Unleashing the potential of mRNA therapeutics for inherited neurological diseases.

Neurological monogenic loss-of-function diseases are hereditary disorders resulting from gene mutations that decrease or abolish the normal function of the encoded protein. These conditions pose significant therapeutic challenges, which may be resolved through the development of innovative therapeutic strategies. RNA-based technologies, such as mRNA replacement therapy, have emerged as promising and increasingly viable treatments. Notably, mRNA therapy exhibits significant potential as a mutation-agnostic approach that can address virtually any monogenic loss-of-function disease. Therapeutic mRNA carries the information for a healthy copy of the defective protein, bypassing the problem of targeting specific genetic variants. Moreover, unlike conventional gene therapy, mRNA-based drugs are delivered through a simplified process that requires only transfer to the cytoplasm, thereby reducing the mutagenic risks related to DNA integration. Additionally, mRNA therapy exerts a transient effect on target cells, minimizing the risk of long-term unintended consequences. The remarkable success of mRNA technology for developing COVID-19 vaccines has rekindled interest in mRNA as a cost-effective method for delivering therapeutic proteins. However, further optimization is required to enhance mRNA delivery, particularly to the central nervous system, while minimizing adverse drug reactions and toxicity. In this comprehensive review, we delve into past, present, and ongoing applications of mRNA therapy for neurological monogenic loss-of-function diseases. We also discuss the promises and potential challenges presented by mRNA therapeutics in this rapidly advancing field. Ultimately, we underscore the full potential of mRNA therapy as a game-changing therapeutic approach for neurological disorders.

Charcot-Marie-Tooth type 2A in vivo models: Current updates.

Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.

Charcot-Marie-tooth disease type 2A: An update on pathogenesis and therapeutic perspectives.

Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad clinical phenotype involving the entire nervous system. MFN2, a dynamin-like GTPase protein located on the outer mitochondrial membrane, is well-known for its involvement in mitochondrial fusion. Numerous studies have demonstrated its participation in a network crucial for various other mitochondrial functions, including mitophagy, axonal transport, and its controversial role in endoplasmic reticulum (ER)-mitochondria contacts. Considerable progress has been made in the last three decades in elucidating the disease pathogenesis, aided by the generation of animal and cellular models that have been instrumental in studying disease physiology. A review of the literature reveals that, up to now, no definitive pharmacological treatment for any CMT2A variant has been established; nonetheless, recent years have witnessed substantial progress. Many treatment approaches, especially concerning molecular therapy, such as histone deacetylase inhibitors, peptide therapy to increase mitochondrial fusion, the new therapeutic strategies based on MF1/MF2 balance, and SARM1 inhibitors, are currently in preclinical testing. The literature on gene silencing and gene replacement therapies is still limited, except for a recent study by Rizzo et al.(Rizzo et al., 2023), which recently first achieved encouraging results in in vitro and in vivo models of the disease. The near-future goal for these promising therapies is to progress to the stage of clinical translation.

Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Regional spreading pattern is associated with clinical phenotype in amyotrophic lateral sclerosis.

Increasing evidence shows that disease spreading in amyotrophic lateral sclerosis (ALS) follows a preferential pattern with more frequent involvement of contiguous regions from the site of symptom onset. The aim of our study was to assess if: (i) the burden of upper (UMN) and lower motor neuron (LMN) involvement influences directionality of disease spreading; (ii) specific patterns of disease progression are associated with motor and neuropsychological features of different ALS subtypes (classic, bulbar, primary lateral sclerosis, UMN-predominant, progressive muscular atrophy, flail arm, flail leg); and (iii) specific clinical features may help identify ALS subtypes, which remain localized to the site of onset for a prolonged time (regionally entrenching ALS). A single-centre, retrospective cohort of 913 Italian ALS patients was evaluated to assess correlations between directionality of the disease process after symptom onset and motor/neuropsychological phenotype. All patients underwent an extensive evaluation including the following clinical scales: Penn Upper Motor Neuron Score (PUMNS), MRC Scale for Muscle Strength and the Edinburgh Cognitive and Behavioural ALS Screen (ECAS). The most frequent initial spreading pattern was that towards adjacent horizontal regions (77.3%), which occurred preferentially in patients with lower MRC scores (P = 0.038), while vertical diffusion (21.1%) was associated with higher PUMNS (P < 0.001) and with reduced survival (P < 0.001). Non-contiguous disease spreading was associated with more severe UMN impairment (P = 0.003), while contiguous disease pattern with lower MRC scores. Furthermore, non-contiguous disease spreading was associated with more severe cognitive impairment in both executive and visuospatial ECAS domains. Individuals with regionally entrenching ALS were more frequently female (45.6% versus 36.9%; P = 0.028) and had higher frequencies of symmetric disease onset (40.3% versus 19.7%; P < 0.001) and bulbar phenotype (38.5% versus 16.4%; P < 0.001). Our study suggests that motor phenotypes characterized by a predominant UMN involvement are associated with a vertical pattern of disease progression reflecting ipsilateral spreading within the motor cortex, while those with predominant LMN involvement display more frequently a horizontal spreading from one side of the spinal cord to the other. These observations raise the hypothesis that one of the mechanisms underlying disease spreading in ALS pathology is represented by diffusion of toxic factors in the neuron microenvironment. Finally, it is possible that in our cohort, regionally entrenching ALS forms are mainly observed in patients with atypical bulbar phenotypes, characterized by a slowly progressive course and relatively benign prognosis.

