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

The spectral features of EEG responses to transcranial magnetic stimulation of the primary motor cortex depend on the amplitude of the motor evoked potentials

Transcranial magnetic stimulation (TMS) of the primary motor cortex (M1) can excite both cortico-cortical and cortico-spinal axons resulting in TMS-evoked potentials (TEPs) and motor-evoked potentials (MEPs), respectively. Despite this remarkable difference with other cortical areas, the influence of motor output and its amplitude on TEPs is largely unknown. Here we studied TEPs resulting from M1 stimulation and assessed whether their waveform and spectral features depend on the MEP amplitude. To this aim, we performed two separate experiments. In experiment 1, single-pulse TMS was applied at the same supra-threshold intensity on primary motor, prefrontal, premotor and parietal cortices and the corresponding TEPs were compared by means of local mean field power and time-frequency spectral analysis. In experiment 2 we stimulated M1 at resting motor threshold in order to elicit MEPs characterized by a wide range of amplitudes. TEPs computed from high-MEP and low-MEP trials were then compared using the same methods applied in experiment 1. In line with previous studies, TMS of M1 produced larger TEPs compared to other cortical stimulations. Notably, we found that only TEPs produced by M1 stimulation were accompanied by a late event-related desynchronization (ERD-peaking at ~300 ms after TMS), whose magnitude was strongly dependent on the amplitude of MEPs. Overall, these results suggest that M1 produces peculiar responses to TMS possibly reflecting specific anatomo-functional properties, such as the re-entry of proprioceptive feedback associated with target muscle activation

Alfa-synuclein oligomers induced by docosahexaenoic acid: a study of activity and molecular characterization

