Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Brain Calcifications: Genetic, Molecular, and Clinical Aspects.

Many conditions can present with accumulation of calcium in the brain and manifest with a variety of neurological symptoms. Brain calcifications can be primary (idiopathic or genetic) or secondary to various pathological conditions (e.g., calcium-phosphate metabolism derangement, autoimmune disorders and infections, among others). A set of causative genes associated with primary familial brain calcification (PFBC) has now been identified, and include genes such as SLC20A2, PDGFB, PDGFRB, XPR1, MYORG, and JAM2. However, many more genes are known to be linked with complex syndromes characterized by brain calcifications and additional neurologic and systemic manifestations. Of note, many of these genes encode for proteins involved in cerebrovascular and blood-brain barrier functions, which both represent key anatomical structures related to these pathological phenomena. As a growing number of genes associated with brain calcifications is identified, pathways involved in these conditions are beginning to be understood. Our comprehensive review of the genetic, molecular, and clinical aspects of brain calcifications offers a framework for clinicians and researchers in the field.

SMN post-translational modifications in spinal muscular atrophy.

Since its first identification as the gene responsible for spinal muscular atrophy (SMA), the range of survival motor neuron (SMN) protein functions has increasingly expanded. This multimeric complex plays a crucial role in a variety of RNA processing pathways. While its most characterized function is in the biogenesis of ribonucleoproteins, several studies have highlighted the SMN complex as an important contributor to mRNA trafficking and translation, axonal transport, endocytosis, and mitochondria metabolism. All these multiple functions need to be selectively and finely modulated to maintain cellular homeostasis. SMN has distinct functional domains that play a crucial role in complex stability, function, and subcellular distribution. Many different processes were reported as modulators of the SMN complex activities, although their contribution to SMN biology still needs to be elucidated. Recent evidence has identified post-translational modifications (PTMs) as a way to regulate the pleiotropic functions of the SMN complex. These modifications include phosphorylation, methylation, ubiquitination, acetylation, sumoylation, and many other types. PTMs can broaden the range of protein functions by binding chemical moieties to specific amino acids, thus modulating several cellular processes. Here, we provide an overview of the main PTMs involved in the regulation of the SMN complex with a major focus on the functions that have been linked to SMA pathogenesis.

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration.

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

Retromer dysfunction in amyotrophic lateral sclerosis.

Retromer is a heteropentameric complex that plays a specialized role in endosomal protein sorting and trafficking. Here, we report a reduction in the retromer proteins-vacuolar protein sorting 35 (VPS35), VPS26A, and VPS29-in patients with amyotrophic lateral sclerosis (ALS) and in the ALS model provided by transgenic (Tg) mice expressing the mutant superoxide dismutase-1 G93A. These changes are accompanied by a reduction of levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA1, a proxy of retromer function, in spinal cords from Tg SOD1G93A mice. Correction of the retromer deficit by a viral vector expressing VPS35 exacerbates the paralytic phenotype in Tg SOD1G93A mice. Conversely, lowering Vps35 levels in Tg SOD1G93A mice ameliorates the disease phenotype. In light of these findings, we propose that mild alterations in retromer inversely modulate neurodegeneration propensity in ALS.

Sumoylation regulates the assembly and activity of the SMN complex.

SMN is a ubiquitously expressed protein and is essential for life. SMN deficiency causes the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. SMN interacts with itself and other proteins to form a complex that functions in the assembly of ribonucleoproteins. SMN is modified by SUMO (Small Ubiquitin-like Modifier), but whether sumoylation is required for the functions of SMN that are relevant to SMA pathogenesis is not known. Here, we show that inactivation of a SUMO-interacting motif (SIM) alters SMN sub-cellular distribution, the integrity of its complex, and its function in small nuclear ribonucleoproteins biogenesis. Expression of a SIM-inactivated mutant of SMN in a mouse model of SMA slightly extends survival rate with limited and transient correction of motor deficits. Remarkably, although SIM-inactivated SMN attenuates motor neuron loss and improves neuromuscular junction synapses, it fails to prevent the loss of sensory-motor synapses. These findings suggest that sumoylation is important for proper assembly and function of the SMN complex and that loss of this post-translational modification impairs the ability of SMN to correct selective deficits in the sensory-motor circuit of SMA mice.

