Novel regenerative medicine approaches with the use of adult mesenchymal stem cells: in vitro and in vivo experimental procedures

Tendon injuries are often associated with skeletal muscle lesions that can originate from a variety of events, including direct trauma, tendon and muscle lacerations and contusions, indirect insults and degenerative diseases as muscular dystrophies. Currently, a complete cure for musculoskeletal diseases is not present and the restitutio ad integrum is difficult to obtain. In the last decade, adult MSCs gained general attention in both human and veterinary medicine and the understanding of MSC function is improved promoting the application of cell therapy and the development of powerful cell-derived therapeutics for regenerative medicine. The first part of this research focused on the reprogramming of stromal cells derived from equine and sheep mesenchymal tissue towards tenogenic and myogenic fate in vitro using new non-viral transfection system. 1) Equine MSCs isolated from peripheral blood (PB-MSCs) can develop the tenogenic pathway using four specific growth factors such as TGFβ3 (transforming growth factor β3), EGF2 (epidermal growth factor 2), bFGF2 (fibroblast growth factor 2) and IGF1 (insulin-like growth factor 1) in presence or without Low Level Laser Technology (LLLT). 2) PB-MSCs were induced to differentiate towards myogenic fate using the complex TAT-MyoD in presence of a conditioned medium obtained from co-culturing PB-MSCs with C2C12 without a direct contact. 3) A novel surface-active maghemite nanoparticles (SAMNs) were tested as vectors for eukaryotic cell transfection of coding gene in PB-MSCs without the application of external magnetic fields. The full characterization of these three techniques was achieved using molecular and immunohistochemistry analysis. Real-time PCR (rt-PCR) was performed to study the expression level of the typical tenogenic genes markers Early Growth Response Protein-1 (EGR1), Tenascin C (TNC) and Decorin (DCN) to discover the best combination of GFs in presence or without LLLT. To evaluate the myoblasts differentiation, rt-PCR analysis was executed to study Myf5 and Myogenin gene expression while immunofluorescence experiments was performed to estimate MyoD, Myf5 and Myogenin protein expression. The cytotoxicity effects of SAMNs nanoparticles was observed with XTT cell proliferation assay and to evaluate SAMNs efficacy as vector for pDNA coding GFP, an immunofluorescence analysis was performed. The second topic of this research project was on skin regeneration studied in vivo. Skin is a soft tissue and covers the entire surface area of body. It is a self-repairing, self-renewing organ that forms an important barrier from the outer environment to the inner environment. Therefore, damage to the skin leads to debilitating wounds that is an impairment of the anatomical structure and function of the skin. In the two papers of the second section, the capability of adult equine and ovine MSCs to regenerate skin injuries has been studied. 1) Wounds were induced in the gluteus region of six horses and treated with autologous epithelial stem cells (EpSCs), allogeneic EpSCs, vehicle treatment or untreated control. 2) Sheep allogeneic PB-MSCs were utilized to treat experimental lesions on the back of six sheep. This project is part of a large scheme where conventional treatments (Manuka Honey, Connettivina and Acemannane) were compared to innovative cures (MSCs and gas-ionized plasma). In this thesis, only the data about skin regeneration with PB-MSCs was reported. In the first work of the second section, rt-PCR was performed on tissue biopsies collected after one and five weeks of treatment and IFN-y, IL-6, VEGF, EGF, IGF-1 and epidermal keratin (eKER) were analyzed to study cellular immune response, neovascularization and the epidermal keratinization. In the second paper, clinical analysis have been performed to analyze the healing time, the presence, the color and the nature of exudate, the aspect of gauze, the hydration of the wound, the percentage of re-epithelization and contraction of the lesions. Tissue biopsies were collected after 15 and 42 days of treatments to conduct molecular analysis, histological and immunohistochemical staining. Molecular analysis were performed to study the expression level of genes such as Collagen 1α1 (Col1α1) and Keratin of hair (hKER). Dermal and subcutaneous inflammation, granulation tissue and skin adnexa were evaluated using histological analysis while the expression of MHCII, von Willebrand factor (vWF) and a cellular proliferation marker (KI67) were estimated with immunohistochemical staining

Repairing folding-defective \u3b1-sarcoglycan mutants by CFTR correctors, a potential therapy for Limb Girdle Muscular Dystrophy 2D