Parkinson disease (PD) is the main neurodegenerative disease that involves motor symptoms. About 1% of population above 65 years is affected by PD. Main symptoms are bradikinesia, resting tremor, postural instability, muscle rigidity, and sometimes, cognitive problems and personality. Neuropathological features of PD are neuronal death in the substantia nigra pars compacta and formation of cytoplasmatic inclusion, named Lewy bodies, constituted by fibrillar form of α-synuclein (aS). aS is a 140 amino acid protein, whose structure and function is yet not well defined. As a consequence of specific genetic mutations or environmental factors, it undergoes aggregation and forms amyloid fibrils. It is highly expressed in neuronal pre-synaptic nerve terminals. Its sequence is characterized by an amphipathic lysine-rich amino terminus, which governs binding to lipids and interactions with membranes and contains seven imperfect repetition of the sequence KTKEGV; by a hydrophobic central region (NAC, non-amyloid component), responsible for protein aggregation and α-sheet formation and a highly acidic C-terminal, rich in Pro and acidic residues. Overexpression of aS and mutations in its gene are associated with a premature development of PD. Mechanism that make aS a toxic protein has not yet been well clarified, but it seems clear that oligomeric forms, and not the final fibrillar forms, are the main responsible for the pathogenesis of PD. The project of this thesis focuses on the characterization of oligomers of aS that form in the presence of docosahexaenoic acid (DHA), and their interaction with membrane, to understand the mechanism of toxicity. DHA is one of the most abundant fatty acids in neuronal membrane and it has been correlated to PD. It has been demonstrated that dopaminergic cell cultures exposed to PUFAs accumulate soluble cytotoxic aS oligomers (Assayag et al., 2007). Indeed, aS seems to be involved in fatty acids metabolism (Golovko et al., 2007). Moreover, it was reported that, in PD patients, DHA concentration is enhanced in those area affected by aS inclusions. In vivo studies demonstrated that a DHA enriched diet enhances formation of aS oligomers (Sharon et al., 2003). In previous studies in this laboratory, it was analyzed the aggregation process of aS in the presence of DHA using different protein to DHA molar ratios (De Franceschi et al., 2009; 2011). Oligomers obtained in these conditions were characterized from a morphological point of view (De Franceschi et al., 2011). The presence of DHA (50:1 lipid:protein molar ratio) leads to the formation of stable oligomers, off-pathway in the aggregation process of aS, that have significant toxic activity on cells, suggesting that they are potentially relevant in the pathogenesis of PD (De Franceschi et al., 2011). In the first part of this thesis a characterization of oligomers have been conducted using several biophysical methods, since these oligomers are sufficiently stable to allow the use of these techniques. In particular, transmission electron (TEM) and atomic force (AFM) microscopy were used to study oligomers morphology and dimension. The secondary structure was evaluated by circular dichroism (CD). This spectroscopic analysis reveals that oligomers have a partial α-helix structure, in contrast with the majority of oligomeric species described in literature. It was analyzed also the ability of oligomers to interact with membrane, using liposomes of different size and composition and cell cultures. Interaction of oligomers with membrane, analyzed by CD measurements and leakage assays, causes the leakage of small molecule, demonstrating their ability to destabilize membranes. Oligomers activity was tested also on dopaminergic cell culture that showed an altered permeabilization after treatment. To determine the mechanism by which oligomers cause membrane permeabilization, different tests were performed. Initially, dynamic light scattering (DLS) and TEM allow to exclude a detergent-like effect. Moreover, aggregation studies and planar lipid membrane (PLM) measurements lead to hypothesize a toxicity mechanism that depends on the formation of a transient aperture or on the enhancement of flip-flop. This part of the thesis is object of a publication (Fecchio et al., 2013). Another aspect faced in this thesis is the study of chemical modification occurring on oligomers after exposition to DHA. Different chemical modification were evidenced by mass spectrometry: carbonylation and the formation of adduct with DHA at the level of His50. To deepen the role of this residue in the interaction with FA, it was used an aS variant, H50Q, that has recently been linked to familiar form of PD. Finally, also the interaction with DHA of other pathological variants of aS (A30P, E46K, A53T) was studied. In particular their secondary structure and oligomerization in the presence of DHA were analyzed, in comparison with results obtained with aS. In conclusion, this study supplied further information about structure and activity of oligomeric species that are potentially relevant in PD pathogenesis. These data can be compared to oligomers produced in different conditions or formed by different amyloidogenic proteins: this knowledge would be fundamental to the development of therapeutic agent that would prevent or defeat these kind of debilitative diseasesIl morbo di Parkinson (PD) è la principale malattia neurodegenerativa riguardante la funzionalità motoria. L'1% della popolazione sopra i 65 anni è affetto da questa malattia. I sintomi principali sono bradichinesia, tremore a riposo, instabilità posturale, rigidità muscolare e, talvolta, problemi cognitivi e della personalità . Le principali caratteristiche neuropatologiche del PD sono la morte dei neuroni dopaminergici a livello