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.

Generation of two hiPSC lines (UMILi027-A and UMILi028-A) from early and late-onset Congenital Central hypoventilation Syndrome (CCHS) patients carrying a polyalanine expansion mutation in the PHOX2B gene.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare disorder of the autonomic nervous system (ANS), characterized by inadequate control of autonomic ventilation and global autonomic dysfunction. Heterozygous polyalanine repeat expansion mutations in exon 3 of the transcription factor Paired-like homeobox 2B (PHOX2B) gene occur in 90% of CCHS cases. In this study, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from female CCHS patients carrying a heterozygous + 5 alanine expansion mutation. The generated iPSC lines show a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Stathmins and Motor Neuron Diseases: Pathophysiology and Therapeutic Targets.

Motor neuron diseases (MNDs) are a group of fatal, neurodegenerative disorders with different etiology, clinical course and presentation, caused by the loss of upper and lower motor neurons (MNs). MNs are highly specialized cells equipped with long, axonal processes; axonal defects are some of the main players underlying the pathogenesis of these disorders. Microtubules are key components of the neuronal cytoskeleton characterized by dynamic instability, switching between rapid polymerization and shrinkage. Proteins of the stathmin family affect microtubule dynamics regulating the assembly and the dismantling of tubulin. Stathmin-2 (STMN2) is one of the most abundantly expressed genes in MNs. Following axonal injury, STMN2 expression is upregulated, and the protein is transported toward the growth cones of regenerating axons. STMN2 has a critical role in axonal maintenance, and its dysregulation plays an important role in neurodegenerative processes. Stathmin-1 (STMN1) is a ubiquitous protein that is highly expressed during the development of the nervous system, and its phosphorylation controls microtubule dynamics. In the present review, we summarize what is currently known about the involvement of stathmin alterations in MNDs and the potential therapeutic effect of their modulation, with a specific focus on the most common forms of MND, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).

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.

Targeting PTB for Glia-to-Neuron Reprogramming In Vitro and In Vivo for Therapeutic Development in Neurological Diseases.

In vivo cell reprogramming of glial cells offers a promising way to generate new neurons in the adult mammalian nervous system. This approach might compensate for neuronal loss occurring in neurological disorders, but clinically viable tools are needed to advance this strategy from bench to bedside. Recently published work has described the successful neuronal conversion of glial cells through the repression of a single gene, polypyrimidine tract-binding protein 1 (Ptbp1), which encodes a key RNA-binding protein. Newly converted neurons not only express correct markers but they also functionally integrate into endogenous brain circuits and modify disease symptoms in in vivo models of neurodegenerative diseases. However, doubts about the nature of "converted" neurons, in particular in vivo, have been raised, based on concerns about tracking reporter genes in converted cells. More robust lineage tracing is needed to draw definitive conclusions about the reliability of this strategy. In vivo reprogramming and the possibility of implementing it with approaches that could be translated into the clinic with antisense oligonucleotides targeting a single gene like Ptbp1 are hot topics. They warrant further investigation with stringent methods and criteria of evaluation for the ultimate treatment of neurological diseases.

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.

Dysregulation of Muscle-Specific MicroRNAs as Common Pathogenic Feature Associated with Muscle Atrophy in ALS, SMA and SBMA: Evidence from Animal Models and Human Patients.

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.

Systematic elucidation of neuron-astrocyte interaction in models of amyotrophic lateral sclerosis using multi-modal integrated bioinformatics workflow.

Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Networks (SEARCHIN) to identify ligand-mediated interactions between distinct cellular compartments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication mechanisms.

Animal Models of CMT2A: State-of-art and Therapeutic Implications.

Charcot-Marie-Tooth disease type 2A (CMT2A), arising from mitofusin 2 (MFN2) gene mutations, is the most common inherited axonal neuropathy affecting motor and sensory neurons. The cellular and molecular mechanisms by which MFN2 mutations determine neuronal degeneration are largely unclear. No effective treatment exists for CMT2A, which has a high degree of genetic/phenotypic heterogeneity. The identification of mutations in MFN2 has allowed the generation of diverse transgenic animal models, but to date, their ability to recapitulate the CMT2A phenotype is limited, precluding elucidation of its pathogenesis and discovery of therapeutic strategies. This review will critically present recent progress in in vivo CMT2A disease modeling, discoveries, drawbacks and limitations, current challenges, and key reflections to advance the field towards developing effective therapies for these patients.

