TRANSPLANTATION OF A SPECIFIC NEURAL STEM CELL SUBPOPULATION AS A THERAPEUTIC APPROACH FOR AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neuromuscular disorder caused by degeneration of motor neurons (MNs) in the spinal cord, brainstem, and cortex. It belongs to a group of heterogeneous disorders called \u201cmotor neuron diseases\u201d, in which ALS is the most common form in adults. The progressive MN degeneration leads to a gradual muscle atrophy and paralysis. Patients affected by ALS usually die 3-5 years after the onset of symptoms due to respiratory failure. Up to now, no effective cure is available for ALS beyond supportive care and Riluzole, which only modestly prolongs Survival. In the early stage of the disease, MN loss and consequent muscle denervation are compensated by axonal sprouting and reinnervation by the remaining MNs, but this mechanism is insufficient in the long term. Thanks to their multiple beneficial mechanisms, stem cell transplantation represents a promising therapeutic strategy for ALS and other neurodegenerative disorders. In fact, transplanted stem cells can provide therapeutic effect by modulating the micro-environment through the production of neurotrophic factors, eliminating toxic molecules, reducing neuroinflammation and generating auxiliary neural networks. Moreover, stem cells can eventually replace degenerating cells. A novel source for stem cell transplantation consists in the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs). Since iPSCs are directly derived from adult tissues, they bypass ethical issue of the embryo manipulation and are patient-specific, potentially reproducing ALS features in vitro. This means that they are a promising tool for stem cells transplantation and to model human pathologies in vitro. In this study, we isolated a specific subpopulation of neural stem cells (NSCs) derived from differentiated iPSCs. Compared to other types of stem cell, NSCs are particularly appropriate for ALS treatment due to their peculiar ability to differentiate into neurons, astrocytes and oligodendrocytes. The rationale of this study consists in the selection of a subpopulation of NSCs able to engraft and migrate through the nervous system parenchyma, to protect degenerating MNs and to improve ALS phenotype. We selected NSCs for the presence of three markers: Lewis X (or LeX), CXCR4 e \u3b21 integrin. Lewis X is a glycoprotein marker of stem cells with a relevant role in cell adhesion and migration. CXCR4 is a chemokine receptor, which increases the sensitivity of the cells to be recruited by the host spinal cord that produces chemoattractant cytokines. \u3b21 integrin is a subunit of VLA4, a receptor that allows cells to cross the blood-brain barrier, particularly in the presence of inflammation as in ALS animal models and human patients. In order to evaluate the ability of LeX+CXCR4+\u3b21+ NSCs to engraft into the nervous system and to improve ALS phenotype, we performed intrathecal injection of these cells in the SOD1G93A mouse model. Transplantation resulted in an efficient engraftment of the cells, which reached central nervous system bypassing blood brain barrier, and in the protection of MNs and their axons from degeneration. This determined a preservation of neuromuscular junction (NMJ) innervations by maintaining their integrity and inducing axonal sprouting. These beneficial effects on neuropathological phenotype correlated with a significant increased survival and improved neuromuscular function of transplanted SOD1G93A mice. We also demonstrated the beneficial effects of LeX+CXCR4+\u3b21+ NSCs in a human in vitro model of ALS. When co-cultured with these cells, iPSC-derived MNs from ALS patients showed an improvement in terms of survival and axonal growth. We then analyzed the molecular mechanisms underlying NSC protection demonstrating that our NSC subpopulation exerted positive effects through neurotrophic factors production, inhibition of the GSK3\u3b2 activity, and limiting astrocytes proliferation through activation of vanilloid receptor. The results of this study suggest that effective protection of MNs and NMJs can be achieved targeting multiple deregulated cellular and molecular mechanisms in both MNs and glial cells in ALS models. This is particularly relevant for ALS because different pathological mechanisms likely contribute to its onset, making NSC transplantation a promising therapeutic approach for ALS

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

IPSC-derived neural stem cells act via kinase inhibition to exert neuroprotective effects in spinal muscular atrophy with respiratory distress type 1