della substantia nigra pars compacta e la formazione di corpi d'inclusione citoplasmatici composti da aggregati proteici fibrillari di tipo amiloide, Lewy bodies (LBs), il cui costituente principale è α-sinucleina (aS) (Spillantini et al., 1998). aS è proteina di 140 amminoacidi, natively unfolded, la cui funzione, nonostante il suo ruolo chiave nel PD, non è ancora completamente chiarita. E' espressa in livelli alti nel sistema nervoso centrale ed è abbondante nei terminali presinaptici neuronali. Strutturalmente è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale con numerosi residui acidi. La sovraespressione di aS e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Il meccanismo con cui un cambiamento nella struttura e nell'espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito, ma è sempre pi๠accreditata l'ipotesi che siano le forme oligomeriche e non gli aggregati finali fibrillari ad essere responsabili della malattia. Il progetto di questa Tesi riguarda la caratterizzazione di oligomeri tossici di α-sinucleina (aS) ottenuti in presenza di acido docosaesaenoico (DHA) e lo studio della loro interazione con membrane lipidiche, allo scopo di comprendere il meccanismo con cui esercitano la loro tossicità . Il DHA è uno dei principali acido grassi cerebrali, strettamente correlato al PD e ad aS. L'esposizione di colture cellulari dopaminergiche a PUFAs porta all'accumulo di oligomeri solubili di aS, responsabili della citotossicità associata alla neurodegenerazione (Assayag et al., 2007). Esistono poi evidenze dell'implicazione di aS nella regolazione del metabolismo degli acidi grassi (Golovko et al., 2007). Inoltre è stato osservato che, nei pazienti affetti da PD, la presenza di DHA è maggiore nelle aree cerebrali contenenti inclusioni di aS. Studi in vivo, infine, hanno dimostrato che il DHA induce la formazione di oligomeri di aS (Sharon et al., 2003). In precedenti studi condotti nel laboratorio dove è stata svolta questa Tesi è stato analizzato il processo di aggregazione di aS in presenza di DHA, utilizzando due diversi rapporti molari proteina/acido grasso) (De Franceschi et al., 2009) e gli aggregati proteici ottenuti in queste condizioni sono stati caratterizzati da un punto di vista morfologico e strutturale (De Franceschi et al., 2011). E' stato osservato che la presenza di DHA in rapporto molare 50:1 rispetto alla proteina, porta alla formazione di oligomeri stabili, off-pathway nel processo di fibrillazione, che presentano una significativa attività tossica sulle cellule rispetto al monomero di aS. Nella prima parte di questa ricerca è stata condotta una caratterizzazione di queste specie oligomeriche che sono sufficientemente stabili nel tempo da consentire l'uso di diverse tecniche biofisiche. In particolare gli oligomeri sono stati analizzati mediante microscopia elettronica a trasmissione (TEM) e a forza atomica (AFM), per studiare le dimensioni e la morfologia. Il tipo di struttura secondaria è stata valutata mediante dicroismo circolare che ha dimostrato un'altra peculiarità di questi oligomeri, ovvero la presenza di struttura parzialmente in α-elica, diversamente dalla maggior parte degli oligomeri descritti in letteratura. E' stata analizzata anche la capacità degli oligomeri di interagire con le membrane, utilizzando liposomi di diversa dimensione e diversa composizione e colture cellulari. L'interazione tra gli oligomeri e i liposomi, studiata mediante CD e saggi di leakage, causa il rilascio di piccole molecole interne alle vescicole, dimostrando così un loro effetto destabilizzante sulle membrane. L'attività degli oligomeri è stata anche testata su cellule in coltura che mostrano un'alterata permeabilità in loro presenza. Per determinare quale sia il meccanismo di destabilizzazione degli oligomeri, sono stati eseguiti vari saggi. Si è dimostrato tramite dynamic light scattering (DLS) e TEM che le vescicole, in seguito al legame con gli oligomeri, non vengono distrutte. Inoltre mediante studi di aggregazione e analisi su planar lipid membrane portano a ipotizzare un meccanismo di tossicità dovuto alla formazione di un'apertura transiente o un aumento di flip-flop a livello delle membrane. I risultati di questa parte di tesi sono oggetto di una pubblicazione (Fecchio et al., 2013). Un altro aspetto che è stato approfondito in questo lavoro di Tesi è lo studio degli oligomeri da un punto di vista chimico, allo scopo di caratterizzare le modifiche chimiche presenti sulla sequenza della proteina in seguito all'esposizione al DHA. Sono state evidenziate diverse modifiche mediante spettrometria di massa tra cui carbonilazioni e presenza di addotti. Quest'ultimo tipo di modifica in particolare avviene a livello dell'istidina in posizione 50. Per approfondire il ruolo di questo aminoacido nell'interazione con gli acidi grassi è stato studiato il mutante H50Q di aS. Questa proteina modificata è tra l'altro responsabile di forme familiari del PD. Infine è stata studiata anche l'interazione di altre varianti patologiche di aS associate a PD, A30P, E46K e A53T con DHA. In particolare, è stata analizzata la loro struttura e la loro tendenza ad aggregare in presenza di DHA, nonchè la loro capacità di formare oligomeri, in confronto ai risultati ottenuti con aS. In conclusione questo studio ha permesso di fornire maggiori informazioni sulla struttura e di studiare l'attività di specie oligomeriche che sono potenzialmente molto rilevanti per la patogenesi del PD. La struttura e l'attività di questi oligomeri potrà essere confrontata con quelle di oligomeri prodotti in diverse condizioni sperimentali o di oligomeri prodotti da altre proteine amiloidogeniche. Questa conoscenza è fondamentale per sviluppare agenti terapeutici che prevengano o debellino queste malattie debilitanti ed in continuo aument