Neural Stem Cell Transplantation for Neurodegenerative Diseases.

Neurodegenerative diseases are disabling and fatal neurological disorders that currently lack effective treatment. Neural stem cell (NSC) transplantation has been studied as a potential therapeutic approach and appears to exert a beneficial effect against neurodegeneration via different mechanisms, such as the production of neurotrophic factors, decreased neuroinflammation, enhanced neuronal plasticity and cell replacement. Thus, NSC transplantation may represent an effective therapeutic strategy. To exploit NSCs' potential, some of their essential biological characteristics must be thoroughly investigated, including the specific markers for NSC subpopulations, to allow profiling and selection. Another key feature is their secretome, which is responsible for the regulation of intercellular communication, neuroprotection, and immunomodulation. In addition, NSCs must properly migrate into the central nervous system (CNS) and integrate into host neuronal circuits, enhancing neuroplasticity. Understanding and modulating these aspects can allow us to further exploit the therapeutic potential of NSCs. Recent progress in gene editing and cellular engineering techniques has opened up the possibility of modifying NSCs to express select candidate molecules to further enhance their therapeutic effects. This review summarizes current knowledge regarding these aspects, promoting the development of stem cell therapies that could be applied safely and effectively in clinical settings.

miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

TDP-43 promotes the formation of neuromuscular synapses through the regulation of Disc-large expression in Drosophila skeletal muscles.

BACKGROUND: The ribonuclear protein TDP-43 has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), with genetic mutations being linked to the neurological symptoms of the disease. Though alterations in the intracellular distribution of TDP-43 have been observed in skeletal muscles of patients suffering from ALS, it is not clear whether such modifications play an active role in the disease or merely represent an expression of muscle homeostatic mechanisms. Also, the molecular and metabolic pathways regulated by TDP-43 in the skeletal muscle remain largely unknown. Here, we analyze the function of TBPH, the Drosophila melanogaster ortholog of TDP-43, in skeletal muscles. RESULTS: We modulated the activity of TDP-43 in Drosophila muscles by means of RNA interference and observed that it is required to promote the formation and growth of neuromuscular synapses. TDP-43 regulated the expression levels of Disc-large (Dlg), and restoring Dlg expression either in skeletal muscles or in motoneurons was sufficient to suppress the locomotive and synaptic defects of TDP-43-null flies. These results were validated by the observation of a decrease in Dlg levels in human neuroblastoma cells and iPSC-differentiated motoneurons derived from ALS patients, suggesting similar mechanisms may potentially be involved in the pathophysiology of the disease. CONCLUSIONS: Our results help to unveil the physiological role of TDP-43 in skeletal muscles as well as the mechanisms responsible for the autonomous and non-autonomous behavior of this protein concerning the organization of neuromuscular synapses.

Current understanding of and emerging treatment options for spinal muscular atrophy with respiratory distress type 1 (SMARD1).

Spinal muscular atrophy (SMA) with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease that is characterized by distal and proximal muscle weakness and diaphragmatic palsy that leads to respiratory distress. Without intervention, infants with the severe form of the disease die before 2 years of age. SMARD1 is caused by mutations in the IGHMBP2 gene that determine a deficiency in the encoded IGHMBP2 protein, which plays a critical role in motor neuron survival because of its functions in mRNA processing and maturation. Although it is rare, SMARD1 is the second most common motor neuron disease of infancy, and currently, treatment is primarily supportive. No effective therapy is available for this devastating disease, although multidisciplinary care has been an essential element of the improved quality of life and life span extension in these patients in recent years. The objectives of this review are to discuss the current understanding of SMARD1 through a summary of the presently known information regarding its clinical presentation and pathogenesis and to discuss emerging therapeutic approaches. Advances in clinical care management have significantly extended the lives of individuals affected by SMARD1 and research into the molecular mechanisms that lead to the disease has identified potential strategies for intervention that target the underlying causes of SMARD1. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to halt or possibly prevent neurodegenerative disease in SMARD1 patients. The recent approval of the first gene therapy approach for SMA associated with mutations in the SMN1 gene may be a turning point for the application of this strategy for SMARD1 and other genetic neurological diseases.