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Multi-omics profiling of CSF from spinal muscular atrophy type 3 patients after nusinersen treatment: a 2-year follow-up multicenter retrospective study.

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene resulting in reduced levels of the SMN protein. Nusinersen, the first antisense oligonucleotide (ASO) approved for SMA treatment, binds to the SMN2 gene, paralogue to SMN1, and mediates the translation of a functional SMN protein. Here, we used longitudinal high-resolution mass spectrometry (MS) to assess both global proteome and metabolome in cerebrospinal fluid (CSF) from ten SMA type 3 patients, with the aim of identifying novel readouts of pharmacodynamic/response to treatment and predictive markers of treatment response. Patients had a median age of 33.5 [29.5; 38.25] years, and 80% of them were ambulant at time of the enrolment, with a median HFMSE score of 37.5 [25.75; 50.75]. Untargeted CSF proteome and metabolome were measured using high-resolution MS (nLC-HRMS) on CSF samples obtained before treatment (T0) and after 2 years of follow-up (T22). A total of 26 proteins were found to be differentially expressed between T0 and T22 upon VSN normalization and LIMMA differential analysis, accounting for paired replica. Notably, key markers of the insulin-growth factor signaling pathway were upregulated after treatment together with selective modulation of key transcription regulators. Using CombiROC multimarker signature analysis, we suggest that detecting a reduction of SEMA6A and an increase of COL1A2 and GRIA4 might reflect therapeutic efficacy of nusinersen. Longitudinal metabolome profiling, analyzed with paired t-Test, showed a significant shift for some aminoacid utilization induced by treatment, whereas other metabolites were largely unchanged. Together, these data suggest perturbation upon nusinersen treatment still sustained after 22 months of follow-up and confirm the utility of CSF multi-omic profiling as pharmacodynamic biomarker for SMA type 3. Nonetheless, validation studies are needed to confirm this evidence in a larger sample size and to further dissect combined markers of response to treatment.

Combined RNA interference and gene replacement therapy targeting MFN2 as proof of principle for the treatment of Charcot-Marie-Tooth type 2A.

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.

Cognitive abnormalities in Becker muscular dystrophy: a mysterious link between dystrophin deficiency and executive functions.

BACKGROUND: Distrophinopathies are a heterogeneous group of neuromuscular disorders due to mutations in the DMD gene. Different isoforms of dystrophin are also expressed in the cerebral cortex and Purkinje cells. Despite cognitive abnormalities in Duchenne muscular dystrophy subjects that have been described in the literature, little is known about a comprehensive cognitive profile in Becker muscular dystrophy patients. AIM: The aim of this study was to assess cognitive functioning in Becker muscular dystrophy patients by using an extensive neuropsychological battery. Our hypothesis is that the most impaired functions are the highly intentional and conscious ones, such as working memory functions, which require a prolonged state of cellular activation. METHODS: We performed an extensive neuropsychological assessment on 28 Becker muscular dystrophy patients from 18 to 65 years old. As control subjects, we selected 20 patients with limb-girdle muscular dystrophy, whose clinical picture was similar except for cognitive integrity. The evaluation, although extended to all areas, was focused on prefrontal control skills, with a distinction between inhibitory processes of selective attention and activating processes of working memory. RESULTS AND CONCLUSIONS: Significant underperformances were found exclusively in the Dual Task and PASAT tests, to demonstrate a selective impairment of working memory that, while not causing intellectual disability, reduces the intellectual potential of patients with Becker muscular dystrophy.

Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the Survival of Motoneuron 1 gene (SMN1) and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a Caenorhabditis elegans SMA model showed less expression of the B-Raf homolog lin-45 Accordingly, motoneuron survival was rescued by a cell autonomous lin-45 expression in a C. elegans SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.

Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy.

Inclusion body myositis (IBM) is a slowly progressive disorder belonging to the idiopathic inflammatory myopathies, and it represents the most common adult-onset acquired myopathy. The main clinical features include proximal or distal muscular asymmetric weakness, with major involvement of long finger flexors and knee extensors. The main histological findings are the presence of fiber infiltrations, rimmed vacuoles, and amyloid inclusions. The etiopathogenesis is a challenge because both environmental and genetic factors are implicated in muscle degeneration and a distinction has been made previously between sporadic and hereditary forms. Here, we describe an Italian patient affected with a hereditary form of IBM with onset in his mid-forties. Next-generation sequencing analysis disclosed a heterozygous mutation c.76C>T (p.Pro26Ser) in the PDZ motif of the LDB3/ZASP gene, a mutation already described in a family with a late-onset myopathy and highly heterogenous degree of skeletal muscle weakness. In the proband's muscle biopsy, the expression of ZASP, myotilin, and desmin were increased. In our family, in addition to the earlier age of onset, the clinical picture is even more peculiar given the evidence, in one of the affected family members, of complete ophthalmoplegia in the vertical gaze. These findings help extend our knowledge of the clinical and genetic background associated with inclusion body myopathic disorders.

Ischemic optic neuropathy as first presentation in patient with m.3243 A > G MELAS classic mutation.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a systemic disorder in which multi-organ dysfunction may occur from mitochondrial metabolism failure. Maternally inherited mutations in the MT-TL1 gene are the most frequent causes for this disorder. Clinical manifestations may include stroke-like episodes, epilepsy, dementia, headache and myopathy. Among these, acute visual failure, usually in association with cortical blindness, can occur because of stroke-like episodes affecting the occipital cortex or the visual pathways. Vision loss due to optic neuropathy is otherwise considered a typical manifestation of other mitochondrial diseases such as Leber hereditary optic neuropathy (LHON). CASE PRESENTATION: Here we describe a 55-year-old woman, sister of a previously described patient with MELAS harbouring the m.3243A > G (p.0, MT-TL1) mutation, with otherwise unremarkable medical history, that presented with subacute, painful visual impairment of one eye, accompanied by proximal muscular pain and headache. Over the next weeks, she developed severe and progressive vision loss limited to one eye. Ocular examination confirmed unilateral swelling of the optic nerve head; fluorescein angiography showed segmental perfusion delay in the optic disc and papillary leakage. Neuroimaging, blood and CSF examination and temporal artery biopsy ruled out neuroinflammatory disorders and giant cell arteritis (GCA). Mitochondrial sequencing analysis confirmed the m.3243A > G transition, and excluded the three most common LHON mutations, as well as the m.3376G > A LHON/MELAS overlap syndrome mutation. Based on the constellation of clinical symptoms and signs presented in our patient, including the muscular involvement, and the results of the investigations, the diagnosis of optic neuropathy as a stroke-like event affecting the optic disc was performed. L-arginine and ubidecarenone therapies were started with the aim to improve stroke-like episode symptoms and prevention. The visual defect remained stable with no further progression or outbreak of new symptoms. CONCLUSIONS: Atypical clinical presentations must be always considered in mitochondrial disorders, even in well-described phenotypes and when mutational load in peripheral tissue is low. Mitotic segregation of mitochondrial DNA (mtDNA) does not allow to know the exact degree of heteroplasmy existent within different tissue, such as retina and optic nerve. Important therapeutic implications arise from a correct diagnosis of atypical presentation of mitochondrial disorders.

Cell-penetrating peptide-conjugated Morpholino rescues SMA in a symptomatic preclinical model.

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)2XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy.

Inhibition of myostatin and related signaling pathways for the treatment of muscle atrophy in motor neuron diseases.