Alfa-synuclein oligomers induced by docosahexaenoic acid: a study of activity and molecular characterization

Parkinson disease (PD) is the main neurodegenerative disease that involves motor symptoms. About 1% of population above 65 years is affected by PD. Main symptoms are bradikinesia, resting tremor, postural instability, muscle rigidity, and sometimes, cognitive problems and personality. Neuropathological features of PD are neuronal death in the substantia nigra pars compacta and formation of cytoplasmatic inclusion, named Lewy bodies, constituted by fibrillar form of α-synuclein (aS). aS is a 140 amino acid protein, whose structure and function is yet not well defined. As a consequence of specific genetic mutations or environmental factors, it undergoes aggregation and forms amyloid fibrils. It is highly expressed in neuronal pre-synaptic nerve terminals. Its sequence is characterized by an amphipathic lysine-rich amino terminus, which governs binding to lipids and interactions with membranes and contains seven imperfect repetition of the sequence KTKEGV; by a hydrophobic central region (NAC, non-amyloid component), responsible for protein aggregation and α-sheet formation and a highly acidic C-terminal, rich in Pro and acidic residues. Overexpression of aS and mutations in its gene are associated with a premature development of PD. Mechanism that make aS a toxic protein has not yet been well clarified, but it seems clear that oligomeric forms, and not the final fibrillar forms, are the main responsible for the pathogenesis of PD. The project of this thesis focuses on the characterization of oligomers of aS that form in the presence of docosahexaenoic acid (DHA), and their interaction with membrane, to understand the mechanism of toxicity. DHA is one of the most abundant fatty acids in neuronal membrane and it has been correlated to PD. It has been demonstrated that dopaminergic cell cultures exposed to PUFAs accumulate soluble cytotoxic aS oligomers (Assayag et al., 2007). Indeed, aS seems to be involved in fatty acids metabolism (Golovko et al., 2007). Moreover, it was reported that, in PD patients, DHA concentration is enhanced in those area affected by aS inclusions. In vivo studies demonstrated that a DHA enriched diet enhances formation of aS oligomers (Sharon et al., 2003). In previous studies in this laboratory, it was analyzed the aggregation process of aS in the presence of DHA using different protein to DHA molar ratios (De Franceschi et al., 2009; 2011). Oligomers obtained in these conditions were characterized from a morphological point of view (De Franceschi et al., 2011). The presence of DHA (50:1 lipid:protein molar ratio) leads to the formation of stable oligomers, off-pathway in the aggregation process of aS, that have significant toxic activity on cells, suggesting that they are potentially relevant in the pathogenesis of PD (De Franceschi et al., 2011). In the first part of this thesis a characterization of oligomers have been conducted using several biophysical methods, since these oligomers are sufficiently stable to allow the use of these techniques. In particular, transmission electron (TEM) and atomic force (AFM) microscopy were used to study oligomers morphology and dimension. The secondary structure was evaluated by circular dichroism (CD). This spectroscopic analysis reveals that oligomers have a partial α-helix structure, in contrast with the majority of oligomeric species described in literature. It was analyzed also the ability of oligomers to interact with membrane, using liposomes of different size and composition and cell cultures. Interaction of oligomers with membrane, analyzed by CD measurements and leakage assays, causes the leakage of small molecule, demonstrating their ability to destabilize membranes. Oligomers activity was tested also on dopaminergic cell culture that showed an altered permeabilization after treatment. To determine the mechanism by which oligomers cause membrane permeabilization, different tests were performed. Initially, dynamic light scattering (DLS) and TEM allow to exclude a detergent-like effect. Moreover, aggregation studies and planar lipid membrane (PLM) measurements lead to hypothesize a toxicity mechanism that depends on the formation of a transient aperture or on the enhancement of flip-flop. This part of the thesis is object of a publication (Fecchio et al., 2013). Another aspect faced in this thesis is the study of chemical modification occurring on oligomers after exposition to DHA. Different chemical modification were evidenced by mass spectrometry: carbonylation and the formation of adduct with DHA at the level of His50. To deepen the role of this residue in the interaction with FA, it was used an aS variant, H50Q, that has recently been linked to familiar form of PD. Finally, also the interaction with DHA of other pathological variants of aS (A30P, E46K, A53T) was studied. In particular their secondary structure and oligomerization in the presence of DHA were analyzed, in comparison with results obtained with aS. In conclusion, this study supplied further information about structure and activity of oligomeric species that are potentially relevant in PD pathogenesis. These data can be compared to oligomers produced in different conditions or formed by different amyloidogenic proteins: this knowledge would be fundamental to the development of therapeutic agent that would prevent or defeat these kind of debilitative diseasesIl morbo di Parkinson (PD) è la principale malattia neurodegenerativa riguardante la funzionalità motoria. L'1% della popolazione sopra i 65 anni è affetto da questa malattia. I sintomi principali sono bradichinesia, tremore a riposo, instabilità posturale, rigidità muscolare e, talvolta, problemi cognitivi e della personalità . Le principali caratteristiche neuropatologiche del