Is spinal muscular atrophy a disease of the motor neurons only: pathogenesis and therapeutic implications?

Spinal muscular atrophy (SMA) is a genetic neurological disease that causes infant mortality; no effective therapies are currently available. SMA is due to homozygous mutations and/or deletions in the survival motor neuron 1 gene and subsequent reduction of the SMN protein, leading to the death of motor neurons. However, there is increasing evidence that in addition to motor neurons, other cell types are contributing to SMA pathology. In this review, we will discuss the involvement of non-motor neuronal cells, located both inside and outside the central nervous system, in disease onset and progression. Even if SMN restoration in motor neurons is needed, it has been shown that optimal phenotypic amelioration in animal models of SMA requires a more widespread SMN correction. It has been demonstrated that non-motor neuronal cells are also involved in disease pathogenesis and could have important therapeutic implications. For these reasons it will be crucial to take this evidence into account for the clinical translation of the novel therapeutic approaches

Neurofascin (NFASC) gene mutation causes autosomal recessive ataxia with demyelinating neuropathy

Introduction: Neurofascin, encoded by NFASC, is a transmembrane protein that plays an essential role in nervous system development and node of Ranvier function. Anti-Neurofascin autoantibodies cause a specific type of chronic inflammatory demyelinating polyneuropathy (CIDP) often characterized by cerebellar ataxia and tremor. Recently, homozygous NFASC mutations were recently associated with a neurodevelopmental disorder in two families. Methods: A combined approach of linkage analysis and whole-exome sequencing was performed to find the genetic cause of early-onset cerebellar ataxia and demyelinating neuropathy in two siblings from a consanguineous Italian family. Functional studies were conducted on neurons from induced pluripotent stem cells (iPSCs) generated from the patients. Results: Genetic analysis revealed a homozygous p.V1122E mutation in NFASC. This mutation, affecting a highly conserved hydrophobic transmembrane domain residue, led to significant loss of Neurofascin protein in the iPSC-derived neurons of affected siblings. Conclusions: The identification of NFASC mutations paves the way for genetic research in the developing field of nodopathies, an emerging pathological entity involving the nodes of Ranvier, which are associated for the first time with a hereditary ataxia syndrome with neuropathy

Gene therapy rescues disease phenotype in a spinal muscular atrophy with respiratory distress type 1 (SMARD1) mouse model

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease affecting children. It is caused by mutations in the IGHMBP2 gene (11q13) and presently has no cure. Recently, adeno-associated virus serotype 9 (AAV9)-mediated gene therapy has been shown to rescue the phenotype of animal models of another lower motor neuron disorder, spinal muscular atrophy 5q, and a clinical trial with this strategy is ongoing. We report rescue of the disease phenotype in a SMARD1 mouse model after therapeutic delivery via systemic injection of an AAV9 construct encoding the wild-type IGHMBP2 to replace the defective gene. AAV9-IGHMBP2 administration restored protein levels and rescued motor function, neuromuscular physiology, and life span (450% increase), ameliorating pathological features in the central nervous system, muscles, and heart. To test this strategy in a human model, we transferred wild-type IGHMBP2 into human SMARD1-induced pluripotent stem cell-derived motor neurons; these cells exhibited increased survival and axonal length in long-term culture. Our data support the translational potential of AAV-mediated gene therapies for SMARD1, opening the door for AAV9-mediated therapy in human clinical trials

DEVELOPMENT OF 3D IN VITRO MODEL TO STUDY MOLECULAR MECHANISMS OF SPINAL MUSCULAR ATROPHY

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease and the leading genetic cause of death during childhood. SMA is caused in the majority of the cases (up to 95%) by mutations in the Survival Motor Neuron 1 (SMN1) gene coding for the SMN protein, resulting in a progressive muscular paralysis due to lower motor neurons degeneration. A deeper knowledge of SMN biology and of its role in different organs/system in reliable models is very important for the optimization of available treatments and the development of complementary therapeutic approaches. In particular, it can be crucial to generate a human model able to recapitulate the complexity of the central nervous system (CNS) and its development. A promising tool to study SMA pathology is the three-dimensional (3D) organoid, obtained starting from induced pluripotent stem cell (iPSCs). Moreover, this model could be used to identify new therapeutic strategy. Rationale: In this project, we exploited CNS organoid technology, which is a novel stem cell-based 3D platform that has the potential to address the limitations of human existing bi-dimensional (2D) cultures improving preclinical testing. We aim to demonstrate that this approach can recapitulate some of the complexity of whole-organism biology overcoming the conventional use of 2D iPSCs-derived motor neurons. Nowadays, several therapeutic strategies have been tested in clinical trials and two compounds have been approved by FDA. Nevertheless, all these approaches are completely efficacious only if administered at pre-symptomatic stages. The generation of a new model for SMA could lead to a better knowledge of the mechanisms underlying the disorder during the development of the CNS and it might contribute to develop new or combined therapeutic options for affected patients. Methods: We obtained iPSCs from human fibroblasts of both healthy subjects and SMA type 1 patients and, using two different protocols that recapitulate the embryonal developmental steps, we generated 3D brain organoids and 3D spinal cord-like spheroids. We performed immunohistochemical and molecular analysis to confirm their differentiation state. Moreover, to verify their basal activity and their capability to response to stimuli, we performed calcium imaging and electrophysiological analysis. Results: CNS organoids derived from healthy subjects and patient have been successfully obtained, as suggested by the protein and gene expression data. In particular, brain organoids gave rise to an early cerebral cortex-like formation containing progenitor cells and more mature neural subtypes. Electrophysiological analysis demonstrated not only their basal activity, but also their ability to respond to stimuli. Concerning spinal cord-like spheroids, we used a modified protocol in order to induce neural caudalization and ventralization. This model gave us a powerful tool to investigate early motor neuron pathology and causes of degeneration. Like brain organoids, spinal cord-like spheroids have been characterized by immunohistochemistry, gene expression analysis and electrophysiological activity. Preliminary results suggested that SMA organoids and spheroids, compared to the controls, exhibited not only an alteration in the proper markers expression, but also in the electrophysiological activity. Conclusion: We successfully generated and characterized healthy and SMA CNS 3D organoids and spheroids that recapitulated human CNS development, showed disease-related features. This model can be used as an innovative in vitro system to study pathogenic mechanisms, identifying therapeutic targets and test potential therapeutic strategies