Myostatin is a negative regulator of skeletal muscle growth secreted by skeletal myocytes. In the past years, myostatin inhibition sparked interest among the scientific community for its potential to enhance muscle growth and to reduce, or even prevent, muscle atrophy. These characteristics make it a promising target for the treatment of muscle atrophy in motor neuron diseases, namely, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), which are rare neurological diseases, whereby the degeneration of motor neurons leads to progressive muscle loss and paralysis. These diseases carry a huge burden of morbidity and mortality but, despite this unfavorable scenario, several therapeutic advancements have been made in the past years. Indeed, a number of different curative therapies for SMA have been approved, leading to a revolution in the life expectancy and outcomes of SMA patients. Similarly, tofersen, an antisense oligonucleotide, is now undergoing clinical trial phase for use in ALS patients carrying the SOD1 mutation. However, these therapies are not able to completely halt or reverse progression of muscle damage. Recently, a trial evaluating apitegromab, a myostatin inhibitor, in SMA patients was started, following positive results from preclinical studies. In this context, myostatin inhibition could represent a useful strategy to tackle motor symptoms in these patients. The aim of this review is to describe the myostatin pathway and its role in motor neuron diseases, and to summarize and critically discuss preclinical and clinical studies of myostatin inhibitors in SMA and ALS. Then, we will highlight promises and pitfalls related to the use of myostatin inhibitors in the human setting, to aid the scientific community in the development of future clinical trials.

Insights into the identification of a molecular signature for amyotrophic lateral sclerosis exploiting integrated microRNA profiling of iPSC-derived motor neurons and exosomes.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by progressive degeneration of motor neurons (MNs). Most cases are sporadic, whereas 10% are familial. The pathological mechanisms underlying the disease are partially understood, but it is increasingly being recognized that alterations in RNA metabolism and deregulation of microRNA (miRNA) expression occur in ALS. In this study, we performed miRNA expression profile analysis of iPSC-derived MNs and related exosomes from familial patients and healthy subjects. We identified dysregulation of miR-34a, miR-335 and miR-625-3p expression in both MNs and exosomes. These miRNAs regulate genes and pathways which correlate with disease pathogenesis, suggesting that studying miRNAs deregulation can contribute to deeply investigate the molecular mechanisms underlying the disease. We also assayed the expression profile of these miRNAs in the cerebrospinal fluid (CSF) of familial (fALS) and sporadic patients (sALS) and we identified a significant dysregulation of miR-34a-3p and miR-625-3p levels in ALS compared to controls. Taken together, all these findings suggest that miRNA analysis simultaneously performed in different human biological samples could represent a promising molecular tool to understand the etiopathogenesis of ALS and to develop new potential miRNA-based strategies in this new propitious therapeutic era.

Advancing Stroke Research on Cerebral Thrombi with Omic Technologies.

Cerebrovascular diseases represent a leading cause of disability, morbidity, and death worldwide. In the last decade, the advances in endovascular procedures have not only improved acute ischemic stroke care but also conceded a thorough analysis of patients' thrombi. Although early anatomopathological and immunohistochemical analyses have provided valuable insights into thrombus composition and its correlation with radiological features, response to reperfusion therapies, and stroke etiology, these results have been inconclusive so far. Recent studies applied single- or multi-omic approaches-such as proteomics, metabolomics, transcriptomics, or a combination of these-to investigate clot composition and stroke mechanisms, showing high predictive power. Particularly, one pilot studies showed that combined deep phenotyping of stroke thrombi may be superior to classic clinical predictors in defining stroke mechanisms. Small sample sizes, varying methodologies, and lack of adjustments for potential confounders still represent roadblocks to generalizing these findings. However, these techniques hold the potential to better investigate stroke-related thrombogenesis and select secondary prevention strategies, and to prompt the discovery of novel biomarkers and therapeutic targets. In this review, we summarize the most recent findings, overview current strengths and limitations, and present future perspectives in the field.

The Profiling of 179 miRNA Expression in Serum from Limb Girdle Muscular Dystrophy Patients and Healthy Controls.

