van der Kooy, Derek
abstract

The use of stem cell progeny for transplantation for retinal regenerative therapy will require techniques to direct the differentiation of these cells toward specific retinal cell types. Adult retinal stem cells (RSCs) represent a promising source of cells for retinal cell therapy to treat blinding eye diseases. Here, we describe the use of combinations of exogenous culture additives to direct RSCs along photoreceptor and retinal pigment epithelial (RPE) lineages with high efficiency.


2015 - Small retinoprotective peptides reveal a receptor-binding region on pigment epithelium-derived factor [Articolo su rivista]
Kenealey, Jason; Subramanian, Preeti; Comitato, Antonella; Bullock, Jeanee; Keehan, Laura; Polato, Federica; Hoover, David; Marigo, Valeria; Becerra, S. Patricia
abstract

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDFR). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78-121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98-114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98-114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.


2014 - Activation of Bax in Three Models of Retinitis Pigmentosa [Articolo su rivista]
Comitato, Antonella; D., Sanges; A., Rossi; M. M., Humphries; Marigo, Valeria
abstract

PURPOSE:The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas. METHODS:Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin. RESULTS:We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina. CONCLUSIONS:Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.


2014 - Photoreceptor Transplantation and Regeneration [Capitolo/Saggio]
Marigo, Valeria; Simona, Casarosa
abstract

A recent study showed that an electronic chip implanted under the human retina restored some extent of vision to a blind patient. Because the device was implanted where the light sensitive cells, the photoreceptors, should have been, this study demonstrated that it is possible to take advantage of the internal circuitry of the retina even in the absence of photoreceptors and in the presence of extensive glial and neuronal reorganization. This result strongly supports the development of cell replacement therapies for the cure of photoreceptor degeneration, provided that the cells are implanted in the same anatomical location. Similarly to other sensory neurons but differently from neurons lost in most degenerative diseases, photoreceptors are the first neurons of the circuit and only have to make efferent connections. Secondly, photoreceptors are histologically located in a restricted region of the organ. These features make them the most immediately transplantable type of neuron and interesting candidates for clinical trials involving cell transplantation. In cell replacement therapies the identification of the source of cells able to integrate and connect to the host tissue needs to be defined. For the retina, cells showing the best survival and integration rates are post-mitotic rod precursors, rather than immature retinal progenitors. Given the difficulty of obtaining human fetal cells, many studies are undergoing to differentiate cells with such features starting from stem cells. Three main classes of stem cells are under investigation to be sources for in vitro photoreceptor generation. They are embryonic stem cells, induced pluripotent stem cells and adult retinal stem cells. This chapter will describe the current preclincal studies for in vitro generation and subsequent transplantation of photoreceptor precursors.


2014 - Protective effects by inhibition of CNG channels and PKG activity in rd1 mutant photoreceptors [Abstract in Rivista]
Bertolotti, E; Hoffmann, D; Rentsch, A; Schwede, F; Marigo, V
abstract

Protective effects by inhibition of CNG channels and PKG activity in rd1 mutant photoreceptors


2014 - Stem cells as source for retinal pigment epithelium transplantation [Articolo su rivista]
Bertolotti, Evelina; A., Neri; M., Camparini; C., Macaluso; Marigo, Valeria
abstract

Inherited maculopathies, age related macular degeneration and some forms of retinitis pigmentosa are associated with impaired function or loss of the retinal pigment epithelium (RPE). Among potential treatments, transplantation approaches are particularly promising. The arrangement of RPE cells in a well-defined tissue layer makes the RPE amenable to cell or tissue sheet transplantation. Different cell sources have been suggested for RPE transplantation but the development of a clinical protocol faces several obstacles. The source should provide a sufficient number of cells to at least recover the macula area. Secondly, cells should be plastic enough to be able to integrate in the host tissue. Tissue sheets should be considered as well, but the substrate on which RPE cells are cultured needs to be carefully evaluated. Immunogenicity can also be an obstacle for effective transplantation as well as tumorigenicity of not fully differentiated cells. Finally, ethical concerns may represent drawbacks when embryo-derived cells are proposed for RPE transplantation. Here we discuss different cell sources that became available in recent years and their different properties. We also present data on a new source of human RPE. We provide a protocol for RPE differentiation of retinal stem cells derived from adult ciliary bodies of post-mortem donors. We show molecular characterization of the in vitro differentiated RPE tissue and demonstrate its functionality based on a phagocytosis assay. This new source may provide tissue for allogenic transplantation based on best matches through histocompatibility testing.


2013 - Differentiation of retinal pigment epithelium from retinal neurospheres of the adult ciliary body [Abstract in Rivista]
Bertolotti, Evelina; Aruta, CLAUDIA GAETANA; Giordano, Francesca; Raposo, Graça; Marigo, Valeria
abstract

Transplantation of retina pigment epithelium (RPE) has great potentials for the cure of several retinal degenerative diseases such as age-related macular degeneration (AMD). The source of cells for this type of therapy is still under evaluation even if clinical trials with RPE derived from human embryonic stem cells have recently started. We evaluated the ciliary body as a source of cells to differentiate RPE in vitro. The ciliary body is an accessible tissue that can be easily obtained from post-mortem donors. Retinal neurospheres can be generated from ciliary body cells after dissociation and growth in clonal conditions. We deeply characterized differentiation of RPE cells from retinal neurospheres derived from murine eyes. We will also discuss the differentiation protocol developed for the human tissue.


2013 - Integrated in silico and in vitro characterization of Rhodopsin mutations and molecular mechanisms activated in photoreceptor cell death [Poster]
Marigo, Valeria; Behnen, PETRA JOHANNA; Comitato, Antonella; DI SALVO, MARIA TERESA; Felline, Angelo Nicola; Fanelli, Francesca
abstract

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were identified in the rhodopsin gene (RHO) as cause of the Autosomal Dominant form of the disease (ADRP). A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants and the cell death pathways activated by mutant RHO are obscure, hampering rational drug design. Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. Subcellular localization and effects of retinal as chaperone in the same mutants were characterized in vitro in COS-7 cells. Molecular death pathways activated by RHO mutation were studied in vivo in transgenic and knock-in mice bearing the P23H mutation. Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO as well as different abilities of the mutated proteins to be affected by 9-cis retinal. We characterized ER-stress pathways and calpain pathway activation in P23H mutant retinas. We defined the different contributions of these pathways by in vivo treatments with specific drugs blocking either ER-stress or calpains. Conclusions: The in silico and in vitro levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also characterized cell death pathways activated by RHO mutants. This knowledge will be our starting point for an in silico screen of chaperone molecules to be tested in vivo.


2013 - Integrated in silico and in vitro characterization of Rhodopsin mutations causing RP4 [Abstract in Rivista]
Marigo, Valeria; Behnen, PETRA JOHANNA; Felline, Angelo Nicola; Fanelli, Francesca
abstract

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were discovered in the rhodopsin gene (RHO). RHO belongs to the G protein Coupled Receptor superfamily of seven-transmembrane proteins. The vast majority of the rhodopsin mutations cause the Autosomal Dominant form (ADRP) of the disease. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants are obscure, which hampers rational drug design. Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. In order to define levels of expression and differences in post-translational modifications of the mutants compared to the wild type, the proteins were analyzed by Western blotting. Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. We could define a number of computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO. We also defined different abilities of the mutated proteins to be affected by 9-cis retinal. Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. This knowledge will be our starting point for the choice of a number of mutations to be used to reveal therapeutic effect of chaperone molecules.


2012 - Cell death pathways activated in rod photoreceptors in different models of retinitis pigmentosa [Poster]
Marigo, Valeria; Comitato, Antonella; Morandi, Paolo
abstract

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. About 50 genes have been linked to this hereditary disease. Heterogeneity of such magnitude in RP represents a major impediment to the development therapies, therefore mutation-independent approaches to target common molecular mechanisms activated during the degenerative process should be exploited. We molecularly characterized mitochondrial and ER pathways activated during rod cell death in three murine models of RP and developed molecules to interfere with their function. Methods: We studied the rd1, the Rho-/- and the P23H transgenic mouse models with molecular biology, immunocytochemical and biochemical approaches. We analyzed Aif, caspase-3 and caspase-7, Bax and Bak, calpain, cathepsin and ER stress markers. We performed in vitro interferences with shRNAs and in vivo treatments with Calpain, Cathepsin D and Bax inhibiting substances. Results: We found that calpains play a key role in the activation of Aif, Bax and cell death in the rd1 retina. shRNA experiments down-regulating either calpain 1 or calpain 2 allowed to define the different contributions of these two proteases. By reduction of Aif expression we confirmed the important role of Aif in apoptosis in the rd1 retina. Aif appears also to be important in the other forms of retinal degeneration. Otherwise, ER stress pathways do not appear to be activated in all models analyzed. Conclusions: The option of exploiting cell death as a therapeutic target is complex. Nevertheless the molecular understanding of the factors activated during degeneration and the identification of common activators are the first step toward this goal. Our study identifies calpains and Aif as a key factors triggering photoreceptor cell demise in the retina of different murine models of RP. The efficacy of interfering molecules targeting the different factors activated during the apoptotic cascade opens new perspectives for designing therapeutic approaches to rescue photoreceptor cell death in this disease.


2012 - Cellule e segnali [Monografia/Trattato scientifico]
Marigo, Valeria; Zappavigna, Vincenzo
abstract

È un libro di testo indirizzato agli studenti delle lauree triennali e magistrali in Scienze Biologiche e Biotecnologie per lo studio dei principali meccanismi di comunicazione tra cellule che controllano la proliferazione cellulare, il differenziamento e la morfogenesi. Il testo tratta le principali vie di segnalazione quali: Vie mediate dai recettori accoppiati a proteine G e recettori tirosina chinasi, Famiglia di proteine TGFb, Wnt, Hedgehog, Recettori nucleari per ormoni, Notch, Efrine. Si propone altresì di discutere i meccanismi molecolari attivati da queste vie di segnalazione e la loro importanza nello sviluppo embrionale e nella proliferazione cellulare. Inoltre illustra il coinvolgimento di queste vie di trasduzione del segnale in patologie come il cancro e le malattie ereditarie.


2012 - Functional and Molecular Characterization of Rod-like Cells from Retinal Stem Cells Derived from the Adult Ciliary Epithelium [Articolo su rivista]
G. C., Demontis; C., Aruta; Comitato, Antonella; A., De Marzo; Marigo, Valeria
abstract

In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.


2012 - Melanoregulin, product of the dsu locus, links the BLOC-pathway and Oa1 in organelle biogenesis. [Articolo su rivista]
R. A., Rachel; K., Nagashima; T. N., O'Sullivan; L. S., Frost; F. P., Stefano; Marigo, Valeria; K., Boesze Battaglia
abstract

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg(dsu) gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg(dsu) locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1(ko/ko) mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.