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a motor neuron disease caused by mutations in the IGHMBP2 gene, without a cure. Here, we demonstrate that neural stem cells (NSCs) from human-induced pluripotent stem cells (iPSCs) have therapeutic potential in the context of SMARD1. We show that upon transplantation NSCs can appropriately engraft and differentiate in the spinal cord of SMARD1 animals, ameliorating their phenotype, by protecting their endogenous motor neurons. To evaluate the effect of NSCs in the context of human disease, we generated human SMARD1-iPSCs motor neurons that had a significantly reduced survival and axon length. Notably, the coculture with NSCs ameliorate these disease features, an effect attributable to the production of neurotrophic factors and their dual inhibition of GSK-3 and HGK kinases. Our data support the role of iPSC as SMARD1 disease model and their translational potential for therapies in motor neuron disorders

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

Induced neural stem cells: methods of reprogramming and potential therapeutic applications

Developmental studies and experimental data have enabled us to assert that the terminal cell differentiation state is reversible, and that altering the balance of specific transcription factors could be a powerful strategy for inducing pluripotency. Due to the risks related to using induced pluripotent cells in clinical applications, biologists are now striving to develop methods to induce a committed differentiated cell type by direct conversion of another cell line. Several reprogramming factors have been discovered, and some cellular phenotypes have been obtained by novel transdifferentiation processes. It has been recently demonstrated that induced neural stem cells (iNSCs) can be obtained from rodent and human somatic cells, like fibroblasts, through the forced expression of defined transcription factors. To date, two different approaches have been successfully used to obtain iNSCs: a direct method and an indirect method that involves an intermediate destabilized state. The possibility to induce characterized iNSCs from human cells, e.g. fibroblasts, has opened new horizons for research in human disease modelling and cellular therapeutic applications in the neurological field. This review focuses on reported reprogramming techniques and innovative techniques that can be further explored in this area, as well as on the criteria for the phenotypic characterization of iNSCs and their use in developing novel therapeutic strategies for neurological diseases

iPSC-derived LewisX+CXCR4+β1-integrin+ neural stem cells improve the amyotrophic lateral sclerosis phenotype by preserving motor neurons and muscle innervation in human and rodent models

Amyotrophic lateral sclerosis (ALS) is a fatal incurable neurodegenerative disease characterized by progressive degeneration of motor neurons (MNs), leading to relentless muscle paralysis. In the early stage of the disease, MN loss and consequent muscle denervation are compensated by axonal sprouting and reinnervation by the remaining MNs, but this mechanism is insufficient in the long term. Here, we demonstrate that induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs), in particular the subpopulation positive for LewisX-CXCR4-\u3b21-integrin, enhance neuronal survival and axonal growth of human ALS-derived MNs co-cultured with toxic ALS astrocytes, acting on both autonomous and non-autonomous ALS disease features. Transplantation of this NSC fraction into transgenic SOD1G93A ALS mice protects MNs in vivo, promoting their ability to maintain neuromuscular junction integrity, inducing novel axonal sprouting and reducing macro- and microgliosis. These effects result in a significant increase in survival and an improved neuromuscular phenotype in transplanted SOD1G93A mice. Our findings suggest that effective protection of MN functional innervation can be achieved by modulation of multiple dysregulated cellular and molecular pathways in both MNs and glial cells. These pathways must be considered in designing therapeutic strategies for ALS patients

Clinical evaluation and cellular electrophysiology of a recessive CLCN1 patient

Here we present the case of a 32-year-old female patient with myotonia congenita. She carried two mutations in the CLCN1 gene that encodes the chloride channel ClC-1: p.Phe167Leu, which was previously identified in several families, and p.Val536Leu, which has been previously reported but not yet characterized by electrophysiological investigations. The patient's symptoms included generalized stiffness, myotonia, and muscle cramps mostly localized in the lower limbs. These symptoms started during childhood and worsened over the following years. The symptoms were exacerbated by low outside temperature, rest, stress, and fasting and were improved by mild exercise, suggesting a warm-up phenomenon. The mutation p.Phe167Leu has previously been associated with a slight shift in the overall open probability. Here we further analysed this mutation to extrapolate the voltage-dependence of the fast and slow gates. In our experimental conditions, p.Phe167Leu exclusively affected the slow gate, increasing the minimum open probability and displacing the voltage-dependence toward depolarized potentials. p.Val536Leu showed more severe effects, dramatically influencing the slow gate as well as modifying properties of the fast gate. Co-expression of the mutants in a human cell line to reproduce the compound heterozygous condition of the patient produced channels with altered voltage-dependence of the slow gate but a restored fast gate. The alteration of the slow mechanism was reflected by the relative open probability, reducing the contribution of ClC-1 channels in maintaining the resting membrane potential of skeletal muscles and thus explaining the myotonic phenotype of the patient