Limb Girdle Muscular Dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for \u3b1-sarcoglycan. Nowadays more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of \u3b1-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of \u3b1-sarcoglycan partners, \u3b2-, \u3b3- and \u3b4-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy.Here, we show that the application of small molecules developed to rescue \u394F508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several \u3b1-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective \u3b1-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations

Efficacy of conventional versus innovative therapies for treating skin wounds in veterinary medicine

open16siINTRODUCTION: The skin is the largest organ of mammals. The loss of skin integrity may induce important dysfunctions or even death. For superficial wounds, the endogenous healing mechanisms in combination with traditional wound care are sufficient to achieve functional repair. In contrast, in larger wounds, like third and fourth degree burns, chronic wound or deep ulcers it is difficult to obtain the restitutio ad integrum and fibrosis and/or scar tissue develops1,2. The aim of this study was to verify the efficacy of conventional and innovative topic treatments on skin regeneration, induced experimentally in sheep. To achieve this goal different types of investigations (clinical, molecular, histological, immunohistochemical) were performed. METHODS: Six skin lesions (4x4cm) were surgically created on the back of six healthy adult sheep; every single wound was destined, in a randomized way, to one of the following treatments: Acemannan gel, Manuka Honey, hyaluronic acid, Plasma3 (ionized gas), allogeneic mesenchymal stem cells isolated from peripheral blood (PB-MSCs). The sixth wound was the placebo. Biopsies were collected with a surgical punch (0,6x0,6 cm) at time T0, T15 and T40 days. Lesions were clinically evaluated considering the presence and color of wound fluid, the state of hydration, the wound surface/surroundings and other parameters. Histological examinations considered crust formation, re-epithelization and epidermal thickness, dermis edema, extension of granulation tissue, acute and chronic inflammation. Immunohistochemistry for evaluation of inflammation, vascularization and cell proliferation was performed using CD3, CD20, MHCII, von Willebrand factor (vWF) and KI67 antibodies. Furthermore, Real time-PCR investigated genes as V ascular endothelial growth factors (VEGF), Transforming growth factor beta 1(TGFβ1), Vimentin (VIM), Collagen 1α1 (Col1α1) and hair Keratin (hKER). RESULTS: Clinically, the lesions treated with plasma healed more rapidly respect to other treatments and a reduced bacterial load was observed. At T7 wounds treated with stem cells and plasma were less macerated than lesions treated with other therapies. At T15 the wounds treated with hyaluronic acid showed a normal state of hydration while lesions treated with Manuka Honey exhibited a normal hydration from the third week only (Acemannan gel at fourth week). From the second week onwards all wounds did not show presence of fluid and exhibited a dry and clean secondary layer. All lesions, excluded wounds treated with acemannan gel, presented a red (hyaluronic acid and plasma) and dark red (Manuka Honey, PB-MSCs) granulation tissue starting from the first week. Molecular analysis showed a correspondence between clinical and molecular/histologic results. For instance, VEGF mRNA expression confirms angiogenetic events observed at histological level while TGF-β, CD3 and CD20 mRNA/protein expression indicated the presence/absence of inflammation in the used treatments. DISCUSSION & CONCLUSIONS: Innovative therapies led to surprising results regarding regeneration of mammalian skin. Indeed, on the basis of clinical analysis, wounds treated with plasma and MSC healed more rapidly. Further examinations are ongoing in order to elucidate possible mechanisms explaining these differences. REFERENCES: 1S.Y. Broeckx, S. Maes, T. Martinello, et al (2014) Equine epidermis: a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities Stem Cells Dev, pp 1134-48. 2J.H. Spaas, C. Gomiero, S.Y. Broeckx, et al (2016) Wound healing markers after autologous and allogeneic epithelial-like stem cell treatment Cytotherapy 2016 (in press). 3E. Martines, M. Zuin, R. Cavazzana, et al. (2009) A novel plasma source for sterilization of living tissues, New J. Phys. 11, 115014.openPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, IlariaPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, Ilari