2012 - TARGETING CELL DEATH PATHWAYS ACTIVATED IN MURINE MODELS OF RETINITIS PIGMENTOSA [Poster]
Comitato, Antonella; Marigo, Valeria
abstract

Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. About 50 genes have been linked to this hereditary disease and this genetic heterogeneity represents a major impediment to the development therapies. Mutation-independent approaches to target common molecular mechanisms activated during the degenerative process should be exploited. We molecularly characterized mitochondrial and ER pathways activated during rod cell death in three murine models of RP and developed molecules to interfere with their function. We studied the rd1, the Rho-/- and the P23H transgenic mouse models with immunocytochemical, molecular and biochemical approaches. We analyzed Aif, caspase-3 and caspase-7, Bax and Bak, calpain, cathepsin and ER stress markers. We performed in vitro interferences with shRNAs and in vivo treatments with Calpain, Cathepsin D and Bax inhibiting substances. We found that calpains play a key role in the activation of Aif, Bax and cell death in the rd1 retina. shRNA experiments down-regulating either calpain 1 or calpain 2 allowed to define the different contributions of these two proteases. Aif appears to play a fundamental role in different forms of retinal degeneration. Otherwise, ER stress pathways do not appear to be activated in all models analyzed. Our study identifies calpains and Aif as a key factors triggering photoreceptor cell demise in the retina of different murine models of RP. The efficacy of interfering molecules targeting the different factors activated during the apoptotic cascade opens new perspectives for designing therapeutic approaches to rescue photoreceptor cell death in this disease.


2011 - In vitro differentiation of retinal pigment epithelium from adult retinal stem cells. [Articolo su rivista]
Aruta, CLAUDIA GAETANA; F., Giordano; DE MARZO, Anna; A., Comitato; G., Raposo; E. F., Nandrot; Marigo, Valeria
abstract

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.


2011 - Mitochondrial and ER pathways interplay in rod photoreceptor cell death [Relazione in Atti di Convegno]
Marigo, Valeria; Comitato, Antonella; Morandi, Paolo; Benassi, Silvia
abstract

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Several genes have been linked to this hereditary disease. While heterogeneity of such magnitude in RP would appear to represent a major impediment to the development of therapeutics, mutation-independent approaches to target common molecular mechanisms activated during the degenerative process have to be exploited. We molecularly characterized mitochondrial and ER pathways activated during rod cell death in three murine models of RP and developed molecules to interfere with their function. Methods: We studied the rd1, the Rho-/- and the P23H transgenic mouse models with immunocytochemical and biochemical approaches. We analyzed mitochondrial Aif, caspase-3 and caspase-7, Bax and Bak, calpain and cathepsin activation, ER caspase-12, and ER stress markers. We performed in vitro interferences with shRNAs and in vivo treatments with Calpain and Bax inhibiting substances. Results: We found that calpains play a key role in the activation of Aif, Bax and cell death in the rd1 retina. shRNA experiments down-regulating either calpain 1 or calpain 2 allowed to define the different contributions of these two proteases. By reduction of Aif expression we confirmed the important role of Aif in apoptosis in the rd1 retina. Aif appears also to be important in the other forms of retinal degeneration. Otherwise, only two ER stress pathways are activated in the degenerating rd1 retina and they appear not to be critical for cell death. Conclusions: The option of exploiting cell death as a therapeutic target is complex. Nevertheless the molecular understanding of the factors activated during degeneration and the identification of common activators are the first step toward this goal. Our study identifies calpains and Aif as a key factors triggering photoreceptor cell demise in the rd1 retina. The efficacy of interfering molecules targeting the different factors activated during the apoptotic cascade opens new perspectives for designing therapeutic approaches to rescue photoreceptor cell death in this disease.


2011 - Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa. [Articolo su rivista]
C., Mussolino; D., Sanges; E., Marrocco; C., Bonetti; U., Di Vicino; Marigo, Valeria; A., Auricchio; G., Meroni; E. M., Surace
abstract

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a 'repression-replacement' strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.


2010 - PEDF Promotes Retinal Neurosphere Formation and Expansion In Vitro [Capitolo/Saggio]
DE MARZO, Anna; Aruta, CLAUDIA GAETANA; Marigo, Valeria
abstract

The retina is subject to degenerative conditions leading to blindness. Although retinal regeneration is possible in lower vertebrates, it does not occur in the adult mammalian retina. Retinal stem cell (RSC) research offers unique opportunities for developing clinical application for therapy. The ciliary body of adult mammals represents a source of quiescent RSC. These neural progenitors have a limited self-renewal potential in vitro but this can be improved by mitogens. Pigment Epithelium Derived Factor (PEDF), a member of the serpin gene family, is synthesized and secreted by retinal pigment epithelium (RPE) cells. We tested combinations of PEDF with fibroblast growth factor (FGF) during RSC growth to evaluate self-renewal and subsequent differentiation into retinal-like neuronal cell types. Medium supplemented with FGF + PEDF enhanced the RSC yield and more interestingly allowed expansion of the culture by increasing secondary retinal neurospheres after the 1st passage. This effect was accompanied by cell proliferation as revealed by BrdU incorporation. PEDF usage did not affect rod-like differentiation potential. This was demonstrated by immunofluorescence analysis of Rhodopsin and Pde6b that were found similarly expressed in cells derived from FGF or FGF + PEDF cultured RSC. Our studies suggest a possible application of PEDF in Retinal Stem Cell culture and transplantation.


2010 - Photoreceptor rescue and toxicity induced by different calpain inhibitors. [Articolo su rivista]
F., Paquet Durand; D. Sanges, D.; J., Mccall; J., Silva; T., van Veen; Marigo, Valeria; P., Ekström
abstract

Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP.


2009 - The Ocular Albinism type 1 (OA1) G-Protein Coupled Receptor functions with MART-1 at early stages of melanogenesis to control melanosome identity and composition [Articolo su rivista]
F., Giordano; C., Bonetti; E. M., Surace; Marigo, Valeria; G., Raposo
abstract

OA1 (GPR143; GPCR, G-protein-coupled receptor), the protein product of the ocular albinism type 1 gene, encodes a pigment-cell-specific GPCR that localizes intracellularly to melanosomes. OA1 mutations result in ocular albinism due to alterations in melanosome formation, suggesting that OA1 is a key player in the biogenesis of melanosomes. To address the function of OA1 in melanosome biogenesis, we have used siRNA inactivation and combined morphological and biochemical methods to investigate melanosome ultrastructure, melanosomal protein localization and expression in human pigmented melanocytic cells. OA1 loss of function leads to decreased pigmentation and causes formation of enlarged aberrant premelanosomes harboring disorganized fibrillar structures and displaying proteins of mature melanosomes and lysosomes at their membrane. Moreover, we show that OA1 interacts biochemically with the premelanosomal protein MART-1. Inactivation of MART-1 by siRNA leads to a decreased stability of OA1 and is accompanied by similar defects in premelanosome biogenesis and composition. These data show for the first time that melanosome composition and identity are regulated at early stages by OA1 and that MART-1 likely acts as an escort protein for this GPCR.


2009 - Transcriptional Repression of the Human Rhodopsin Gene by Artificial Zinc Finger Protein Recovers Retinal Function in a Model of Autosomal Dominant Retinitis Pigmentosa [Abstract in Rivista]
C., Mussolino; D., Sanges; C., Bonetti; Marigo, Valeria; G., Meroni; E. M., Surace
abstract

Retinitis Pigmentosa (RP) represents a heterogeneous group of severe retinal degenerations. Rhodopsin (Rho) is the gene most commonly associated to the autosomal dominant form of RP (ADRP) with over 150 mutations identifi ed. The objective of this study is to treat ADRP by silencing the Rho gene expression at the transcriptional level in a mutation independent fashion to potentially treat all ADRP due to rhodopsin mutations. To this end we have used artifi cial zinc fi nger transcription factors (ZF-TFs) targeted to human Rhodospin promoter. Through the modular assembly method we generated 10 DNA Binding Domains (DBDs) targeted to hRho promoter and we fused them to the KRAB or VP64 domains to assemble specifi c Rho transcriptional repressors (ZFRs) and activators (ZFAs), respectively. We selected the most effi cient and selective hRho transcription inhibitor using the luciferase reporter system. Twenty percent success rate (2 out of 10) was obtained in generating artifi cial ZF-TFs that robustly and specifi cally repress expression of luciferase driven by hRho promoter in vitro. As further demonstration of specifi city, the 2 selected ZF-TFs, when fused to VP64 domain, are also able to effi ciently transactivate the targeted promoter as well as directly bind it as assessed through Electromobility shift assay (EMSA) experiments. The therapeutic potential of the two selected hRho specifi c repressors (ZFR-2 and ZFR-6) was assessed by transduction experiments on Retinal Stem Cells (RSC) explanted from adult P347S ADRP transgenic mouse model. These cells die once differentiated in vitro because of the expression of the mutated copy of hRho gene. Retroviral delivery of the selected ZFRs to RSC resulted in selective repression of the mutated human Rhodopsin as measured by quantitative Real Time PCR analysis, and remarkably in 90% reduction of apoptotic cells compared to controls. To assess the feasibility of such approach in vivo, we generated an AAV 2/8 harbouring the ZFR-6 driven by a Rhodopsin kinase promoter. One month after vector delivery RT-PCR analysis showed specifi c repression of RHO transcript. Remarkably a significant retinal morphological and functional recovery was observed. Taken together these results demonstrate that artifi cial Zinc Finger transcription factors repress hRho gene in vitro and in vivo, underscoring the potential impact of this novel strategy to treat ADRP.


2008 - A high-resolution RNA expression atlas of Retinitis Pigmentosa genes in the human and mouse retinas [Articolo su rivista]
Trifunovic, D; Karali, M; Camposampiero, D; Ponzin, D; Banfi, S; Marigo, Valeria
abstract

PURPOSE. Retinitis pigmentosa (RP) is one of the leading causes of visual handicap in the world population and is characterized by high genetic heterogeneity. The study of the disease mechanisms and the development of efficient therapeutic approaches have mostly relied on the availability of animal models for this condition, so far. Nevertheless, little information is available about the RNA expression profiles of RP genes in the human retina. An expression atlas of 34 known RP genes in human and murine retinas was generated to overcome this lack of information. METHODS. Appropriate templates were retrieved for 34 RP genes that were used to perform RNA in situ hybridization studies on human and murine adult eyes. RESULTS. Most of the genes displayed similar patterns between human and mouse retina. Different expression patterns were observed for the CNGB1, USH2A, and FSCN2 genes, compared with those in previously reported profiles. In addition, different expression profiles were detected for the RPGR, CA4, PAP1, RGR, and RLBP1 genes in human and mouse retinas. CONCLUSIONS. The first gene expression atlas has been generated of RP genes in human and murine retinas. Differences observed in the expression patterns of some genes in humans and mice, will open new perspectives on the function of these genes and their putative roles in disease pathogenesis.


2008 - Embryonic stem cells as a source of photoreceptors for retinal transplantation [Articolo su rivista]
Marigo, Valeria
abstract

Retinitis pigmentosa (RP) is a degenerative disease causing blindness in later life. More than 30 genes have been linked to this hereditary disease. Heterogeneity of such magnitude in RP represents a major impediment to the development of therapeutics hence mutation-independent approaches have to be exploited. The loss of retinal neurons is generally regarded to be the irreversible cause and the end stage of blindness. Any strategy to restore sight in these cases would almost certainly require cell replacement or transplantation. The paper under evaluation offers methods for in vitro differentiation of murine, monkey and human embryonic stem cells into photoreceptor-like cells. This is an important step forward in the generation of cells apt for retinal transplants.


