Disease Mechanisms and Therapeutic Approaches in SMARD1-Insights from Animal Models and Cell Models.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal childhood motoneuron disease caused by mutations in the IGHMBP2 gene. It is characterized by muscle weakness, initially affecting the distal extremities due to the degeneration of spinal α-motoneurons, and respiratory distress, due to the paralysis of the diaphragm. Infantile forms with a severe course of the disease can be distinguished from juvenile forms with a milder course. Mutations in the IGHMBP2 gene have also been found in patients with peripheral neuropathy Charcot-Marie-Tooth type 2S (CMT2S). IGHMBP2 is an ATP-dependent 5'→3' RNA helicase thought to be involved in translational mechanisms. In recent years, several animal models representing both SMARD1 forms and CMT2S have been generated to initially study disease mechanisms. Later, the models showed very well that both stem cell therapies and the delivery of the human IGHMBP2 cDNA by AAV9 approaches (AAV9-IGHMBP2) can lead to significant improvements in disease symptoms. Therefore, the SMARD1 animal models, in addition to the cellular models, provide an inexhaustible source for obtaining knowledge of disease mechanisms, disease progression at the cellular level, and deeper insights into the development of therapies against SMARD1.

RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis.

RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export. Accordingly, failure of IGHMBP2 regulation of THOC causes perturbations of the transcriptome and its encoded proteome, and ablation of THOC subunits phenocopies these changes. Thus, IGHMBP2 is an upstream regulator of THOC. Of note, IGHMBP2-dependent regulation of THOC is also observed in astrocytes derived from patients with SMARD1 disease, suggesting that deregulated mRNA metabolism contributes to SMARD1 etiology and may enable alternative therapeutic avenues.

The SMA Modifier Plastin 3 Targets Cell Membrane-Associated Proteins in Motoneurons.

Loss of the Survival Motor Neuron (SMN) gene inevitably leads to spinal muscular atrophy (SMA), one of the most common fatal neuromuscular diseases in children with FDA and EMA approved therapies. However, the cellular mechanisms leading to neuromuscular junction (NMJ) dysfunction due to impaired Ca2+ homeostasis in the presynaptic compartment remain largely unexplained. In the last decade, the so-called SMA modifiers have gained attention. The F-actin bundler Plastin 3 (PLS3) is one of them and counteracts neurotransmission defects, including altered vesicle endocytosis, in Smn-deficient NMJs. Properly bundled F-actin is the basis for the translocation and arrangement of transmembrane proteins at the cell surface. Our recently published data by Hennlein et al., J Cell Biol. (2023) clearly showed that Smn deficiency impairs the F-actin dependent translocation of the high-affinity BDNF receptor TrkB to the cell surface resulting in reduced BDNF-mediated TrkB activation in motor axon terminals. Strikingly, the overexpression of PLS3 restores TrkB availability, and significantly improves the clustering of the active zone-associated voltage-gated calcium channel Cav2.2 in growth cones of Smn-deficient motoneurons. These observations raise the question of how PLS3 mediates the proper cell surface localization and cluster-like formation of Cav2.2 in motor axon terminals.

Nifedipine Ameliorates Cellular Differentiation Defects of Smn-Deficient Motor Neurons and Enhances Neuromuscular Transmission in SMA Mice.

In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca2+ transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands.

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy.

Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 "cluster-like" formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.

Pegylated Insulin-Like Growth Factor 1 attenuates Hair Cell Loss and promotes Presynaptic Maintenance of Medial Olivocochlear Cholinergic Fibers in the Cochlea of the Progressive Motor Neuropathy Mouse.