Muscle spindles of the rat sternomastoid muscle

The sternomastoid (SM) muscle in rodents presents a peculiar distribution of fiber types with a steep gradient from the ventral, superficial, white portion to the dorsal, deep, red region, where muscle spindles are restricted. Cross section of the medial longitudinal third of the rat SM contains around 10,000 muscle fibers with a mean diameter of 51.28±12.62 (μm +/- SD). Transverse sections stained by Succinate Dehydrogenase (SDH) reaction clearly presents two distinct regions: the dorsal deep red portion encompassing a 40% cross section area contains a high percentage of packed SDH-positive muscle fibers, and the ventral superficial region which contains mainly SDH-negative muscle fibers. Indeed, the ventral superficial region of the rat SM muscle contains mainly fast 2B muscle fibers. These acidic ATPase pH 4.3-negative and SDH-negative 2B muscle fibers are the largest of the SM muscle, while the acidic ATPase pH 4.3-positive and SDH-positive Type 1 muscle fibers are the smallest. Here we show that in thin transverse cryosections only 2 or 3 muscle spindle are observed in the central part of the dorsal deep red portion of the SM muscle. Azan Mallory stained sections allow at the same time to count the spindles and to evaluate aging fibrosis of the skeletal muscle tissue. Though restricted in the muscle red region, SM spindles are embedded in perimysium, whose changes may influence their reflex activity. Our findings confirm that any comparisons of changes in number and percentage of muscle spindles and muscle fibers of the rat SM muscle will require morphometry of the whole muscle cross-section. Muscle biopsies of SM muscle from large mammals will only provide partial data on the size of the different types of muscle fibers biased by sampling. Nonetheless, histology of muscle tissue continue to provide practical and low-cost quantitative data to follow-up translational studies in rodents and beyond

Novel regenerative medicine approaches with the use of adult mesenchymal stem cells: in vitro and in vivo experimental procedures