2008 - Stem cells as prospective therapeutic tools for retinal degeneration [Articolo su rivista]
Marigo, Valeria
abstract

In the recent years stem cells are attracting muchinterest as prospective therapies for treating humandiseases and injury. Degenerative retinal diseasessuch as Retinitis Pigmentosa (RP) and Age RelatedMacular Dystrophy (AMD) have a dramatic socioeconomicimpact in our society. RP is a geneticallyheterogeneous disease and, up to now, around 34genes have been associated with it. On the otherhand, both genetic and environmental factors cooperateas cause of AMD. Due to genetic and functionaldiversity of the involved proteins, the molecularmechanisms underlying the different forms ofretinal degeneration are still not well understood.Limited knowledge on molecular processes leadingto photoreceptor loss hampers therapeutic interventionthat is still lagging behind and mutation independentapproaches are therefore seen as morepracticable approaches to restore vision. Amongthese methods, transplantation of in vitro culturedphotoreceptors is drawing a lot of attention.Cells with retinal progenitor characteristics havebeen isolated with the objective to develop replacementtherapies for the retina. Retinal progenitorscan be purified from the embryonic retina and inducedto differentiate into photoreceptors and otherretinal neuronal cell types. Importantly, stem cellshave been identified in the marginal region of theadult eye and retinal stem cells can be derived andcultured in vitro as pigmented neurospheres fromthe adult murine and human ciliary body (1) and iris(2). Of note, isolation of stem cells from the ciliarybody has been reported from eyes of donors aged upto 70 years old, indicating the possibility of usingeye bank tissues to obtain stem cells for regenerativetherapy. Secondly, stem cells derived from theadult ciliary body possess very good potentialitiesto give rise to high percentages of photoreceptorlikecells (3). In the last years several importantissues have been the focus of research in this field.A first aspect is the source of suitable human cells.Secondly, the cell source should be cultured underappropriate conditions in the absence of feederlayers, FBS, uncharacterized matrigel that can induceexpression of immunogenic antigens. Third, thecultures should be as less heterogeneous as possibleavoiding cells taking different fates other than photoreceptors.This opens several problems that needto be solved in order to develop a system that can betranslated to patients: priorities will be amplificationof the cells in culture to increase the amount ofphotoreceptor progenitors apt for cell transplantation.Secondly, we need to devise a differentiationand selection system to purify photoreceptors fromRPE and other cell types that may differentiate invitro. The most important obstacle to a therapeuticallyefficient retinal cells transplant is the availabilityof high amounts of cells necessary to restoresight. Because of the limited availability of cellsfrom human adult tissues, embryonic stem (ES)cells are, therefore, also seen as a possible source ofphotoreceptors. Several attempts to generate retinalneurons from ES cells have been reported. Themost recent of these papers showed differentiationof Rhodopsin expressing cells from human ES cellsby treatment with a defined formulated medium (4).Previous studies reported differentiation of ES cellsinto photoreceptor-like cells, however they werebased on co-culture experiments with embryonic oradult retinal tissue. These methods were far fromtherapeutic applicability because avoidance of animalderived additives to the culture medium is arequirement to translate protocols to patients.ARCH SOC ESP OFTALMOL 2008; 83: 397-400To date, transplantation studies have not providedcompelling evidence on the maturation of engraftedcells to photoreceptors and their integration into retinalnetworks to support vision. However, very promisingtransplantation achievements suggest that thisfield is advancing and that cell engraftment is apotential therapy that needs to be exploited for retinaldegenerations. T


2008 - The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells [Articolo su rivista]
Palmisano, I.; Bagnato, P.; Palmigiano, A.; Innamorati, G.; Rotondo, G.; Altimare, D.; Venturi, C.; Sviderskaya, E. V.; Piccirillo, R.; Coppola, M.; Marigo, V.; Incerti, B.; Ballabio, A.; Surace, E. M.; Tacchetti, C.; Bennett, D. C.; Schiaffino, M. V.
abstract

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.


2007 - FGF and EGF differently affect differentiation of murine retinal stem cells in vitro [Articolo su rivista]
Giordano, F; DE MARZO, Anna; Vetrini, F; Marigo, Valeria
abstract

Purpose: The developmental processes that mediate differentiation from retinal stem cells (RSC) to different retinal neuronal types remain unclear. During retinal development, progenitor cells modify expression of growth factor (GF) receptors and their differentiation potentials. Similarly, RSC in culture may exhibit alternative molecular characteristics in response to different GF stimuli.Methods: RSC were purified from the adult ciliary margin and exposed to fibroblast growth factor (FGF), epidermal growth factor (EGF), or FGF+EGF. Proliferation was analyzed by bromodeoxyuridine (BrdU) labeling. Differentiation was evaluated by immunofluorescence with antibodies recognizing specific markers of different retinal cell types.Results: In the absence of GF stimuli, RSC in culture expressed FGFR1, similar to early progenitors in vivo. Treatment with GFs up-regulated the expression of both fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Exposure to either FGF, EGF, or FGF+EGF strongly affected retinal stem cell-renewal and differentiation. Specifically, expression of progenitor/stem cell markers and stem cell-renewal was higher in the presence of FGF than in that of EGF. FGF favored differentiation of RSC into photoreceptor-like cells. Finally, we showed that the treatment of the primary culture with FGF+EGF imprinted the cells and limited plasticity in subsequent differentiation.Conclusions: We provide evidence that conditions of the primary culture have a strong influence on cell-renewal and differentiation potentials of RSC.


2007 - Identification and characterization of MicroRNAs expressed in the mouse eye [Articolo su rivista]
M., Karali; I., Peluso; Marigo, Valeria; S., Banfi
abstract

PURPOSE. MicroRNAs ( miRNAs) are a class of small, endogenous RNAs that negatively regulate gene expression post-transcriptionally by binding to target sites in the 3' untranslated region (UTR) of messenger RNAs. Although they have been found to regulate developmental and physiological processes in several organs and tissues, their role in the eye transcriptome is completely unknown. This study was conducted to gain understanding of their eye-related function in mammals, by looking for miRNAs significantly expressed in the mouse eye by means of high-resolution expression analysis. METHODS. The spatiotemporal localization of miRNAs was analyzed in the murine embryonic and postnatal eye by RNA in situ hybridization ( ISH) using LNA-modified oligonucleotide probes. RESULTS. Seven miRNAs were expressed in the eye with diverse and partially overlapping patterns, which may reflect their role in controlling cell differentiation of the retina as well as of other ocular structures. Most eye-expressed miRNAs overlap with or are in the near vicinity of transcripts derived predominantly from eye cDNA libraries. We found that these transcripts share very similar cellular distribution with their corresponding miRNAs, suggesting that miRNAs may share common expression regulatory elements with their host genes. CONCLUSIONS. The data provide a detailed characterization of expression of eye-enriched miRNAs. Knowledge of the spatiotemporal distribution of miRNAs is an essential step toward the identification of their targets and eventually the elucidation of their biological role in eye development and function.


2007 - Mutations in splicing factor PRPF3, causing retinal degeneration, form detrimental aggregates in photoreceptor cells. [Articolo su rivista]
Comitato, A; Spampanato, C; Chakarova, C; Sanges, D; Bhattacharya, S. S.; Marigo, Valeria
abstract

PRPF3 is an element of the splicing machinery ubiquitously expressed, yet mutations in this gene are associated with a tissue-specific phenotype: autosomal dominant retinitis pigmentosa (RP). Here, we studied the subcellular localization of endogenous- and mutant-transfected PRPF3. We found that (i) subcellular distribution of the endogenous wild-type protein co-localizes with small nuclear ribonucleoproteins, partially with a nucleolar marker and accumulates in speckles labeled by SC35; (ii) in human retinas, PRPF3 does not show a distinctive abundance in photoreceptors, the cells affected in RP and (iii) the RP causing mutant PRPF3, differently from the wild-type protein, forms abnormally big aggregates in transfected photoreceptor cells. Aggregation of T494M mutant PRPF3 inside the nucleus triggers apoptosis only in photoreceptor cells. On the basis of the observation that mutant PRPF3 accumulates in the nucleolus and that transcriptional, translational and proteasome inhibition can induce this phenomenon in non-photoreceptor cells, we hypothesize that mutation affects splicing factor recycling. Noteworthy, accumulation of the mutant protein in big aggregates also affects distribution of some other splicing factors. Our data suggest that the mutant protein has a cell-specific dominant effect in rod photoreceptors while appears not to be harmful to epithelial and fibroblast cells.


2007 - Mutations in TOPORS Cause Autosomal Dominant Retinitis Pigmentosa with Perivascular Retinal Pigment Epithelium Atrophy [Articolo su rivista]
CHAKAROVA C., F; PAPAIOANNOU M., G; Khanna, H; Lopez, I; Waseem, N; Shah, A; Theis, T; Friedman, J; Maubaret, C; Bujakowska, K; Veraitch, B; ABD EL AZIZ M., M; PRESCOTT D., Q; Parapuram, S; BICKMORE W., A; MUNRO P. M., G; Gal, A; Hamel, C; Marigo, Valeria; PONTING C., P; Wissinger, B; Zrenner, E; Matter, K; Swaroop, A; KOENEKOOP R., K; Bhattacharya, S. S.
abstract

We report mutations in the gene for topoisomerase I–binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families—one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain–containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.


2007 - Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors [Articolo su rivista]
Allocca, M; Mussolino, C; Hoyos, M. G.; Sanges, D; Iodice, C; Petrillo, M; Vandenberghe, L. H.; Wilson, J. M.; Marigo, Valeria; Surace, E. M.; Auricchio, A.
abstract

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.


2007 - Programmed cell death in retinal degeneration - Targeting apoptosis in photoreceptors as potential therapy for retinal degeneration [Articolo su rivista]
Marigo, Valeria
abstract

Retinal degenerations are the major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Several genes causing these genetic diseases have been identified, however the molecular characterization of a high percentage of patients affected by retinitis pigmentosa (RP), a common form of retinal degeneration, is still unknown. The high genetic heterogeneity of these diseases hampers the comprehension of the pathogenetic mechanism causing photoreceptor cell death. Therapies are not available yet and for this reason there is a lot of interest in understanding the etiology and the pathogenesis of these disorders at a cellular and molecular level. Some common features have been identified in different forms of RP. Apoptosis was reported to be the final outcome in all RP animal models and patients analyzed so far. We recently identified two apoptotic pathways coactivated in photoreceptors undergoing cell death in the retinal degeneration (rd1) mouse model of autosomal recessive RP. Our studies opened new perspectives together with many questions that require deeper analyses in order to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise may represent a promising curing strategy that needs to be exploited for retinal degeneration.