The progressive motor neuropathy (PMN) mouse is a model of an inherited motor neuropathy disease with progressive neurodegeneration. Axon degeneration associates with homozygous mutations of the TBCE gene encoding the tubulin chaperone E protein. TBCE is responsible for the correct dimerization of alpha and beta-tubulin. Strikingly, the PMN mouse also develops a progressive hearing loss after normal hearing onset, characterized by degeneration of the auditory nerve and outer hair cell (OHC) loss. However, the development of this neuronal and cochlear pathology is not fully understood yet. Previous studies with pegylated insulin-like growth factor 1 (peg-IGF-1) treatment in this mouse model have been shown to expand lifespan, weight, muscle strength, and motor coordination. Accordingly, peg-IGF-1 was evaluated for an otoprotective effect. We investigated the effect of peg-IGF-1 on the auditory system by treatment starting at postnatal day 15 (p15). Histological analysis revealed positive effects on OHC synapses of medial olivocochlear (MOC) neuronal fibers and a short-term attenuation of OHC loss. Peg-IGF-1 was able to conditionally restore the disorganization of OHC synapses and maintain the provision of cholinergic acetyltransferase in presynapses. To assess auditory function, frequency-specific auditory brainstem responses and distortion product otoacoustic emissions were recorded in animals on p21 and p28. However, despite the positive effect on MOC fibers and OHC, no restoration of hearing could be achieved. The present work demonstrates that the synaptic pathology of efferent MOC fibers in PMN mice represents a particular form of "efferent auditory neuropathy." Peg-IGF-1 showed an otoprotective effect by preventing the degeneration of OHCs and efferent synapses. However, enhanced efforts are needed to optimize the treatment to obtain detectable improvements in hearing performances.

Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus-response in spinal muscular atrophy.

BACKGROUND: Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA. METHODS: Using super-resolution microscopy, proximity ligation assay (PLA) and live imaging of cultured motoneurons from a mouse model of SMA, we investigated the dynamics of the axonal ER and ribosome distribution and activation. RESULTS: We observed that the dynamic remodeling of ER was impaired in axon terminals of Smn-deficient motoneurons. In addition, in axon terminals of Smn-deficient motoneurons, ribosomes failed to respond to the brain-derived neurotrophic factor stimulation, and did not undergo rapid association with the axonal ER in response to extracellular stimuli. CONCLUSIONS: These findings implicate impaired dynamic interplay between the ribosomes and ER in axon terminals of motoneurons as a contributor to the pathophysiology of SMA and possibly also other motoneuron diseases.

Therapy development for spinal muscular atrophy: perspectives for muscular dystrophies and neurodegenerative disorders.

BACKGROUND: Major efforts have been made in the last decade to develop and improve therapies for proximal spinal muscular atrophy (SMA). The introduction of Nusinersen/Spinraza™ as an antisense oligonucleotide therapy, Onasemnogene abeparvovec/Zolgensma™ as an AAV9-based gene therapy and Risdiplam/Evrysdi™ as a small molecule modifier of pre-mRNA splicing have set new standards for interference with neurodegeneration. MAIN BODY: Therapies for SMA are designed to interfere with the cellular basis of the disease by modifying pre-mRNA splicing and enhancing expression of the Survival Motor Neuron (SMN) protein, which is only expressed at low levels in this disorder. The corresponding strategies also can be applied to other disease mechanisms caused by loss of function or toxic gain of function mutations. The development of therapies for SMA was based on the use of cell culture systems and mouse models, as well as innovative clinical trials that included readouts that had originally been introduced and optimized in preclinical studies. This is summarized in the first part of this review. The second part discusses current developments and perspectives for amyotrophic lateral sclerosis, muscular dystrophies, Parkinson's and Alzheimer's disease, as well as the obstacles that need to be overcome to introduce RNA-based therapies and gene therapies for these disorders. CONCLUSION: RNA-based therapies offer chances for therapy development of complex neurodegenerative disorders such as amyotrophic lateral sclerosis, muscular dystrophies, Parkinson's and Alzheimer's disease. The experiences made with these new drugs for SMA, and also the experiences in AAV gene therapies could help to broaden the spectrum of current approaches to interfere with pathophysiological mechanisms in neurodegeneration.