PD sono la morte dei neuroni dopaminergici a livello della substantia nigra pars compacta e la formazione di corpi d'inclusione citoplasmatici composti da aggregati proteici fibrillari di tipo amiloide, Lewy bodies (LBs), il cui costituente principale è α-sinucleina (aS) (Spillantini et al., 1998). aS è proteina di 140 amminoacidi, natively unfolded, la cui funzione, nonostante il suo ruolo chiave nel PD, non è ancora completamente chiarita. E' espressa in livelli alti nel sistema nervoso centrale ed è abbondante nei terminali presinaptici neuronali. Strutturalmente è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale con numerosi residui acidi. La sovraespressione di aS e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Il meccanismo con cui un cambiamento nella struttura e nell'espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito, ma è sempre pi๠accreditata l'ipotesi che siano le forme oligomeriche e non gli aggregati finali fibrillari ad essere responsabili della malattia. Il progetto di questa Tesi riguarda la caratterizzazione di oligomeri tossici di α-sinucleina (aS) ottenuti in presenza di acido docosaesaenoico (DHA) e lo studio della loro interazione con membrane lipidiche, allo scopo di comprendere il meccanismo con cui esercitano la loro tossicità . Il DHA è uno dei principali acido grassi cerebrali, strettamente correlato al PD e ad aS. L'esposizione di colture cellulari dopaminergiche a PUFAs porta all'accumulo di oligomeri solubili di aS, responsabili della citotossicità associata alla neurodegenerazione (Assayag et al., 2007). Esistono poi evidenze dell'implicazione di aS nella regolazione del metabolismo degli acidi grassi (Golovko et al., 2007). Inoltre è stato osservato che, nei pazienti affetti da PD, la presenza di DHA è maggiore nelle aree cerebrali contenenti inclusioni di aS. Studi in vivo, infine, hanno dimostrato che il DHA induce la formazione di oligomeri di aS (Sharon et al., 2003). In precedenti studi condotti nel laboratorio dove è stata svolta questa Tesi è stato analizzato il processo di aggregazione di aS in presenza di DHA, utilizzando due diversi rapporti molari proteina/acido grasso) (De Franceschi et al., 2009) e gli aggregati proteici ottenuti in queste condizioni sono stati caratterizzati da un punto di vista morfologico e strutturale (De Franceschi et al., 2011). E' stato osservato che la presenza di DHA in rapporto molare 50:1 rispetto alla proteina, porta alla formazione di oligomeri stabili, off-pathway nel processo di fibrillazione, che presentano una significativa attività tossica sulle cellule rispetto al monomero di aS. Nella prima parte di questa ricerca è stata condotta una caratterizzazione di queste specie oligomeriche che sono sufficientemente stabili nel tempo da consentire l'uso di diverse tecniche biofisiche. In particolare gli oligomeri sono stati analizzati mediante microscopia elettronica a trasmissione (TEM) e a forza atomica (AFM), per studiare le dimensioni e la morfologia. Il tipo di struttura secondaria è stata valutata mediante dicroismo circolare che ha dimostrato un'altra peculiarità di questi oligomeri, ovvero la presenza di struttura parzialmente in α-elica, diversamente dalla maggior parte degli oligomeri descritti in letteratura. E' stata analizzata anche la capacità degli oligomeri di interagire con le membrane, utilizzando liposomi di diversa dimensione e diversa composizione e colture cellulari. L'interazione tra gli oligomeri e i liposomi, studiata mediante CD e saggi di leakage, causa il rilascio di piccole molecole interne alle vescicole, dimostrando così un loro effetto destabilizzante sulle membrane. L'attività degli oligomeri è stata anche testata su cellule in coltura che mostrano un'alterata permeabilità in loro presenza. Per determinare quale sia il meccanismo di destabilizzazione degli oligomeri, sono stati eseguiti vari saggi. Si è dimostrato tramite dynamic light scattering (DLS) e TEM che le vescicole, in seguito al legame con gli oligomeri, non vengono distrutte. Inoltre mediante studi di aggregazione e analisi su planar lipid membrane portano a ipotizzare un meccanismo di tossicità dovuto alla formazione di un'apertura transiente o un aumento di flip-flop a livello delle membrane. I risultati di questa parte di tesi sono oggetto di una pubblicazione (Fecchio et al., 2013). Un altro aspetto che è stato approfondito in questo lavoro di Tesi è lo studio degli oligomeri da un punto di vista chimico, allo scopo di caratterizzare le modifiche chimiche presenti sulla sequenza della proteina in seguito all'esposizione al DHA. Sono state evidenziate diverse modifiche mediante spettrometria di massa tra cui carbonilazioni e presenza di addotti. Quest'ultimo tipo di modifica in particolare avviene a livello dell'istidina in posizione 50. Per approfondire il ruolo di questo aminoacido nell'interazione con gli acidi grassi è stato studiato il mutante H50Q di aS. Questa proteina modificata è tra l'altro responsabile di forme familiari del PD. Infine è stata studiata anche l'interazione di altre varianti patologiche di aS associate a PD, A30P, E46K e A53T con DHA. In particolare, è stata analizzata la loro struttura e la loro tendenza ad aggregare in presenza di DHA, nonchè la loro capacità di formare oligomeri, in confronto ai risultati ottenuti con aS. In conclusione questo studio ha permesso di fornire maggiori informazioni sulla struttura e di studiare l'attività di specie oligomeriche che sono potenzialmente molto rilevanti per la patogenesi del PD. La struttura e l'attività di questi oligomeri potrà essere confrontata con quelle di oligomeri prodotti in diverse condizioni sperimentali o di oligomeri prodotti da altre proteine amiloidogeniche. Questa conoscenza è fondamentale per sviluppare agenti terapeutici che prevengano o debellino queste malattie debilitanti ed in continuo aument