2006 - Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides [Articolo su rivista]
F., Vetrini; R., Tammaro; S., Bondanza; Em, Surace; A., Auricchio; DE LUCA, Michele; A., Ballabio; Marigo, Valeria
abstract

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G > A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino, oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.


2006 - Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors [Articolo su rivista]
D., Sanges; A., Comitato; R., Tammaro; Marigo, Valeria
abstract

Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum. in a degenerating neuron.


2006 - Cross-talk between two apoptotic pathways activated by endoplasmic reticulum stress: differential contribution of caspase-12 and AIF [Articolo su rivista]
D., Sanges; Marigo, Valeria
abstract

Co-activation and cross-talk of different apoptotic pathways have been described in several systems however, the differential contributions of the different executors have not been well characterized. Here we report the co-translocation to the nucleus of caspase-12 and AIF in response to two endoplasmic reticulum (ER) stresses: protein misfolding and disruption of calcium homeostasis. As seen by treatment with pan-caspase inhibitor and calpain inhibitors, apoptosis is not mediated by executor caspases but by calpains. By reduction of AIF or caspase-12 expression we unraveled that AIF primarily controls apoptosis caused by changes in calcium homeostasis while caspase-12 has a main role in programmed cell death induced by protein misfolding. Nevertheless, the two apoptotic factors appear to reinforce each other during the apoptotic process, confirming that while the first response primarily involves one organelle, mitochondria and ER can influence each other in the apoptotic event.


2005 - Amelioration of both Functional and Morphological Abnormalities in the Retina of a Mouse Model of Ocular Albinism Following AAV-Mediated Gene Transfer. [Articolo su rivista]
Surace, E. M.; Domenici, L.; Cortese, K.; Cotugno, G.; DI VICINO, U.; Venturi, C.; Cellerino, A.; Marigo, Valeria; Tacchetti, C.; Ballabio, A.; Auricchio, A.
abstract

X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1-/-) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1-/- mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1-/- mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1-/- mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.


2005 - Functional and Morphological Rescue of the Type I Ocular Albinism Murine Retina Following AAV-Mediated Gene Transfer. [Abstract in Rivista]
E. M., Surace; L., Domenici; K., Cortese; C., Venturi; G., Cotugno; U., Di Vicino; A., Cellerino; Marigo, Valeria; C., Tacchetti; A., Ballabio; A., Auricchio
abstract

X-linked recessive Type I Ocular Albinism (OA1) is a developmental disorder of the retina due to mutations in the OA1 gene expressed in the retinal pigment epithelium (RPE). The disease is characterized by foveal hypoplasia, misrouting of the optic fibers at the chiasm and RPE ultrastructural abnormalities. The Oa1 -/- mouse recapitulates many of the OA1 retinal morphological anomalies including a lower number of melanosomes of increased size in the RPE. To test the functionality of the Oa1 -/- retina we measured by electrophysiological methods its ability to dark adapt, which is impaired in forms of albinism other than OA1. Control C57/BL6 mice showed a characteristic kinetic of recovery of the bwave amplitude within 60 min following exposure to a pre-adapting saturating light. In contrast, the recovery of photoreceptor responses was significantly delayed in Oa1-/- mice. To test whether Adeno- Associated-Vector-(AAV) mediated Oa1 gene transfer was able to revert this functional abnormality and the characteristic melanosome landmarks of the Oa1 phenotype, two groups of animals at different ages were injected subretinally with a mixture of AAV2/1-CMVOa1+ AAV2/1-EGFP in the right eye and the same amount of AAV2/ 1-CMV-EGFP in the left eye. Dark adaptation in both eyes and groups was analyzed 1 month after gene transfer. The Oa1 -/- eyes that received only EGFP showed a delayed ability to dark adapt similar to the eyes of untreated Oa1 mutant mice. In the eyes that received the therapeutic vector the recovery was complete in 60 min: as in wild type animals. Electron microscopy analysis revealed a significant increase in the number of melanosomes in the RPE of the retina treated precociously with AAV. Our results suggest that post-natal Oa1 gene transfer in mice rescues some of the functional and morphological abnormalities that develop in the Oa1 -/- retina.


2005 - Functional studies of mutations found in PRPF3 pre-MRNA splicing factor causing RP18 [Abstract in Rivista]
Marigo, Valeria; C., Spampanato; C. F., Chakarova; S. S., Bhattacharya
abstract

Purpose: Genes encoding pre–mRNA splicing factors (PRPF31, PRPF8, PRPF3) were found mutated in three forms of autosomal dominant RP (adRP, RP11, RP13 and RP18). The link between mutant splicing factors and retinal degeneration is completely obscure. So far, only 2 amino acids (P493S and T494M) were found mutated in PRPF3. Analysis of the subcellular localization and interaction with other splicing factors will be fundamental in defining why mutation of PRPF3 causes RP and whether RP18 is due to a dominant effect of the mutant protein or haploinsufficiency. Methods: Analyses of the subcellular localization of the endogenous protein by immunofluorescence using specific polyclonal and monoclonal antibodies in cell lines and in the murine retina were undertaken. Transfection experiments using cell lines to define subcellular localization of the mutant forms of PRPF3. Interaction of wild type and mutant PRPF3 with known partner proteins and snRNA was performed by co–immunoprecipitation experiments. Results: We analyzed expression of the Prpf3 gene in the developing and adult murine eye by RNA in situ hybridization. We found that Prpf3 transcripts are ubiquitously distributed but higher amounts could be detected in the central nervous system and neural retina. Using in vitro transfection in HeLa, NIH3T3, Cos7 cells and primary retinal cultures, PRPF3 localizes in the nuclei. Using similar approaches we found the two different mutations (P493S and T494M) have similar intracellular distribution as found for the wild–type protein. Mutant PRPF3 also maintains its co–localization with spliceosome specific proteins such as SC35. Conclusions: We analyzed Prpf3 transcript distribution in vivo and found no direct correlation with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. Co–localization with splicing factors confirms that the protein is part of the spliceosome. Finally, mutant PRPF3 has a subcellular distribution similar to the wild type, suggesting that RP18 is caused either by a dominant effect of the mutation or by haploinsufficiency due to lack of function.


2005 - Molecular characterization of a T(2;6) balaced translocation associated with complex phenotype and leading to the truncation of the TCBA1 gene. [Articolo su rivista]
Bocciardi, R.; Giorda, R.; Marigo, Valeria; Zordan, P.; Montanaro, D.; Gimelli, S.; Seri, M.; Lerone, M.; Ravazzolo, R.; Gimelli, G.
abstract

The molecular characterization of balanced chromosomal rearrangements has often been a powerful tool for the positional identification of genes associated with specific diseases. In some instances, these rearrangements may be associated with a variety of different phenotypes, and thus establishing a genotype-phenotype correlation may be a complex process. However, molecular characterization of the rearrangement remains a useful tool for diagnoses or prognoses, or for identifying new genes and establishing a gene-to-function relationship. In this work we describe the characterization of a de novo balanced translocation t(2;6) (q24.3;q22.31) found in a patient with a complex phenotype. The major clinical finding was a severe neurological involvement. Thanks to the molecular characterization of this translocation we found that the rearrangement led to the truncation of the TCBA1 gene on chromosome 6q. We found that the gene is transcribed in different splice variants and is highly specific for the central nervous system. TCBA1 does not show any similarity with other known genes, and no information is available about its function. However, the gene appears to be well conserved among species, and we were able to infer the sequence of a putative mouse homolog of TCBA1. This allowed us to perform a more detailed expression study in mice, thus confirming its specificity for the nervous system. This finding is of particular interest because it suggests that TCBA1 may be correlated with the neurological phenotype of our patient, and possibly mutated in genetic diseases with a neurological phenotype.


2005 - The Ocular albinism type 1 (OA1) gene controls melanosome maturation and size. [Articolo su rivista]
Cortese, K.; Giordano, F.; Surace, E. M.; Venturi, C.; Ballabio, A.; Tacchetti, C.; Marigo, Valeria
abstract

PURPOSE. The authors took advantage of the Oa1 mutant mouse in combination with other albinism mouse models (i.e., Tyrosinase and membrane-associated transporter protein [Matp]) to study the function of Oa1, the gene mutated in ocular albinism type 1, in the RPE during development and after birth. METHODS. Enzyme activity and protein localization were analyzed by immunohistochemistry of tyrosinase (Tyr) in Oa1-null mice. Ultrastructural analysis and morphometry were performed by electron microscopy, of the RPE in Oa1-knockout mouse and double-mutant mice of Oa1 with either Tyr or Matp. RESULTS. Differently from other albinism models, Tyr activity was not impaired in Oa1–/– eyes. Hypopigmentation of the RPE in Oa1–/– mice is due to a reduced number of melanosomes. Analysis of Oa1–/–;Tyrc-2J/Tyrc-2J and Oa1–/–;Matpuw/Matpuw double-knockout mice, which display a block at stages II and III of melanosome maturation, respectively, revealed that Oa1 controls the rate of melanosome biogenesis at early stages of the organellogenesis, whereas the control on the organelle size is exerted at the final stage of melanosome development (stage IV). CONCLUSIONS. The findings indicate that Oa1 is involved in the regulation of melanosome maturation at two steps. Acting at early maturation stages, Oa1 controls the abundance of melanosomes in RPE cells. At later stages, Oa1 has a function in the maintenance of a correct melanosomal size. This study helps to define ocular albinism type 1 as a defect in melanosome organellogenesis and not in melanin production.


2004 - An expression atlas of connexin genes in the mouse. [Articolo su rivista]
Buniello, A.; Montanaro, D.; Volinia, S.; Gasparini, P.; Marigo, Valeria
abstract

Connexin genes are involved in several human diseases such as hearing and dermatological and peripheral nerve disorders. Connexins are protein units of gap junctions and form homotypic, heterotypic, or heteromeric complexes known as connexons. Data on the expression patterns of members of this family are partial and fragmentary. We therefore cloned all the identifiable murine homologs of human CONNEXIN genes and analyzed their expression patterns in embryonic and neonatal mouse tissues. We found that connexins are preferentially expressed in tissues derived from ectoderm and/or endoderm. Our data provide a comprehensive and detailed atlas of expression of connexin genes and in some cases suggest possible interactions of proteins that are coexpressed in the same tissue. Knowledge of temporal and spatial distribution of connexins also allows the identification of candidate genes for human diseases and provides important insight into mechanisms that lead to human disorders due to mutations in CONNEXIN genes.


2004 - Correlation between the clinical phenotype of MYH9-related disease and tissue distribution of class II non-muscle myosin heavy chains. [Articolo su rivista]
Marigo, Valeria; Nigro, A.; Pecci, A.; Montanaro, D.; DI STAZIO, M. T.; Balduini, C. L.; Savoia, A.
abstract

Nonmuscle myosin heavy chain II-A is responsible for MYH9-related disease, which is characterized by macrothrombocytopenia, granulocyte inclusions, deafness, cataracts, and renal failure. Since another two highly conserved nonmuscle myosins, II-B and II-C, are known, an analysis of their tissue distribution is fundamental for the understanding of their biological roles. In mouse, we found that all forms are ubiquitously expressed. However, megakaryocytic and granulocytic lineages express only II-A, suggesting that congenital features, macrothrombocytopenia, and leukocyte inclusions correlate with its exclusive presence. In kidney, eye, and ear, where clinical manifestations have a late onset, as well as in other tissues apparently not affected in patients, II-A and at least one of the other two isoforms are expressed, suggesting that II-B and II-C can partially compensate for each other. We hypothesize that cells expressing only II-A manifest the congenital defects, while tissues expressing additional myosin II isoforms show either late onset of abnormalities or no pathological sign.