The wide spectrum of clinical phenotypes of spinal muscular atrophy with respiratory distress type 1: a systematic review

Spinal muscular atrophy with respiratory distress type 1 (SMARD1), also known as distal spinal-muscular atrophy 1 (DSMA10), is an autosomal recessive type of spinal muscular atrophy that is related to mutations in the IGHMBP2 gene, which encodes for the immunoglobulin \u3bc-binding protein. SMARD1 patients usually present low birth weight, diaphragmatic palsy and distal muscular atrophy. Clinical features are still the most important factor that leads to the diagnosis of SMARD1, due to the fact that IGHMBP2 gene mutations are characterized by significant phenotypic heterogeneity. In the present review, we will systematically discuss the genetic, clinical and neuropathological features of SMARD1 in order to provide a complete overview of SMARD1 variable clinical presentations and of the most important diagnostic tools which can be used to identify and properly manage affected individuals. This background is crucial also in the perspective of the development of novel therapeutic strategies for this still orphan disorder

Peptide-conjugated Morpholino Oligomers for treatment of Spinal Muscular Atrophy

The use of Antisense oligonucleotide(ASO) represents a promising treatment for Spinal Muscular Atrophy (SMA) by its ability to increase the production of a functional SMN protein and rescue the phenotype in SMA animal models. However, there are several hurdles to overcome. To increase the cellular and tissue uptake and pharmacological profile of Morpholino Oligomers (MO), an ASO variants, one possible strategy is the conjugation with cell-penetrating peptides (CPPs). In this study we investigated the efficacy of different CPPs linked to our validated MO sequence (Tat, R6, r6 and (RXRRBR)2XB) and of novel MOs sequences in in vitro and in vivo SMA models. In vitro, we nucleofected induced pluripotent stem cells (iPSCs) derived from SMA patients with the four MOs. The treatment with MO B and D, and in particular their combination, showed a consistent increase of SMN protein levels and a significant upregulation of SMN GEMS in the cell nuclei. The same increment was obtained in vivo in the SMA\uf0447 mouse model. Moreover, we administered our alreadyvalidated MO sequence (MO-10-34) conjugated with four CPPsin a small pilot group of pre-symptomatic SMA mice, using the protocol already established for unconjugated MO (Nizzardo et al., 2014).The best conjugated was selected for next studies in presymptomatic and symptomatic SMA mice to assess its therapeutic potential. We will assess the feasibility of this strategy to: 1) cross the blood brain barrier, allowing MO non-invasive systemic delivery, and 2) treat the disease in a symptomatic phase, expanding the therapeutic window

Gene therapy rescues disease phenotype in a spinal muscular atrophy with respiratory distress type 1 (SMARD1) mouse model

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease affecting children that is caused by mutations in the IGHMBP2 gene (11q13) and lacks a cure. Recently, adeno-associated virus serotype 9 (AAV9)-mediated gene therapy rescued the phenotype of animal models of another lower motor neuron disorder, spinal muscular atrophy 5q, and a clinical trial with this strategy is ongoing. In this study, we report rescue of the disease phenotype in a SMARD1 mouse model following therapeutic delivery of an AAV9 construct encoding the wild-type IGHMBP2 via systemic injection to replace the defective gene. AAV9-IGHMBP2 administration restored protein levels and rescued motor function, neuromuscular physiology, and lifespan (450% increase), ameliorating pathological features in the CNS, muscles, and heart. To test this strategy in a human model, we transferred wild-type IGHMBP2 into human SMARD1 induced pluripotent stem cell-derived motor neurons; these cells exhibited increased survival and axonal length in long-term culture. Our data support the translational potential of AAV-mediated gene therapies for SMARD1, opening the door for AAV9-mediated therapy in human clinical trials