Loss of full-length hnRNP R isoform impairs DNA damage response in motoneurons by inhibiting Yb1 recruitment to chromatin.

Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.

Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons.

In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation. This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.

Interaction of 7SK with the Smn complex modulates snRNP production.

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.

Loss of Tdp-43 disrupts the axonal transcriptome of motoneurons accompanied by impaired axonal translation and mitochondria function.

Protein inclusions containing the RNA-binding protein TDP-43 are a pathological hallmark of amyotrophic lateral sclerosis and other neurodegenerative disorders. The loss of TDP-43 function that is associated with these inclusions affects post-transcriptional processing of RNAs in multiple ways including pre-mRNA splicing, nucleocytoplasmic transport, modulation of mRNA stability and translation. In contrast, less is known about the role of TDP-43 in axonal RNA metabolism in motoneurons. Here we show that depletion of Tdp-43 in primary motoneurons affects axon growth. This defect is accompanied by subcellular transcriptome alterations in the axonal and somatodendritic compartment. The axonal localization of transcripts encoding components of the cytoskeleton, the translational machinery and transcripts involved in mitochondrial energy metabolism were particularly affected by loss of Tdp-43. Accordingly, we observed reduced protein synthesis and disturbed mitochondrial functions in axons of Tdp-43-depleted motoneurons. Treatment with nicotinamide rescued the axon growth defect associated with loss of Tdp-43. These results show that Tdp-43 depletion in motoneurons affects several pathways integral to axon health indicating that loss of TDP-43 function could thus make a major contribution to axonal pathomechanisms in ALS.

R-Roscovitine Improves Motoneuron Function in Mouse Models for Spinal Muscular Atrophy.

Neurotransmission defects and motoneuron degeneration are hallmarks of spinal muscular atrophy, a monogenetic disease caused by the deficiency of the SMN protein. In the present study, we show that systemic application of R-Roscovitine, a Cav2.1/Cav2.2 channel modifier and a cyclin-dependent kinase 5 (Cdk-5) inhibitor, significantly improved survival of SMA mice. In addition, R-Roscovitine increased Cav2.1 channel density and sizes of the motor endplates. In vitro, R-Roscovitine restored axon lengths and growth cone sizes of Smn-deficient motoneurons corresponding to enhanced spontaneous Ca2+ influx and elevated Cav2.2 channel cluster formations independent of its capability to inhibit Cdk-5. Acute application of R-Roscovitine at the neuromuscular junction significantly increased evoked neurotransmitter release, increased the frequency of spontaneous miniature potentials, and lowered the activation threshold of silent terminals. These data indicate that R-Roscovitine improves Ca2+ signaling and Ca2+ homeostasis in Smn-deficient motoneurons, which is generally crucial for motoneuron differentiation, maturation, and function.

Impaired Local Translation of ß-actin mRNA in Ighmbp2-Deficient Motoneurons: Implications for Spinal Muscular Atrophy with respiratory Distress (SMARD1).

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal motoneuron disorder in children with unknown etiology. The disease is caused by mutations in the IGHMBP2 gene, encoding a Super Family 1 (SF1)-type RNA/DNA helicase. IGHMBP2 is a cytosolic protein that binds to ribosomes and polysomes, suggesting a role in mRNA metabolism. Here we performed morphological and functional analyses of isolated immunoglobulin µ-binding protein 2 (Ighmbp2)-deficient motoneurons to address the question whether the SMARD1 phenotype results from de-regulation of protein biosynthesis. Ighmbp2-deficient motoneurons exhibited only moderate morphological aberrations such as a slight increase of axonal branches. Consistent with the rather mild phenotypic aberrations, RNA sequencing of Ighmbp2-deficient motoneurons revealed only minor transcriptome alterations compared to controls. Likewise, we did not detect any global changes in protein synthesis using pulsed SILAC (Stable Isotope Labeling by Amino acids in Cell culture), FUNCAT (FlUorescent Non-Canonical Amino acid Tagging) and SUnSET (SUrface SEnsing of Translation) approaches. However, we observed reduced ß-actin protein levels at the growth cone of Ighmbp2-deficient motoneurons which was accompanied by reduced level of IMP1/ZBP1, a known interactor of ß-actin mRNA. Fluorescence Recovery after Photobleaching (FRAP) studies revealed translational down-regulation of an eGFP-myr-ß-actin 3'UTR mRNA in growth cones. Local translational regulation of ß-actin mRNA was dependent on the 3' UTR but independent of direct Ighmbp2-binding to ß-actin mRNA. Taken together, our data indicate that Ighmbp2 deficiency results in local but modest disruption of protein biosynthesis which might partially contribute to the motoneuron defects seen in SMARD1.