2004 - Functional studies of PRP3 pre-RNA splicing factor mutated in patients affected by retinitis pigmentosa. [Abstract in Rivista]
Marigo, Valeria; C., Spampanato; A., Comitato; D., Sanges
abstract

Purpose: Many genes mutated in retinitis pigmentosa (RP) are components of the phototransduction cascade. However, in the last 2 years, genes encoding pre–RNA splicing factors were found mutated in three forms of autosomal dominant RP (adRP, RP11, RP13 and RP18). The slicing factors involved in adRP are PRPF31, PRP3, PRPF8. The link between mutant splicing factors and retinal degeneration is completely obscure. Methods: RNA in situ hybridization studies for the analysis of the expression pattern of PRP3 in embryonic and adult eyes. Subcellular localization of the protein using transfection experiment. Subcellular localization of the mutant forms of Prp3 as found in patients. Results: We analyzed expression of the Prp3 gene in the developing and adult murine eye by RNA in situ hybridization. We found that Prp3 transcripts are ubiquitously distributed but higher amounts could be detected in the central nervous system and neural retina. Expression does not seem to be strictly correlated with the photoreceptor cell layer. Using in vitro transfection in HeLa, NIH3T3 and Cos7 cells, Prp3 localizes into the nuclei. Using similar approaches we found the two different mutations (P493S and T494M) have similar intracellular distribution as found for the wild–type protein. We then wanted to study Prp3 protein distribution compared to splicing proteins. Co–localization analysis of transfected cells using antibodies recognizing a protein of the splicing complex suggests that Prp3 only partially co–localizes with the spliceosome. Conclusions: We analyzed Prp3 transcript distribution in vivo and found no direct correlation with photoreceptors, the cells that undergo degeneration in patients with mutations in this gene. Co–localization with splicing factors confirms that the protein is part of the spliceosome. However the protein shows a more complex subcellular distribution suggesting additional roles of this protein. Finally, mutant Prp3 is translocated into the nucleus like the wild type, ruling out the possibility of mis–folding and degradation of the mutant protein.


2004 - Sintesi del colesterolo e Sonic hedgehog: convergenza di queste vie metaboliche come causa di malformazioni congenite. [Articolo su rivista]
Marigo, Valeria
abstract

Durante gli ultimi anni è risultato sempre più chiara l’associazione tra difetti nella biosintesi del colesterolo e dismorfogenesi di vari organi. Infatti sia malattie genetiche dovute a mutazioni in enzimi della biosintesi del colesterolo che alcune molecole teratogene che interferiscono con questa via metabolica causano simili malformazioni al sistema nervoso centrale e agli arti. Tuttavia non si era riusciti a spiegare a livello molecolare quali fossero gli elementi comuni in queste patologie. Ricerche recenti hanno fatto luce su alcuni fattori con attività chiave durante lo sviluppo embrionale in particular modo sul sistema nervoso centrale e sull’arto. Ricerche di base di embriologia, biochimica e genetica hanno svelato che la molecola segnale Sonic hedgehod (SHH), per diventare attiva, deve venire covalentemente legata al colesterolo. Ridotta attività di SHH causa malformazioni simili alle patologie da carenza di colesterolo. Una comprensione dettagliata dei meccanismi mediante cui agisce SHH e come viene trasdotto il segnale SHH sono di fondamentale importanza per potere sviluppare nuovi approcci terapeutici per queste patologie. Questo articolo si propone di descrivere in modo dettagliato la via del segnale di SHH. Inoltre mette in evidenza gli organi che vengono principalmente interessati dal segnale SHH. Infine discute i meccanismi mediante i quali la carenza di colesterolo interferisce con l’attività di SHH e i comuni fenotipi nelle patologie genetiche derivanti da mutazioni in queste due vie metaboliche.


2004 - The amino acid transporter asc-1 is not involved in cystinuria. [Articolo su rivista]
Pineda, M.; Font, M.; Bassi, M. T.; Manzoni, M.; Borsani, G.; Marigo, Valeria; Fernandez, E.; Rio, R. M.; Purroy, J.; Zorzano, A.; Nunes, V.; Palacin, M.
abstract

BACKGROUND: The human amino acid transporter asc-1 (SLC7A10) exhibits substrate selectivity for small neutral amino acids, including cysteine, is expressed in kidney, is located close to the cystinuria B gene and presents sequence variants (e.g., E112D) in some cystinuria patients. We have cloned human asc-1, assessed its transport characteristics, localized its expression in kidney, searched for mutations in cystinuria patients, and tested the transport function of variant E112D. METHODS: We used an EST-based homology cloning strategy. Transport characteristics of asc-1 were assessed by coexpression with 4F2hc in Xenopus oocytes and HeLa cells. Localization of asc-1 mRNA in kidney was assessed by in situ hybridization. Exons and intron-exon boundaries were polymerase chain reaction (PCR)-amplified from blood cell DNA and mutational screening was performed by single-stranded conformational polymorphism (SSCP). RESULTS: Asc-1 reaches the plasma membrane in HeLa cells, unlike in oocytes, most probably by interaction with endogenous 4F2hc and presents similar transport characteristics to those in oocytes coexpressing asc-1/4F2hc. Asc-1 mediates a substantial efflux of alanine in a facilitated diffusion mode of transport. Expression of asc-1 mRNA localized to Henle's loop, distal tubules, and collecting ducts. Finally, SLC7A10 polymorphisms were identified in cystinuria probands and the SLC7A10 sequence variant E112D showed full transport activity. CONCLUSION: The lack of expression of asc-1 in the proximal tubule indicates that it plays no role in the bulk of renal reabsorption of amino acids. No mutations causing cystinuria have been found in SLC7A10. The facilitated diffusion mode of transport and the expression in distal nephron suggest a role for asc-1 in osmotic adaptation.


2004 - The microphthalmia transcription factor (Mitf) controls expression of the ocular albinism type 1 gene: link between melanin synthesis and melanosome biogenesis. [Articolo su rivista]
Vetrini, F.; Auricchio, A.; Du, J.; Angeletti, B.; Fisher, D. E.; Ballabio, A.; Marigo, Valeria
abstract

Melanogenesis is the process that regulates skin and eye pigmentation. Albinism, a genetic disease causing pigmentation defects and visual disorders, is caused by mutations in genes controlling either melanin synthesis or melanosome biogenesis. Here we show that a common transcriptional control regulates both of these processes. We performed an analysis of the regulatory region of Oa1, the murine homolog of the gene that is mutated in the X-linked form of ocular albinism, as Oa1's function affects melanosome biogenesis. We demonstrated that Oa1 is a target of Mitf and that this regulatory mechanism is conserved in the human gene. Tissue-specific control of Oa1 transcription lies within a region of 617 bp that contains the E-box bound by Mitf. Finally, we took advantage of a virus-based system to assess tissue specificity in vivo. To this end, a small fragment of the Oa1 promoter was cloned in front of a reporter gene in an adeno-associated virus. After we injected this virus into the subretinal space, we observed reporter gene expression specifically in the retinal pigment epithelium, confirming the cell-specific expression of the Oa1 promoter in the eye. The results obtained with this viral system are a preamble to the development of new gene delivery approaches for the treatment of retinal pigment epithelium defects.


2004 - URB expression in human bone marrow stromal cells and during mouse development. [Articolo su rivista]
Liu, Y.; Monticone, M.; Tonachini, L.; Mastrogiacomo, M.; Marigo, Valeria; Cancedda, R.; Castagnola, P.
abstract

Seven genes preferentially expressed in undifferentiated human bone marrow stromal cells (BMSC) with respect to BMSC-derived osteoblasts were previously identified by differential display. Here we characterize the expression of one of these genes, URB, belonging to the sushi-repeat-containing protein superfamily. In culture, URB is expressed in both human primary and cloned BMSC, and is drastically downregulated during osteoblastic differentiation of these cells. Here we show that in mouse tissues a single 3.8kb Urb transcript is detected and that the mouse Urb protein is secreted as a 150kDa glycoprotein. During mouse development Urb RNA is barely detectable in 9dpc embryos and increases at later stages. Both in situ hybridization and immunohistochemistry analysis show Urb expression in mouse embryos starting from 14dpc mostly in cartilage. The temporal and spatial expression pattern of Urb suggests its role in mouse skeletogenesis.


2003 - A Slc7a7 null mouse model of lysinuric protein intolerance. [Abstract in Rivista]
M. P., Sperandeo; V., Ammendola; V., Fiorito; A., Pepe; M., D'Armiento; R., Vecchione; G., Andria; G., Borsani; Marigo, Valeria; S., Banfi; A., Ballabio; G. A., Sebastio
abstract

Lysinuric protein intolerance (LPI, MIM 222700) is an autosomal recessive defect of cationic amino acids transport at the basolateral membrane of epithelial cells in the intestine and kidney, caused by mutations of the SLC7A7 gene.The unknown pathogenesis of the multisystem involvement of LPI, which includes a life-threatening alveolar proteinosis, and the lack of an effective treatment has prompted the creation of a LPI animal model. A constitutive knockout of Slc7a7 was generated by random insertional mutagenesis in embryonic stem cells (Lexicon Genetics Inc, Texas). More than 400 Slc7a7+/- intercrosses led to only one Slc7a7-/- live male. Since birth, this null animal showed a severe failure to thrive compared to siblings. The null animal, still alive at 12 months of age, shows also a strong reduced fertility compared with +/- and +/+ littermates. It is currently fed on a low protein diet and citrulline supplementation. Five additional Slc7a7-/- died within the first day of life, all showing severe growth failure. To establish the timing of the apparent lethality of Slc7a7-/- mice, embryos were collected at E 16.5 and E 18.5 stages, respectively. The proportions of Slc7a7 genotypes were found as expected for an autosomal recessive transmission. These data suggest that Slc7a7-/- pups die early in the perinatal period and not during the embryogenesis. Most of Slc7a7-/- pups were probably lost at birth because of cannibalism. At E 16.5 stage, the Slc7a7-/- embryos were already smaller than +/- or +/+ sibs. None of the null embryos showed gross morphological abnormalities. In humans, LPI does not cause clinical manifestations until weaning is started, neither increased rate of spontaneous abortion is known. In mice the absence of Slc7a7 gene might cause an early severe metabolic derangement underlying the intrauterine growth failure.