Tendon injuries are often associated with skeletal muscle lesions that can originate from a variety of events, including direct trauma, tendon and muscle lacerations and contusions, indirect insults and degenerative diseases as muscular dystrophies. Currently, a complete cure for musculoskeletal diseases is not present and the restitutio ad integrum is difficult to obtain. In the last decade, adult MSCs gained general attention in both human and veterinary medicine and the understanding of MSC function is improved promoting the application of cell therapy and the development of powerful cell-derived therapeutics for regenerative medicine. The first part of this research focused on the reprogramming of stromal cells derived from equine and sheep mesenchymal tissue towards tenogenic and myogenic fate in vitro using new non-viral transfection system. 1) Equine MSCs isolated from peripheral blood (PB-MSCs) can develop the tenogenic pathway using four specific growth factors such as TGFβ3 (transforming growth factor β3), EGF2 (epidermal growth factor 2), bFGF2 (fibroblast growth factor 2) and IGF1 (insulin-like growth factor 1) in presence or without Low Level Laser Technology (LLLT). 2) PB-MSCs were induced to differentiate towards myogenic fate using the complex TAT-MyoD in presence of a conditioned medium obtained from co-culturing PB-MSCs with C2C12 without a direct contact. 3) A novel surface-active maghemite nanoparticles (SAMNs) were tested as vectors for eukaryotic cell transfection of coding gene in PB-MSCs without the application of external magnetic fields. The full characterization of these three techniques was achieved using molecular and immunohistochemistry analysis. Real-time PCR (rt-PCR) was performed to study the expression level of the typical tenogenic genes markers Early Growth Response Protein-1 (EGR1), Tenascin C (TNC) and Decorin (DCN) to discover the best combination of GFs in presence or without LLLT. To evaluate the myoblasts differentiation, rt-PCR analysis was executed to study Myf5 and Myogenin gene expression while immunofluorescence experiments was performed to estimate MyoD, Myf5 and Myogenin protein expression. The cytotoxicity effects of SAMNs nanoparticles was observed with XTT cell proliferation assay and to evaluate SAMNs efficacy as vector for pDNA coding GFP, an immunofluorescence analysis was performed. The second topic of this research project was on skin regeneration studied in vivo. Skin is a soft tissue and covers the entire surface area of body. It is a self-repairing, self-renewing organ that forms an important barrier from the outer environment to the inner environment. Therefore, damage to the skin leads to debilitating wounds that is an impairment of the anatomical structure and function of the skin. In the two papers of the second section, the capability of adult equine and ovine MSCs to regenerate skin injuries has been studied. 1) Wounds were induced in the gluteus region of six horses and treated with autologous epithelial stem cells (EpSCs), allogeneic EpSCs, vehicle treatment or untreated control. 2) Sheep allogeneic PB-MSCs were utilized to treat experimental lesions on the back of six sheep. This project is part of a large scheme where conventional treatments (Manuka Honey, Connettivina and Acemannane) were compared to innovative cures (MSCs and gas-ionized plasma). In this thesis, only the data about skin regeneration with PB-MSCs was reported. In the first work of the second section, rt-PCR was performed on tissue biopsies collected after one and five weeks of treatment and IFN-y, IL-6, VEGF, EGF, IGF-1 and epidermal keratin (eKER) were analyzed to study cellular immune response, neovascularization and the epidermal keratinization. In the second paper, clinical analysis have been performed to analyze the healing time, the presence, the color and the nature of exudate, the aspect of gauze, the hydration of the wound, the percentage of re-epithelization and contraction of the lesions. Tissue biopsies were collected after 15 and 42 days of treatments to conduct molecular analysis, histological and immunohistochemical staining. Molecular analysis were performed to study the expression level of genes such as Collagen 1α1 (Col1α1) and Keratin of hair (hKER). Dermal and subcutaneous inflammation, granulation tissue and skin adnexa were evaluated using histological analysis while the expression of MHCII, von Willebrand factor (vWF) and a cellular proliferation marker (KI67) were estimated with immunohistochemical staining.Nell’ultima decade, le cellule staminali mesenchimali adulte (CSM) sono state considerate una cura innovativa per la medicina umana e veterinaria. Questo progetto di ricerca supporta l’efficacia delle cellule staminali nella rigenerazione dei tessuti muscolo-scheletrici e cutanei. In particolare è stata analizzata la loro potenzialità in vitro nel differenziare in tenociti e mioblasti e la loro capacità, in vivo, nel riparare danni alla cute. Le lesioni tendinee e muscolari sono frequenti e altamente debilitanti, possono originare da diversi eventi come traumi, lacerazioni, contusioni e malattie degenerative (distrofie muscolari). Attualmente una cura efficace non è ancora nota per cui risulta molto difficile riacquisire la restitutio ad integrum del tessuto lesionato. La prima parte di questa tesi di dottorato si è focalizzata sulla riprogrammazione in vitro di cellule stromali isolate da tessuti mesenchimali di cavalli e pecore verso la via tenogenica e miogenica usando nuove metodologie di trasfezione senza l’uso di vettori virali. 1) CSM isolate da sangue periferico di cavallo sono state indotte a differenziare verso la via tenogenica in presenza di quattro fattori di crescita come il TGFβ3 (fattore di crescita trasformante-3), EGF2 (fattore di crescita dell’epidermide-2), bFGF2 (fattore di crescita dei fibroblasti-2) e IGF-1 (fattore di crescita insulino-1) combinati tra loro in presenza o in assenza della Tecnologia Low Level Laser (LLLT). 2) CSM isolate da sangue periferico di cavallo sono state indotte a differenziare verso la via miogenica usando il complesso TAT-MyoD in presenza di un terreno di crescita condizionato ottenuto dalla co-coltura tra CSM e le cellule C2C12 senza il loro diretto contatto. 3) Nanoparticelle di maghemite (SAMNs) sono state testate come vettori di trasfezione in CSM isolate da sangue periferico di cavallo senza l’impiego di campi magnetici esterni. La caratterizzazione di queste tre tecniche è stata effettuata usando analisi molecolari ed immunoistochimiche. Per individuare la miglior combinazione di fattori di crescita nel differenziamento verso la linea tenogenica, in presenza o assenza della tecnologia LLLT, sono state effettuate real-time PCR (rt-PCR) analizzando i livelli di espressione genica dei markers tenogenici Early Growth Response Protein-1 (EGR1), Tenascina C (TNC) e Decorina (DCN). Per valutare il differenziamento delle CSM verso la miogenesi, sono state effettuate analisi con rt-PCR valutando l’espressione genica di Myf5 e Miogenina, mentre saggi di immunofluorescenza sono stati eseguiti per stimare l’espressione delle proteine MyoD, Myf5 e Miogenina nelle cellule staminali mesenchimali adulte. Infine, saggi di immunofluorescenza sono stati effettuati per valutare l’efficienza di trasfezione delle SAMNs mentre gli effetti citotossici delle nanoparticelle sono stati osservati con il saggio di proliferazione cellulare XTT. La seconda sezione di questa tesi di dottorato si è focalizzata sulla rigenerazione della cute in vivo. La cute è un organo che ricopre l’intera superficie del corpo e possiede la capacità di auto-riparazione e di auto-rinnovo formando un’importante barriera tra l’ambiente esterno ed interno. Danni alla pelle possono creare ferite debilitanti che intaccano la struttura anatomica e la funzione della cute stessa. Nei due lavori presenti nella seconda parte di questa tesi, è stata studiata la capacità delle CSM isolate da sangue periferico di cavallo e di pecora di rigenerare lesioni cutanee sperimentali. 1) Lesioni cutanee sono state indotte in corrispondenza dei muscoli dei glutei di sei cavalli e trattate con CSM epiteliali autologhe (Ep-MSCs), allogeniche, soluzione salina o non trattate. 2) CSM allogeniche isolate da sangue periferico di pecora sono state utilizzate per trattare lesioni effettuate sul dorso di sei pecore. Questo progetto rientra in uno studio molto più ampio dove trattamenti convenzionali come Miele di Manuka, Connettivina e Acemannano, sono stati comparati a trattamenti innovativi come le CSM e il gas ionizzato plasma. In questa tesi di dottorato, è stato riportato solo l’articolo inerente la rigenerazione della cute utilizzando CSM allogeniche. Il primo lavoro di questa sezione riporta i risultati ottenuti con analisi molecolari (rt-PCR) su tessuto cutaneo bioptico equino dopo una e cinque settimane di trattamento con Ep-MSCs. I livelli di espressione dei geni Interferone-y (IFN-y), Interleuchina-6 (IL-6), fattore di crescita dell’endotelio vascolare (VEGF), fattore di crescita dell’epidermide-2 (EGF2), fattore di crescita insulino-1 (IGF-1) e cheratina dell’epidermide (eKER) sono stati studiati per analizzare la risposta immunitaria, la neo vascolarizzazione e la cheratinizzazione epidermica. Nel secondo lavoro, sono state effettuate analisi cliniche per analizzare il tempo di guarigione, la presenza, il colore e la natura dell’essudato, l’aspetto della garza, l’idratazione della ferita, la percentuale di ri-epitelizzaione e di contrazione delle lesioni. Il tessuto bioptico prelevato dopo 15 e 42 giorni di trattamento è stato utilizzato per analisi molecolari, istologiche ed immunoistochimiche. Mediante le analisi molecolari sono stati valutati i livelli di espressione dei geni Collagene 1α1 (Col1α1) e cheratina del pelo (hKER). L’infiammazione dermica e sottocutanea, il tessuto di granulazione immaturo e maturo e gli annessi cutanei sono stati valutati mediante analisi istologiche mentre il complesso maggiore di istocompatibilità (MHCII), il fattore di von Willebrand e il marker di proliferazione cellulare KI67 sono stati studiati con saggi di immunoistochimica