Limb girdle muscular dystrophies (LGMDs) are a group of genetically inherited neuromuscular diseases with a very variable clinical presentation and overlapping traits. Over the last few years there has been an increasing interest in the use of non-invasive circulating biomarkers to monitor disease progression and to evaluate the efficacy of therapeutic approaches. Our aim was to identify the miRNA signature with potential value for LGMD patient screening and stratification. Using miRCURY LNA miRNA qPCR Serum/Plasma Panel, we analyzed 179 miRNAs from 16 patients, divided in four pools based on their genetic diagnosis, and from healthy controls. The miRNAs analysis showed a total of 107 dysregulated miRNAs in LGMD patients when compared to the healthy controls. After filtering via skeletal tissue expression and gene/pathways target analysis, the number of dysregulated miRNAs drastically reduced. Six selected miRNAs-let-7f-5p (in LGMDR1), miR-20a-5p (in LGMDR2), miR-130b-5p, miR-378a-5p (both in LGMDR3), miR-376c-3p and miR-382-5p (both in LGMDR4)-whose expression was significantly lower compared to controls in the different LGMD pools, were further investigated. The bioinformatic analysis of the target genes in each selected miRNA revealed ECM-receptor interaction and TGF-beta signaling as the most involved pathways. The correlation analysis showed a good correlation of let-7f-5p with fibrosis and with the cross sectional area of type I and type II fibers, while miR-130b-5p showed a good correlation with the age of onset of the disease. The receiver operating characteristic curves showed how single miRNAs were able to discriminate a specific group of LGMD patients and how the combination of six miRNAs was able to discriminate LGMD patients from controls.

Extracellular Matrix Disorganization and Sarcolemmal Alterations in COL6-Related Myopathy Patients with New Variants of COL6 Genes.

Collagen VI is a heterotrimeric protein expressed in several tissues and involved in the maintenance of cell integrity. It localizes at the cell surface, creating a microfilamentous network that links the cytoskeleton to the extracellular matrix. The heterotrimer consists of three chains encoded by COL6A1, COL6A2 and COL6A3 genes. Recessive and dominant molecular defects cause two main disorders, the severe Ullrich congenital muscular dystrophy and the relatively mild and slowly progressive Bethlem myopathy. We analyzed the clinical aspects, pathological features and mutational spectrum of 15 COL6-mutated patients belonging to our cohort of muscular dystrophy probands. Patients presented a heterogeneous phenotype ranging from severe forms to mild adult-onset presentations. Molecular analysis by NGS detected 14 different pathogenic variants, three of them so far unreported. Two changes, localized in the triple-helical domain of COL6A1, were associated with a more severe phenotype. Histological, immunological and ultrastructural techniques were employed for the validation of the genetic variants; they documented the high variability in COL6 distribution and the extracellular matrix disorganization, highlighting the clinical heterogeneity of our cohort. The combined use of these different technologies is pivotal in the diagnosis of COL6 patients.

MicroRNAs as serum biomarkers in Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is an X-linked neuromuscular disorder due to mutation in the DMD gene, encoding dystrophin. Despite a wide clinical variability, BMD is characterized by progressive muscle degeneration and proximal muscle weakness. Interestingly, a dysregulated expression of muscle-specific microRNAs (miRNAs), called myomirs, has been found in patients affected with muscular dystrophies, although few studies have been conducted in BMD. We analysed the serum expression levels of a subset of myomirs in a cohort of 29 ambulant individuals affected by BMD and further classified according to the degree of alterations at muscle biopsy and in 11 age-matched healthy controls. We found a significant upregulation of serum miR-1, miR-133a, miR-133b and miR-206 in our cohort of BMD patients, supporting the role of these miRNAs in the pathophysiology of the disease, and we identified serum cut-off levels discriminating patients from healthy controls, confiming the potential of circulating miRNAs as promising noninvasive biomarkers. Moreover, serum levels of miR-133b were found to be associated with fibrosis at muscle biopsy and with patients' motor performances, suggesting that miR-133b might be a useful prognostic marker for BMD patients. Taken together, our data showed that these serum myomirs may represent an effective tool that may support stratification of BMD patients, providing the opportunity of both monitoring disease progression and assessing the treatment efficacy in the context of clinical trials.

A Novel Homozygous VPS11 Variant May Cause Generalized Dystonia.

In this work, we describe the association of a novel homozygous VPS11 variant with adult-onset generalized dystonia, providing a detailed clinical report and biological evidence of disease mechanism. Vps11 is a subunit of the homotypic fusion and protein sorting (HOPS) complex, which promotes the fusion of late endosomes and autophagosomes with the lysosome. Functional studies on mutated fibroblasts showed marked lysosomal and autophagic abnormalities, which improved after overexpression of the wild type Vps11 protein. In conclusion, a deleterious VPS11 variant, damaging the autophagic and lysosomal pathways, is the probable genetic cause of a novel form of generalized dystonia. ANN NEUROL 2021;89:834-839.