2003 - An in vivo doxycycline-controlled expression system for functional studies of the retina. [Articolo su rivista]
Angeletti, B.; Loester, J.; Auricchio, A.; Gekeler, F.; Shinoda, K.; Ballabio, A.; Graw, J.; Marigo, Valeria
abstract

PURPOSE: Transgenic mice were developed that express tetracycline-controlled transactivator 1 (tTA1) specifically in photoreceptor cells. In these mice the transcription of the gene of interest can be easily inactivated in the retina in a short time frame. METHODS: A construct was prepared containing tTA1 under control of the murine rhodopsin regulatory region. This construct was used for the generation of transgenic mice. In situ hybridization was performed to study the distribution of the transactivator in the retina. The activity of the transactivator was analyzed by mating the lines with a luciferase reporter transgenic mouse. tTA1 activity and doxycycline's ability to block it were analyzed by luciferase assay. The effects of tTA1 on the retina were assessed by histology and electrophysiology. RESULTS: Two transgenic lines were developed that specifically express tTA1 in photoreceptor cells. The time course of transgene expression replicated transcription of endogenous rhodopsin. tTA1 was not toxic to the retina. Transactivator activity was blocked readily by doxycycline. CONCLUSIONS: An expression system for photoreceptor cells was generated to drive transcription in a cell-specific and time-controllable manner. This system is suitable for the study of factors involved in retinal biology and of mutant forms of genes involved in retinal diseases.


2003 - Expression atlas of the mouse orthologues of the Williams-Beuren syndrome critical region genes [Abstract in Rivista]
A., Reymond; M., Yaylaoglu; G., Merla; C., Thaller; C., Caccioppoli; C., Ucla; D., Montanaro; T. M., Chen; N., Scamuffa; A., Ballabio; S. E., Antonarakis; G., Eichele; Marigo, Valeria
abstract

The Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder characterized by mental retardation with unique cognitive and personality profile, distinctive facial features, supravalvular aortic stenosis, short stature, connective tissue anomalies, hypertension, infantile hypercalcemia, dental and kidney abnormalities and premature aging of the skin. Its incidence is estimated at 1/15,000 and sporadic de novo inheritance is usual. Molecular basis of the syndrome is a heterozygous ~1.5 Mb microdeletion or inversion at chromosome band 7q11.23. While our understanding of the etiology of WBS has improved greatly, we are still ignorant as to the molecular basis of all except the cardiovascular phenotype. To define where the 31 WBS critical region genes exert their function and identify their possible role in the WBS phenotypes we performed a systematic analysis of the expression profile of all their murine orthologues. To obtain an high resolution expression pattern several complementary methods were combined: RT-PCR on a mouse cDNA panel of 12 adult tissues and 4 developmental stages; wholemount in situ of E9.5 and E10.5 embryos, section in situ of E14.5 embryos, brain section in situ of E15.5 embryos, P7 and P56 mouse. These stages correspond to mid and late embryonic and fetal human periods, when the major organs and body regions are organized, while the brain sections allow to correlate expression with neuroanatomical changes described in WBS patients.The topographical catalogue of expression of the murine orthologues of WBS genes will be instrumental to the understanding of the pathogenesis of this contiguous gene syndrome. The entire data set will be comprehensively documented on a freely accessible web page, which will lists the WBS genes, their murine orthologs and the probes used for ISH and displays images as well as annotation tables.


2002 - Expression Atlas of the mouse orthologues of the human chromosome 21 genes [Abstract in Rivista]
A., Reymond; Marigo, Valeria; M., Yaylaoglu; A., Leoni; C., Ucla; N., Scamuffa; S., Banfi; R., Lyle; C., Caccioppoli; G., Eichele; S. E., Antonarakis; A., Ballabio
abstract

Down syndrome (DS), due to an extra copy of human chromosome 21 (HC21), is the most common genetic cause of mental retardation. To define where HC21 genes exert their function and identify their possible role in the DS phenotypes we performed a systematic analysis of the expression profile of 170 murine genes which represent (almost all) the orthologues of the HC21 genes. To obtain an high resolution expression pattern several complementary methods were combined: RT-PCR on a mouse cDNA panel of 12 adult tissues and 4 developmental stages; wholemount in situ of E9.5 and E10.5 embryos and section in situ of E14.5 embryos. These stages correspond to mid and late embryonic and fetal human periods, when the major organs and body regions are organized. Genes showing interesting and/or restricted expression pattern were analyzed further on appropriate sections at more developmental timepoints. 91% of the tested genes showed a clear expression pattern during murine development. In 37% of the cases expression was ubiquitous while 50% of the cDNAs showed a differential expression pattern in the embryonal tissues. The topographical catalogue of expression of the murine orthologues of human chromosome 21 genes will be instrumental to the understanding of the pathogenesis of trisomy 21 as well as of other chromosome 21 linked disorders. Some of analyzed genes display a pattern of expression relevant to the congenital heart disease and/or to the mental retardation observed in DS. The entire data set will be made available to the scientific community via a web site.


2002 - Human chromosome 21 gene expression atlas in the mouse. [Articolo su rivista]
Reymond, A.; Marigo, Valeria; Yaylaoglu, M. B.; Leoni, A.; Ucla, C.; Scamuffa, N.; Caccioppoli, C.; Dermitzakis, E. T.; Lyle, R.; Banfi, S.; Eichele, G.; Antonarakis, S. E.; Ballabio, A.
abstract

Genome-wide expression analyses have a crucial role in functional genomics. High resolution methods, such as RNA in situ hybridization provide an accurate description of the spatiotemporal distribution of transcripts as well as a three-dimensional 'in vivo' gene expression overview. We set out to analyse systematically the expression patterns of genes from an entire chromosome. We chose human chromosome 21 because of the medical relevance of trisomy 21 (Down's syndrome). Here we show the expression analysis of all identifiable murine orthologues of human chromosome 21 genes (161 out of 178 confirmed human genes) by RNA in situ hybridization on whole mounts and tissue sections, and by polymerase chain reaction with reverse transcription on adult tissues. We observed patterned expression in several tissues including those affected in trisomy 21 phenotypes (that is, central nervous system, heart, gastrointestinal tract, and limbs). Furthermore, statistical analysis suggests the presence of some regions of the chromosome with genes showing either lack of expression or, to a lesser extent, co-expression in specific tissues. This high resolution expression 'atlas' of an entire human chromosome is an important step towards the understanding of gene function and of the pathogenetic mechanisms in Down's syndrome.


2002 - Human chromosome 21 gene expression atlas in the mouse. [Abstract in Rivista]
Marigo, Valeria; A., Reymond; M. B., Yaylaoglu; A., Leoni; C., Ucla; N., Scamuffa; C., Caccioppoli; E. T., Dermitzakis; R., Lyle; S., Banfi; G., Eichele; A., Ballabio; S. E., Antonarakis
abstract

The study of gene expression patterns is of crucial importance to the understanding of gene function. We set out to systematically analyze the expression patterns of genes from an entire chromosome. We chose human chromosome 21 (HC21) because its entire finished nucleotide sequence was available. Furthermore trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation. This chromosome-based approach has advantages over the study of an arbitrary set of genes, as it provides information on the consequences of aneuploidy, and may allow the identification of clusters of co-regulated genes. All identifiable murine orthologs of human chromosome 21 genes (161 out of 178 confirmed human genes) were analyzed by RNA in situ hybridization on whole-mounts (E9.5, E10.5) and tissue sections (E14.5), and by RT-PCR on adult tissues. These stages correspond to mid and late embryonic and fetal human periods, when the major organs and body regions are organized. Patterned expression was observed in several tissues including those affected in trisomy 21 phenotypes (i.e. CNS, heart, gastrointestinal tract, and limbs). Furthermore, statistical analysis suggests the presence of clusters of genes with significant patterns of either coexpression or lack of expression in specific tissues. The entire data set was incorporated in a web site that will be made available to the scientific community. This high resolution expression "atlas" of an entire human chromosome is a new level of gene annotation, which is likely to advance our knowledge on gene function and regulation and to the understanding of human aneuploidies, such as DS.


2002 - Molecular characterization and expression analysis of Mtmr2, mouse homologue of MTMR2, the myotubularin-related 2 gene, mutated in CMT4B. [Articolo su rivista]
Bolino, A.; Marigo, Valeria; Ferrara, F.; Loader, J.; Romio, L.; Leoni, A.; DI DUCA, M.; Cinti, R.; Cecchi, C.; Feltri, M. L.; Wrabetz, L.; Ravazzolo, R.; Monaco, A.
abstract

Charcot-Marie-Tooth type 4B (CMT4B) is caused by mutations in the myotubularin-related 2 gene, MTMR2, on chromosome 11q22. To date, six loss of function mutations and one missense mutation have been demonstrated in CMT4B patients. It remains to be determined how dysfunction of a ubiquitously expressed phosphatase causes a demyelinating neuropathy. An animal model for CMT4B would provide insights into the pathogenesis of this disorder. We have therefore characterized the mouse homologue of MTMR2 by reconstructing the full-length Mtmr2 cDNA as well as the genomic structure. The 1932 nucleotide open reading frame corresponds to 15 coding exons, spanning a genomic region of approximately 55 kilobases, on mouse chromosome 9 as demonstrated by fluorescence in situ hybridization analysis. A comparison between the mouse and human genes revealed a similar genomic structure, except for the number of alternatively spliced exons in the 5'-untranslated region, two in mouse and three in man. In situ hybridization analysis of mouse embryos showed that Mtmr2 was ubiquitously expressed during organogenesis at E9.5, with some areas of enriched expression. At E14.5, Mtmr2 mRNA was more abundant in the peripheral nervous system, including in dorsal root ganglia and spinal roots.


2002 - The mouse model for ocular albinisin type 1 (OA1) as a tool to study Oa1 function in RPE development [Abstract in Rivista]
Marigo, Valeria; E. M., Surace; F., Vetrini; K., Cortese; F., Giordano; A., Ballabio; C., Tacchetti
abstract

Purpose: Ocular Albinism type 1 (OA1) is an X-linked form of albinism isolated to the eye. The disease causes a severe visual handicap in affected males, manifesting foveal hypoplasia, horizontal and rotatory nystagmus, strabismus, photophobia and lack of stereoscopic vision. Histological analysis of patient melanocytes from the RPE and the skin reveals giant melanosomes (macromelanosomes). Mutations causing this disease were identified in OA1 gene. OA1 is specifically expressed in the retinal pigment epithelium (RPE) and in skin melanocytes . The protein is localized on the melanosomal membrane and displays characteristics typical of the seven transmembrane G protein coupled receptors. In order to understand the biological function of OA1, we generated a mouse model for the disease. Methods: To generate Oa1 null mice, we deleted the first exon of Oa1 by homologous recombination in ES cells. Histological studies were performed on RPE of wild-type and mutant mice. Interactions of Oa1 with other melanosomal proteins was investigated by mating Oa1 null mice with p and c mice. Ultrastructural and biochemical analyses were conducted in double mutant mice. Results: Histological analysis of the eyes in Oa1 mutants showed that the RPE was more lightly pigmented. Abnormally large melanosomes (macromelanosomes) could be identified by electron microscopy within the RPE. Interestingly, macromelanosomes could be seen just after birth in mutant mice. Oa1 knock-out mice display a misrouting of the optic fibers as in OA1 patients. A significant decrease of ipsilateral optic fibers could be measured in Oa1 knock-out mice as found in other albino mice. To investigate the function of Oa1 on the melanosomal membrane and its interaction with other genes causing different forms of albinism, we mated Oa1 null mice with p and c mice. Ultrastructural and biochemical analyses were performed in double mutant mice, finding a correlation of the macromelanosomal phenotype and level of pigmentation. We have also studied genes regulating Oa1 expression during development (e.i. Mitf) and we have started experiments aimed to the identification of genes downstream to it. Conclusion: We developed a mouse model for OA1. Our data demonstrate that the Oa1 knock-out mouse is indeed a good model for the study of OA1 pathology. Furthermore the mouse model will be valuable for the identification of factors mediating RPE influence on retinal development.