Proposta di un questionario per l'analisi di aspetti socio-cognitivi e resilienza per le professioni di cura della disabilit\ue0\ua0 intellettiva: uno studio preliminare.

Lo scopo di questa ricerca \ue8 stato quello di predisporre uno strumento per la rilevazione degli aspetti socio-cognitivi degli operatori che operano con persone con grave disabilit\ue0 e di individuare quali di questi aspetti sono maggiormente in relazione con alcuni indici lavorativi. Gli aspetti socio-cognitivi presi in esame sono: l\u2019autoregolazione, stile attributivo, convinzioni sul s\ue9 (teoria e fiducia nella propria intelligenza e personalit\ue0, obiettivi di competenza nel lavoro, percezione di abilit\ue0) e autoefficacia percepita (collettiva e personale). Gli indici lavorativi considerati sono i giorni di assenza del lavoro per malattia, retribuzione mensile, tempo per la formazione professionale, turn-over, lo stress e il burnout lavorativo. E\u2019 stato predisposto uno strumento di 117 item somministrato a 30 operatori occupati in servizi della Provincia di Trento. I risultati hanno evidenziato che complessivamente le aree dello strumento presentano buone caratteristiche psicometriche e l\u2019analisi della relazione tra aspetti socio-cognitivi e indici lavorativi ha evidenziato che la percezione di scelta, l\u2019autoregolazione e lo stile attributivo sono in relazione significativa con lo stress, il burnout, giorni di assenza per malattia ed esperienze lavorative precedenti. Le analisi hanno consentito di identificare il livello di bont\ue0 degli item per poter predisporre una versione pi\uf9 breve del questionario