SETX (senataxin), the helicase mutated in AOA2 and ALS4, functions in autophagy regulation.

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.Abbreviations: 3'READS: 3' region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5'-3' exoribonuclease 2.

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.

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.

MicroRNA Metabolism and Dysregulation in Amyotrophic Lateral Sclerosis.

MicroRNAs (miRNAs) are a subset of endogenous, small, non-coding RNA molecules involved in the post-transcriptional regulation of eukaryotic gene expression. Dysregulation in miRNA-related pathways in the central nervous system (CNS) is associated with severe neuronal injury and cell death, which can lead to the development of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). ALS is a fatal adult onset disease characterized by the selective loss of upper and lower motor neurons. While the pathogenesis of ALS is still largely unknown, familial ALS forms linked to TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) gene mutations, as well as sporadic forms, display changes in several steps of RNA metabolism, including miRNA processing. Here, we review the current knowledge about miRNA metabolism and biological functions and their crucial role in ALS pathogenesis with an in-depth analysis on different pathways. A more precise understanding of miRNA involvement in ALS could be useful not only to elucidate their role in the disease etiopathogenesis but also to investigate their potential as disease biomarkers and novel therapeutic targets.

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.

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.

Clinical and molecular features and therapeutic perspectives of spinal muscular atrophy with respiratory distress type 1.

Spinal muscular atrophy with respiratory distress (SMARD1) is an autosomal recessive neuromuscular disease caused by mutations in the IGHMBP2 gene, encoding the immunoglobulin µ-binding protein 2, leading to motor neuron degeneration. It is a rare and fatal disease with an early onset in infancy in the majority of the cases. The main clinical features are muscular atrophy and diaphragmatic palsy, which requires prompt and permanent supportive ventilation. The human disease is recapitulated in the neuromuscular degeneration (nmd) mouse. No effective treatment is available yet, but novel therapeutical approaches tested on the nmd mouse, such as the use of neurotrophic factors and stem cell therapy, have shown positive effects. Gene therapy demonstrated effectiveness in SMA, being now at the stage of clinical trial in patients and therefore representing a possible treatment for SMARD1 as well. The significant advancement in understanding of both SMARD1 clinical spectrum and molecular mechanisms makes ground for a rapid translation of pre-clinical therapeutic strategies in humans.

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 µ-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.

Motor neuron derivation from human embryonic and induced pluripotent stem cells: experimental approaches and clinical perspectives.

Motor neurons are cells located in specific areas of the central nervous system, such as brain cortex (upper motor neurons), brain stem, and spinal cord (lower motor neurons), which maintain control over voluntary actions. Motor neurons are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs): these disorders share symptoms related to muscular atrophy and paralysis leading to death. No effective treatments are currently available. Stem cell-derived motor neurons represent a promising research tool in disease modeling, drug screening, and development of therapeutic approaches for MNDs and spinal cord injuries. Directed differentiation of human pluripotent stem cells - human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) - toward specific lineages is the first crucial step in order to extensively employ these cells in early human development investigation and potential clinical applications. Induced pluripotent stem cells (iPSCs) can be generated from patients' own somatic cells (for example, fibroblasts) by reprogramming them with specific factors. They can be considered embryonic stem cell-like cells, which express stem cell markers and have the ability to give rise to all three germ layers, bypassing the ethical concerns. Thus, hiPSCs constitute an appealing alternative source of motor neurons. These motor neurons might be a great research tool, creating a model for investigating the cellular and molecular interactions underlying early human brain development and pathologies during neurodegeneration. Patient-specific iPSCs may also provide the premises for autologous cell replacement therapies without related risks of immune rejection. Here, we review the most recent reported methods by which hESCs or iPSCs can be differentiated toward functional motor neurons with an overview on the potential clinical applications.