2002 - Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibers and eye coloboma [Abstract in Rivista]
A., Barbieri; G., Alfano; V., Broccoli; A., Bulfone; Marigo, Valeria; P., Bovolenta; A., Ballabio; S., Banfi
abstract

Vax2 is a homeobox gene whose expression is confined to the ventral portion of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralization of the retina, thus suggesting its role in the molecular pathway underlying eye development. We have generated and characterized a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalization of the expression of mid-late (EphB2 and ephrin-B2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.


2002 - Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibers and eye coloboma. [Articolo su rivista]
Barbieri, A. M.; Broccoli, V.; Bovolenta, P.; Alfano, G.; Marchitiello, A.; Mocchetti, C.; Crippa, L.; Bulfone, A.; Marigo, Valeria; Ballabio, A.; Banfi, S.
abstract

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.


2001 - Cloning and expression of a novel Cysteine-Rich Secreted Protein family member expressed in thyroid and pancreatic mesoderm within the chicken embryo. [Articolo su rivista]
Smith, D. M.; COLLINS RACIE, L. A.; Marigo, Valeria; Roberts, D. J.; Davis, N. M.; Hartmann, C.; Schweitzer, R.; Lavallie, E. R.; Gamer, L.; Mccoy, J.; Tabin, C. J.
abstract

We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.


2001 - The topographical expression map of chromosome 21 genes. [Abstract in Rivista]
A., Reymond; Marigo, Valeria; M., Yaylaoglu; A., Leoni; R., Lyle; C., Caccioppoli; C., Ucla; M., Guipponi; S., Banfi; G., Eichele; S., Antonarakis; A., Ballabio
abstract

Down syndrome (DS), caused by the presence of an extra copy of human chromosome 21 (HC21), is the most common genetic cause of mental retardation with an incidence of approximately 1/700 live births. The sequence of the human chromosome 21 provided evidence for the presence of approximately 220 genes. An important challenge is now to understand how three normal copies of these genes are associated with the various DS phenotypes (mental retardation, congenital heart disease, early onset Alzheimers disease and an increased risk of leukemia). To define where HC21 genes exert their function and identify their possible role in the DS phenotype we have decided to perform a systematic analysis of the expression profile of their murine homologues . We collected 150 murine cDNA clones from several sources. To obtain a high resolution expression pattern several complementary methods were combined: (i) RT-PCR on a mouse cDNA panel of 12 adult tissues and 4 developmental stages; (ii) wholemount in situ hybridisation of E9.5 and E10.5 embryos and (iii) section in situhybridisation of E14.5 embryos and of P7 brains. This set of whole embryos and sections corresponds to mid and late embryonic and foetal human periods, when most of the major organs and body regions are organised. Genes showing an interesting and/or restricted expression pattern were analysed further on appropriate sections at more developmental timepoints. 87% of the tested genes showed a clear expression pattern during murine development. In 37% of the cases expression was ubiquitous while 50% of the cDNAs showed a differential expression pattern in the embryonal tissues. The topographical catalogue of expression of the murine homologues of human chromosome 21 genes will be instrumental to the understanding of the pathogenesis of trisomy 21. Currently, approximately 100 genes have already been tested some of which display a pattern of expression relevant to the congenital heart disease and to the mental retardation observed in DS. The entire data set will be made available to the scientific community via a web site.


2000 - Expression pattern of the ocular albinism type 1 (OA1) gene in the murine retinal pigment epithelium. [Articolo su rivista]
Surace, E. M.; Angeletti, B.; Ballabio, A.; Marigo, Valeria
abstract

PURPOSE: Mutations in the OA1 gene cause ocular albinism type 1 (OA1), an X-linked form of albinism affecting only the eye, with skin pigmentation appearing normal. To better understand the pathogenesis of this disease the time of onset and the pattern of expression of the mouse homolog of the OA1 gene were monitored during eye development. The localization of Oa1 mRNA was studied and compared with the expression of other genes involved in melanosomal biogenesis. METHODS: The Oa1 expression pattern during eye development and after birth was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Localization of Oa1 mRNA was compared with TYROSINASE: (TYR:), pink-eyed dilution (p), and Pax2 expression patterns. RESULTS: RT-PCR revealed that Oa1 expression began at embryonic day (E)10.5 and was maintained until adulthood. By in situ hybridization analysis Oa1 transcripts were detected in the retinal pigment epithelium (RPE) beginning at E10.5 in the dorsal part of the eyecup and in the same area where transcripts of other genes involved in pigmentation are found. Of note, the expression pattern of these genes was complementary to Pax2 expression, which was restricted to the ventral side of the optic cup. At later stages, expression of Oa1, TYR:, and p expanded to the entire RPE and ciliary body. CONCLUSIONS: Oa1 expression can be detected at early stages of RPE development, together with other genes involved in pigmentation defects. Oa1 is likely to play an important function in melanosomal biogenesis in the RPE beginning during the earliest steps of melanosome formation.


2000 - Oa1 knock-out: new insights on the pathogenesis of Ocular Albinism type 1. [Articolo su rivista]
Incerti, B.; Cortese, K.; Pizzigoni, A.; Surace, E. M.; Varani, S.; Coppola, M.; Jeffery, G.; Seeliger, M.; Jaissle, G.; Bennett, D. C.; Marigo, Valeria; Schiaffino, M. V.; Tacchetti, C.; Ballabio, A.
abstract

Ocular albinism type I (OA1) is an X-linked disorder characterized by severe reduction of visual acuity, strabismus, photophobia and nystagmus. Ophthalmologic examination reveals hypopigmentation of the retina, foveal hypoplasia and iris translucency. Microscopic examination of both retinal pigment epithelium (RPE) and skin melanocytes shows the presence of large pigment granules called giant melanosomes or macromelanosomes. In this study, we have generated and characterized Oa1-deficient mice by gene targeting (KO). The KO males are viable, fertile and phenotypically indistinguishable from the wild-type littermates. Ophthalmologic examination shows hypopigmentation of the ocular fundus in mutant animals compared with wild-type. Analysis of the retinofugal pathway reveals a reduction in the size of the uncrossed pathway, demonstrating a misrouting of the optic fibres at the chiasm, as observed in OA1 patients. Microscopic examination of the RPE shows the presence of giant melanosomes comparable with those described in OA1 patients. Ultrastructural analysis of the RPE cells, suggests that the giant melanosomes may form by abnormal growth of single melanosomes, rather than the fusion of several, shedding light on the pathogenesis of ocular albinism.


1999 - Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development [Articolo su rivista]
Bulfone, A.; Martinez, S.; Marigo, Valeria; Campanella, M.; Basile, A.; Quaderi, N.; Gattuso, C.; Rubenstein, J. L. R.; Ballabio, A.
abstract

The members of the T-box gene family share a highly conserved DNA binding domain named the T-domain, and important developmental functions. Here we report the cloning of chicken Tbr1 and of murine and chicken Tbr2 (orthologs of the Xenopus eomesodermin gene), the mapping of the murine Tbr2 to chromosome 9, and their pattern of expression during mouse and chick embryogenesis. Both Tbr 1 and 2 have a restricted and conserved domain of expression in the telencephalic pallium of the two species. Chick Tbr2 has a specific and dynamic expression in the gastrulating embryo.


1997 - The Smoothened gene and Hedgehog signal transduction in Drosophila and vertebrate development. [Articolo su rivista]
Quirk, J.; VAN DEN HEUVEL, M.; Henrique, D.; Marigo, Valeria; Jones, T. A.; Tabin, C.; Ingham, P. W.
abstract

During the past decade, a small number of families of secreted proteins have emerged as key regulators of many of the important inductive interactions that control the development of animal embryos. Members of these families - the fibroblast growth factors (FGFs), transforming growth factor-βs (TGF-βs), Wnts, and Hedgehogs (Hhs) - have been highly conserved during evolution and in some cases have remarkably similar roles in the development of species from different phyla.


1996 - Biochemical evidence that Patched is the Hedgehog receptor. [Articolo su rivista]
Marigo, Valeria; Davey, R.; Zuo, Y.; Cunningham, J. M.; Tabin, C.
abstract

The protein Sonic hedgehog (Shh) is essential for a variety of patterning events during development. It is the signal from the notochord that induces ventral cell fate in the neural tube and somites, and is the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. Because of these and other inductive functions of Shh, it is important to understand how the Hedgehog (Hh) signal is received by the target cells. Here we describe binding studies using labelled Shh that strongly suggest that the Hh receptor is encoded by patched (ptc), a gene first identified in genetic screens in Drosophila.


1996 - Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. [Articolo su rivista]
Marigo, Valeria; Scott, M. P.; Johnson, R. L.; Goodrich, L. V.; Tabin, C. J.
abstract

Hedgehog genes have been implicated in inductive signaling during development in a variety of organisms. A key element of the hedgehog signaling system is encoded by the gene patched. In Drosophila hedgehog regulates gene expression by antagonizing the action of patched. In addition, patched is itself a transcriptional target of hedgehog signaling. We have isolated a chicken patched homolog and find it to be strongly expressed adjacent to all tissues where members of the hedgehog family are expressed. As in Drosophila, ectopic expression of Sonic hedgehog leads to ectopic induction of chicken Patched. Based on this regulatory conservation, vertebrate Patched is likely to be directly downstream of Sonic hedgehog signaling. An important role of Sonic hedgehog is the regulation of anterior/posterior pattern in the developing limb bud. Since Patched is directly downstream of the hedgehog signal, the extent of high level Patched expression provides a measure of the distance that Sonic hedgehog diffuses and directly acts. On this basis, we find that Sonic hedgehog directly acts as a signal over only the posterior third of the limb bud. During limb patterning, secondary signals are secreted in both the mesoderm (e.g. Bone Morphogenetic Protein-2) and apical ectodermal ridge (e.g. Fibroblast Growth Factor-4) in response to Sonic hedgehog. Thus knowing which is the direct target tissue is essential for unraveling the molecular patterning of the limb. The expression of Patched provides a strong indication that the mesoderm and not the ectoderm is the direct target of Sonic hedgehog signaling in the limb bud. Finally we demonstrate that induction of Patched requires Sonic hedgehog but, unlike Bone Morphogenetic Protein-2 and Hox genes, does not require Fibroblast Growth Factor as a co-inducer. It is therefore a more direct target of Sonic hedgehog than previously reported patterning genes.