Rescue of folding-defective alpha-sarcoglycan mutants by means of protein folding correctors

Sarcoglycans (SG) are glycosylated proteins (alpha-, beta-, delta- or gamma-SG) forming a key structural complex, essential for the sarcolemma integrity of striated muscles during contraction. It is well known that defects in any one of the genes coding for sarcoglycans lead to the severe reduction or even the complete loss of SG-complex. Most of the mutations associated to sarcoglycanopathy are missense mutations and the disease severity is strictly related to the residual amount of sarcoglycans found at sarcolemma. We have recently proven that the primary event in these cases is the premature degradation of a folding-defective sarcoglycan, followed by the secondary loss of the wild-type partners, operated by the Endoplasmic Reticulum-Associated Degradation. We have also demonstrated that many missense mutants retain their function and that the entire complex can be properly rescued by targeting the degradative pathway. The knowledge of the pathogenic mechanism of sarcoglycanopathy has been also essential to design novel therapeutic strategies for this neglected and still incurable disease. These strategies intend not only to merely inhibit degradation of sarcoglycan mutants, but particularly to help their folding so that, structurally stabilized, mutants can skip disposal and traffic at the proper site of action. We tested several protein folding correctors, screened for the treatment of cystic fibrosis and called CFTR correctors. These small molecules were effective in recovering different mutants of alpha-sarcoglycan in cellular models, and notably in primary myotubes from a patient suffering of alpha-sarcoglycanopathy. In the latter case, the whole sarcoglycan complex was properly rescued at the plasma membrane, suggesting that a sort of \u201cprotein repair strategy\u201d can be adopted to treat sarcoglycanopathy. Although the mechanism by which CFTR correctors act on sarcoglycan mutants need to be clarified, these data represent the proof o principle of a novel pharmacological strategy aiming at correcting mutant folding by using well-known and available small molecules

Novel therapeutic perspectives for sarcoglycanopathy: rescue of folding-defective mutants by means of protein folding correctors

Sarcoglycans (SG) are glycosylated proteins (\u3b1-, \u3b2-, \u3b3- or \u3b4-SG) forming a key structural complex, essential for the sarcolemma integrity of striated muscles during contraction. In sarcoglycanopathies, it is well known that defects in any one of the sarcoglycan genes lead to the strong reduction or even the loss of the SG-complex. Most of the reported cases are due to missense mutations originating a full length but folding-defective proteins. We proved that the primary pathological event in sarcoglycanopathy occurs in the Endoplasmic Reticulum, where the quality control system, by proof-reading newly synthesized sarcoglycans, recognizes and deliver to proteasomal degradation the folding-defective mutants. This results in secondary loss of the wild-type partners. We also demonstrated that many missense mutants retain their function as the entire complex can be properly rescued by reducing the mutant degradation. These findings opened new perspectives for therapy of this neglected disease allowing to design small molecule-based approaches aimed either to inhibit sarcoglycan mutants degradation, or to help their folding so that, skipping disposal, they can assemble and traffic at the proper site of action. We tested several small molecules known as CFTR correctors which were effective in recovering different mutants of alpha-sarcoglycan in cellular models and, notably, the whole SG-complex in primary myotubes from a patient suffering of \u3b1-sarcoglycanopathy. To confirm in vivo this successful strategy we need animal models expressing folding-defective sarcoglycans. As the SG-null mice are unsuitable to our purposes, and considering the large number of reported sarcoglycan missense mutants, our aim is now the generation and characterization of novel \u3b1-sarcoglycanopathy models by the transduction of the null mice with rAAVs (recombinant adeno associated viruses) expressing different missense mutants of the human \u3b1-SG. We are confident that, once fully characterized, these animals will become suitable sarcoglycanopathy models to test in vivo our therapeutic strategy