Spinal muscular atrophy with respiratory distress type 1: Clinical phenotypes, molecular pathogenesis and therapeutic insights.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare autosomal recessive neuromuscular disorder caused by mutations in the IGHMBP2 gene, which encodes immunoglobulin µ-binding protein 2, leading to progressive spinal motor neuron degeneration. We review the data available in the literature about SMARD1. The vast majority of patients show an onset of typical symptoms in the first year of life. The main clinical features are distal muscular atrophy and diaphragmatic palsy, for which permanent supportive ventilation is required. No effective treatment is available yet, but novel therapeutic approaches, such as gene therapy, have shown encouraging results in preclinical settings and thus represent possible methods for treating SMARD1. Significant advancements in the understanding of both the SMARD1 clinical spectrum and its molecular mechanisms have allowed the rapid translation of preclinical therapeutic strategies to human patients to improve the poor prognosis of this devastating disease.

CSF transplantation of a specific iPSC-derived neural stem cell subpopulation ameliorates the disease phenotype in a mouse model of spinal muscular atrophy with respiratory distress type 1.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a genetic motor neuron disease affecting infants. This condition is caused by mutations in the IGHMBP2 gene and currently has no cure. Stem cell transplantation is a potential therapeutic strategy for motor neuron diseases such as SMARD1, exerting beneficial effects both by replacing cells and by providing support to endogenous motor neurons. In this work, we demonstrate that human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) selected for the expression of specific markers, namely, Lewis X, CXCR4 and beta 1 integrin, and pretreated with neurotrophic factors and apoptosis/necroptosis inhibitors were able to effectively migrate and engraft into the host parenchyma after administration into the cerebrospinal fluid in a SMARD1 mouse model. We were able to detect donor cells in the ventral horn of the spinal cord and observe improvements in neuropathological features, particularly preservation of the integrity of the motor unit, that were correlated with amelioration of the SMARD1 disease phenotype in terms of neuromuscular function and lifespan. This minimally invasive stem cell approach can confer major advantages in the context of cell-mediated therapy for patients with neurodegenerative diseases.

Key role of SMN/SYNCRIP and RNA-Motif 7 in spinal muscular atrophy: RNA-Seq and motif analysis of human motor neurons.

Spinal muscular atrophy is a motor neuron disorder caused by mutations in SMN1. The reasons for the selective vulnerability of motor neurons linked to SMN (encoded by SMN1) reduction remain unclear. Therefore, we performed deep RNA sequencing on human spinal muscular atrophy motor neurons to detect specific altered gene splicing/expression and to identify the presence of a common sequence motif in these genes. Many deregulated genes, such as the neurexin and synaptotagmin families, are implicated in critical motor neuron functions. Motif-enrichment analyses of differentially expressed/spliced genes, including neurexin2 (NRXN2), revealed a common motif, motif 7, which is a target of SYNCRIP. Interestingly, SYNCRIP interacts only with full-length SMN, binding and modulating several motor neuron transcripts, including SMN itself. SYNCRIP overexpression rescued spinal muscular atrophy motor neurons, due to the subsequent increase in SMN and their downstream target NRXN2 through a positive loop mechanism and ameliorated SMN-loss-related pathological phenotypes in Caenorhabditis elegans and mouse models. SMN/SYNCRIP complex through motif 7 may account for selective motor neuron degeneration and represent a potential therapeutic target.

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

MicroRNA expression analysis identifies a subset of downregulated miRNAs in ALS motor neuron progenitors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder that is characterized by a progressive degeneration of motor neurons (MNs). The pathomechanism underlying the disease is largely unknown, even though increasing evidence suggests that RNA metabolism, including microRNAs (miRNAs) may play an important role. In this study, human ALS induced pluripotent stem cells were differentiated into MN progenitors and their miRNA expression profiles were compared to those of healthy control cells. We identified 15 downregulated miRNAs in patients' cells. Gene ontology and molecular pathway enrichment analysis indicated that the predicted target genes of the differentially expressed miRNAs were involved in neurodegeneration-related pathways. Among the 15 examined miRNAs, miR-34a and miR504 appeared particularly relevant due to their involvement in the p53 pathway, synaptic vesicle regulation and general involvement in neurodegenerative diseases. Taken together our results demonstrate that the neurodegenerative phenotype in ALS can be associated with a dysregulation of miRNAs involved in the control of disease-relevant genetic pathways, suggesting that targeting entire gene networks can be a potential strategy to treat complex diseases such as ALS.

Investigation of New Morpholino Oligomers to Increase Survival Motor Neuron Protein Levels in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2. Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.