Emerging therapeutic strategies for sarcoglycanopathy

Introduction: Sarcoglycanopathy is the name shared by four rare autosomal recessive muscular dystrophies (LGMD2 C-F) that are usually characterized by early onset and rapid progression and an accompanying loss of independent walking since adolescence. Respiratory problems are frequent, and dilated cardiomyopathy may occur, although milder forms have also been described. However, sarcoglycanopathy is currently incurable, and we herein aim to describe the state of the art in the field of treatments for this disease. Areas covered: We summarize the pathogenesis of sarcoglycanopathy, with particular emphasis on the molecular mechanism(s) underlying the disease. We describe the very few published cases of symptomatic treatment with steroids and the gene therapy approaches that have entered phase I/II clinical trials. We then present emerging novel therapeutic strategies explored at the preclinical stage that are expected to replace the defective gene (cell therapy), address general effects of the disease, or address the primary events of the pathogenic mechanism (small molecule-based therapy). Expert opinion: Anti-inflammatory strategies, which are at present empirically applied, warrant further exploration. Although promising and currently being evaluated in clinical trials, gene therapy remains associated with concerns and requires additional confirmation. Thus, novel strategies targeting different aspects of the disease pathogenic mechanism are highly anticipated

From the molecular mechanism to novel pharmacological approaches, the story of a muscular dystrophy.

Sarcoglycans (SG) are glycosylated proteins (\u3b1-, \u3b2-, \u3b3- and \u3b4-SG) which form a key structural complex, essential for the membrane integrity of the striated muscle during contraction. Defects in any one of the sarcoglycan coding genes lead to the severe reduction or even the complete loss of the SG-complex and are responsible for a rare genetic disease known as sarcoglycanopathy1. Most of the reported cases are due to missense mutations that originate a full length but folding-defective protein. We proved that the primary event in these cases is the premature degradation of the folding-defective sarcoglycan, followed by the secondary loss of the wild-type partners, operated by the endoplasmic reticulum-associated degradation. We also demonstrated that many missense mutants retain their function because the whole complex can be properly rescued by skipping the degradation of the mutant2. The knowledge of the pathogenic mechanism of sarcoglycanopathy opened new perspectives for the therapy of this neglected disease allowing to design small molecule-based approaches aimed either to inhibit the degradation of sarcoglycan mutants, or to help their folding. By skipping the disposal process, sarcoglycan mutants can assemble into a functional complex that traffics toward the proper site of action. We successfully tested several of such small molecules in cellular models and, notably, in primary myotubes deriving form patients with sarcoglycanopathy3. We are now developing mouse and zebrafish models for the expression of folding-defective sarcoglycans. These novel vertebrate models of sarcoglycanopathy will be of fundamental help to test in vivo efficacy and safety of the proposed therapeutic approaches. Reference: 1. M. Carotti, C. Fecchio and D. Sandona\u2019, Expert Opin in Orphan Drug, 5, 381 (2017) 2. S. Gastaldello, S. D'Angelo, S. Franzoso, M. Fanin, C. Angelini, R. Betto, D. Sandon\ue0 Am J Pathol, 173, 170 (2008) 3. E. Bianchini, M. Fanin, K. Mamchaoui, R. Betto, D. Sandon\ue0, Hum Mol Genet, 23, 3746 (2014