1996 - Regulation of Patched by Sonic hedgehog in the developing neural tube. [Articolo su rivista]
Marigo, Valeria; Tabin, Clifford
abstract

Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog of hedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically downstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling.


1996 - Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. [Articolo su rivista]
Marigo, Valeria; Johnson, R. L.; Vortkamp, A. TABIN C. J.
abstract

Sonic hedgehog is a secreted factor regulating patterning of the anterior-posterior axis in the developing limb. The signaling pathway mediating the transduction of the signal is still poorly understood. In Drosophila several genes are known to act downstream of hedgehog, the fly homolog of Sonic hedgehog. An important gene epistatic to hedgehog is cubitus interruptus, which encodes the fly homolog of a family of vertebrate putative transcription factors, the GLI genes. We have isolated two members of the GLI family from chick, called GLI and GLI3. Their expression patterns in a variety of tissues during embryogenesis suggest that these genes may be targets of the Sonic hedgehog signal. We demonstrate that the two GLI genes are differentially regulated by Sonic hedgehog during limb development. Sonic hedgehog up-regulates GLI transcription, while down-regulating GLI3 expression in the mesenchymal cells of the developing limb bud. Finally, we demonstrate that an activated form of GLI can induce expression of Patched, a known target of Sonic hedgehog, thus implicating GLI as a key transcription factor in the vertebrate hedgehog signaling pathway. In conjunction with evidence from a mouse Gli3 mutant, our data suggest that GLI and GLI3 may have taken two different functions of their Drosophila homolog cubitus interruptus.


1996 - Sonic hedgehog regulates patterning in early embryos. [Capitolo/Saggio]
Marigo, Valeria; Laufer, E.; Nelson, C. E.; Riddle, R. D.; Johnson, R. L.; Tabin, C.
abstract

Recently, a new family of genes, homologues of the Drosophila segment polarity gene hedgehog, has been cloned in vertebrates. One of them, Sonic hedgehog, is expressed in tissues that are known to be inductive centres for patterning early embryos, implicating Sonic hedgehog as an important signal in development. Functional experiments have demonstrated that Sonic hedgehog acts as a signalling molecule in patterning the anterior-posterior axis of the limb. By misexpression of Sonic hedgehog we show that Sonic hedgehog induces expression of Hoxd genes, known to be involved in patterning of the anterior-posterior axis of the limb, and Bmp-2, which might act as a secondary signal. We also demonstrate that Sonic hedgehog is not sufficient for these inductions. In fact, a signal from the mesoderm, Sonic hedgehog, and a signal from the ectoderm, Fgf-4, are required for the induction of Hoxd genes and Bmp-2.


1995 - Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog. [Articolo su rivista]
Marigo, Valeria; Roberts, D. J.; Lee, S. M. K.; Tsukurov, O.; Levi, T.; Gastier, J. M.; Epstein, D. J.; Gilbert, D. J.; Copeland, N. G.; Seidman, C. E.; Jenkins, N. A.; Seidman, J. G.; Mcmahon, A. P.; Tabin, C.
abstract

The hedgehog genes encode signaling molecules that play a role in regulating embryonic morphogenesis. We have cloned and sequenced human cDNA copies of two of these genes, SHH and IHH. The SHH clone includes the full coding sequence and encodes a protein 92.4% identical to its murine homologue. The IHH clone is 89% complete and encodes a protein 94.6% identical to its murine homologue. IHH is expressed in adult kidney and liver. SHH expression was not detected in adult tissues examined; however, it is expressed in fetal intestine, liver, lung, and kidney. SHH mapped to chromosome 7q and IHH to chromosome 2 by PCR with DNA from a panel of rodent-human somatic cell hybrids. To identify the chromosomal location of SHH more precisely, a P1 genomic clone of SHH was isolated. This phage contained a CA repeat sequence tagged site that was used to map SHH relative to a polysyndactyly disease locus, using DNA prepared from affected and unaffected members of a large pedigree. SHH is closely linked, but distinct from the polysyndactyly disease locus at 7q36 (maximum lod score = 4.82, theta = 0.05) tightly linked to the EN2 locus. The murine homologues Shh, Ihh, and Dhh were mapped using (C57BL/6J x Mus spretus)F1 x C57BL/6J interspecific backcross. Shh mapped to a position 0.6 cM distal to En2 and 1.9 cM proximal to Il6 on mouse chromosome 5. This location is closely linked but distinct from the murine limb mutation Hx and syntenic to human chromosome 7q36.


1995 - Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter. [Articolo su rivista]
Piccolo, S.; Marigo, Valeria; Girotto, D.; Volpin, D.; Bressan, G. M.
abstract

DNase I footprinting experiments with a DNA fragment of the human elastin promoter have revealed a protected segment comprised between -156 and -172 nucleotides from the translation start site. Various types of gel retardation experiments indicate that the protected element binds different members of the C/EBP family of transcription factors. CAT (chloramphenicol acetyltransferase) fusion constructs carrying the wild type or a mutated promoter sequence were transfected into NIH3T3 and chick embryo aorta cells. The mutation significantly lowered CAT expression in NIH3T3 cells, but was ineffective in aorta cells. Cotransfection of the CAT promoter constructs with eucaryotic vectors expressing C/EBPs, did not affect the production of the reporter gene in NIH3T3 cells; on the contrary a several-fold increase of CAT activity was observed in aortic cells. This increase, however, was identical for the wild type and the mutated constructs. Taken together the data indicate that the elastin promoter contains a recognition site for proteins of the C/EBP family and that the function of this cis-acting element on basal elastin transcription varies with the cell type.


1995 - Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein. [Articolo su rivista]
Wang, M. Z.; Jin, P.; Bumcrot, D. A.; Marigo, Valeria; Mcmahon, A. P.; Wang, E. A.; Woolf, T.; Pang, K.
abstract

Loss of substantia nigra dopaminergic neurons, which develop from the ventral region of the midbrain, is associated with Parkinson's disease. During embryogenesis, induction of these and other ventral neurons is influenced by interactions with the induction of mesoderm of the notochord and the floor plate, which lies at the ventral midline of the developing CNS. Sonic hedgehog encodes a secreted peptide, which is expressed in notochord and floor plate cells and can induce appropriate ventral cell types in the basal forebrain and spinal cord. Here we demonstrate that Sonic hedgehog is sufficient to induce dopaminergic and other neuronal phenotypes in chick mesencephalic explants in vitro. We find that Sonic hedgehog is a general ventralizing signal in the CNS, the specific response being determined by the receiving cells. These results suggest that Sonic hedgehog may have utility in the induction of clinically important cell types.


1994 - Evolution in developmental biology: of morphology and molecules. [Articolo su rivista]
Laufer, Ed; Marigo, Valeria
abstract

Experimental embryologists, molecular biologists, evolutionary morphologists, paleontologists, and most modern practitioners of developmental biology met recently (British Society for Developmental Biology Spring Meeting, Edinburgh, UK; organized by M. Akam, P. Holland and G. Wray) to discuss the evolution of development.


1994 - Identification of a TGF-beta responsive element in the human elastin promoter. [Articolo su rivista]
Marigo, Valeria; Volpin, D.; Vitale, G.; Bressan, G. M.
abstract

In a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-beta in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-beta responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-beta activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine.


1993 - Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface. [Articolo su rivista]
Bressan, G. M.; DAGA GORDINI, D.; Colombatti, A.; Castellani, I.; Marigo, Valeria; Volpin, D.
abstract

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).


1993 - Murine alpha1(VI) collagen chain. Complete amino acid sequence and identification of the gene promoter region. [Articolo su rivista]
Bonaldo, P.; Piccolo, S.; Marvulli, D.; Volpin, D.; Marigo, Valeria; Bressan, G. M.
abstract

The entire primary structure of the murine alpha 1(VI) collagen chain was deduced from cloned cDNA. The predicted polypeptide consists of 1025 amino acids and shows extensive homology with the corresponding human and chicken chains. A genomic clone isolated with a cDNA probe was found to contain about 13 kilobases of the 5'-flanking region and the first and second exon, coding for the 5'-untranslated sequence and signal peptide and part of the N-terminal portion of the mature protein, respectively. Polymerase chain reaction and primer extension analyses revealed two major and several minor transcription start sites distributed over 76 base pairs (bp). The region just upstream of the transcription initiation sites lacks canonical TATA and CAAT boxes and Sp1 binding sites, but contains putative binding sites for other transcription factors and a 90-bp polypyrimidine tract with elements of dyad symmetry. Chimeric constructs were derived from different fragments of the 5'-flanking genomic region and the chloramphenicol acetyltransferase (CAT) gene and expression of the reporter gene was assayed following transfection of various cell types. A construct containing sequences extending from -215 to +41 directed high levels of CAT expression. The data indicate that this region harbours a functional promoter.


1993 - Regulation of the human elastin promoter in chick embryo cells. Tissue-specific effect of TGF-beta. [Articolo su rivista]
Marigo, Valeria; Volpin, D.; Bressan, G. M.
abstract

The function of the 5' flanking region of the human elastin gene on transcription regulation has been investigated in chick embryo aorta cells by transient DNA transfer experiments with elastin-chloramphenicol acetyltransferase (CAT) fusions. The results have shown that the region comprised within -129 and -12 bp from the translation start site is essential for transcription and probably contains different control sequences. Expression of the reporter gene was increased 2-4-fold by addition of TGF-beta to the cell cultures. Analysis of CAT expression from different deletion constructs suggests that sequences in the region -196 to -12 play a major role in TGF-beta induction. The stimulating effect of the growth factor could not be observed when transfections were performed with chick embryo tendon fibroblasts. This suggests that transcriptional regulation of elastin by TGF-beta is tissue specific.


1992 - Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro. [Articolo su rivista]
Marigo, Valeria; DAGA GORDINI, D.; Sitta, A.; Volpin, D.; Bressan, G. M.
abstract

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.


1992 - Mapping of binding sites for monoclonal antibodies to chick tropoelastin by recombinant DNA techniques. [Articolo su rivista]
Marigo, Valeria; Sitta, A.; Volpin, D.; Bressan, G. M.
abstract

A fusion molecule consisting of the entire coding sequence of mature chicken tropoelastin preceded by 14 amino acids of the signal peptide and 9 amino acids of vector origin has been expressed in a recombinant bacterial system and purified. The molecule has been used as immunogen for the production of hybridomas. Monoclonal antibodies which bound specifically the immunogen were also reactive with tropoelastin purified from chick aorta and stained elastic fibers in aorta sections by immunofluorescence. The region of tropoelastin containing the antigenic determinant recognized by each antibody has been identified by a recombinant DNA expression strategy based on the use of cDNA clones spanning different portions of the coding sequence. It could be shown that several antibodies were directed against unique epitopes; among these, a group of antibodies bound specifically to the sequence (PGVGV)n. Other antibodies were found to recognize antigenic determinants present more than once in the molecule. The monoclonal antibodies thus characterized will be useful reagents in studying the function of the different domains of tropoelastin.