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

\u3b1-Synuclein and polyunsaturated fatty acids: Molecular basis of the interaction and implication in neurodegeneration

\u3b1-Synuclein (\u3b1-syn) is a 140-amino acid protein, the physiological function of which has yet to be clarified. It is involved in several neurodegenerative disorders, and the interaction of the protein with brain lipids plays an important role in the pathogenesis of Parkinson's disease (PD). Polyunsaturated fatty acids (PUFA) are highly abundant in the brain where they play critical roles in neuronal membrane fluidity and permeability, serve as energy reserves and function as second messengers in cell signaling. PUFA concentration and composition in the brain are altered with age when also an increase of lipid peroxidation is observed. Considering that PD is clearly correlated with oxidative stress, PUFA abundance and composition became of great interest in neurodegeneration studies because of PUFA's high propensity to oxidize. The high levels of the PUFA docosahexaenoic acid (DHA) in brain areas containing \u3b1-syn inclusions in patients with PD further support the hypothesis of possible interactions between \u3b1-syn and DHA. Additionally, a possible functional role of \u3b1-syn in sequestering the early peroxidation products of fatty acids was recently proposed. Here, we provide an overview of the current knowledge regarding the molecular interactions between \u3b1-syn and fatty acids and the effect exerted by the protein on their oxidative state. We highlight recent findings supporting a neuroprotective role of the protein, linking \u3b1-syn, altered lipid composition in neurodegenerative disorders and PD development

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

Small molecules to rescue folding-defective sarcoglycans: in vivo assessment of novel therapeutic strategies

LGMD2C-F, or sarcoglycanopathies, are rare genetic diseases that, disrupting the sarcoglycan (SG) complex, affect the sarcolemma stability during muscle contraction. Most of the reported cases are due to missense mutations originating a folding-defective sarcoglycan that is eliminated, although potentially functional, by the quality control of the cell. To prevent mutant degradation and sarcoglycan complex disruption, we have designed and proven in vitro two small molecule-based pharmacological approaches. The first one utilizes inhibitors of the E3 ubiquitin ligase HRD1 to reduce the degradation of alpha-SG mutants (protein rescue), whereas the second approach, CFTR correctors to foster the folding process of the mutants (protein repair). Our successful in vitro strategy needs now to be confirmed in vivo. However, the animal models of sarcoglycanopathy, at present available, are unsuitable to our purposes as we need animals expressing a folding-defective sarcoglycan. Therefore, also considering the large number of reported sarcoglycan missense mutants, the first aim of our project is the generation and characterization of novel LGMD2D mouse models by the transduction of sgca null mice with rAAVs (recombinant adeno associated viruses) expressing different missense mutants of the human alpha-SG. To this intent, during the first year, we cloned the human alpha-SG cDNA, either wild type or carrying missense mutations, under the control of the truncated muscle creatine kinase promoter/enhancer to assure muscle specific expression of the transgene. The cassette was then utilized to engineer AAVs of the serotype 2/1, especially targeting skeletal muscle tissue. For the generation of the animal models, AAVs are intraperitoneally injected in 1-2 day old pups of the sgca null mouse to achieve whole body-muscle transduction. No major effects on survival, body weight and general behavior have been observed after AAV-injection and during animal growth. At 6, 10 and 16 weeks post transduction, the expression of the human transgene is at first quantified by qRT-PCR and western blot analysis of different muscles of the legs, diaphragm, as well as heart. The development of dystrophic phenotype is evaluated in muscle by histological analysis, whereas SG localization is monitored by immunofluorescence analysis. Evaluation of muscle performance of these \u201chumanized\u201d mice (muscle strength and fatigability) will be estimated in vivo, and also ex vivo. We are confident that, once fully characterized, these animals will become suitable sarcoglycanopathy models to test in vivo our therapeutic strategy. Important aim of the project is also the assessment of efficacy of selected small molecules in human pathological samples. The SG-complex has been successfully rescued in primary myotubes from a patient carrying the R34H and V247M amino acid substitution on alpha-SG, whereas experiments with myogenic cells from a homozygote R77C-alpha-SG patient are ongoing