Comprehensive analysis of the mutation spectrum in 301 German ALS families.

OBJECTIVES: Recent advances in amyotrophic lateral sclerosis (ALS) genetics have revealed that mutations in any of more than 25 genes can cause ALS, mostly as an autosomal-dominant Mendelian trait. Detailed knowledge about the genetic architecture of ALS in a specific population will be important for genetic counselling but also for genotype-specific therapeutic interventions. METHODS: Here we combined fragment length analysis, repeat-primed PCR, Southern blotting, Sanger sequencing and whole exome sequencing to obtain a comprehensive profile of genetic variants in ALS disease genes in 301 German pedigrees with familial ALS. We report C9orf72 mutations as well as variants in consensus splice sites and non-synonymous variants in protein-coding regions of ALS genes. We furthermore estimate their pathogenicity by taking into account type and frequency of the respective variant as well as segregation within the families. RESULTS: 49% of our German ALS families carried a likely pathogenic variant in at least one of the earlier identified ALS genes. In 45% of the ALS families, likely pathogenic variants were detected in C9orf72, SOD1, FUS, TARDBP or TBK1, whereas the relative contribution of the other ALS genes in this familial ALS cohort was 4%. We identified several previously unreported rare variants and demonstrated the absence of likely pathogenic variants in some of the recently described ALS disease genes. CONCLUSIONS: We here present a comprehensive genetic characterisation of German familial ALS. The present findings are of importance for genetic counselling in clinical practice, for molecular research and for the design of diagnostic gene panels or genotype-specific therapeutic interventions in Europe.

An open source tool for automatic spatiotemporal assessment of calcium transients and local 'signal-close-to-noise' activity in calcium imaging data.

Local and spontaneous calcium signals play important roles in neurons and neuronal networks. Spontaneous or cell-autonomous calcium signals may be difficult to assess because they appear in an unpredictable spatiotemporal pattern and in very small neuronal loci of axons or dendrites. We developed an open source bioinformatics tool for an unbiased assessment of calcium signals in x,y-t imaging series. The tool bases its algorithm on a continuous wavelet transform-guided peak detection to identify calcium signal candidates. The highly sensitive calcium event definition is based on identification of peaks in 1D data through analysis of a 2D wavelet transform surface. For spatial analysis, the tool uses a grid to separate the x,y-image field in independently analyzed grid windows. A document containing a graphical summary of the data is automatically created and displays the loci of activity for a wide range of signal intensities. Furthermore, the number of activity events is summed up to create an estimated total activity value, which can be used to compare different experimental situations, such as calcium activity before or after an experimental treatment. All traces and data of active loci become documented. The tool can also compute the signal variance in a sliding window to visualize activity-dependent signal fluctuations. We applied the calcium signal detector to monitor activity states of cultured mouse neurons. Our data show that both the total activity value and the variance area created by a sliding window can distinguish experimental manipulations of neuronal activity states. Notably, the tool is powerful enough to compute local calcium events and 'signal-close-to-noise' activity in small loci of distal neurites of neurons, which remain during pharmacological blockade of neuronal activity with inhibitors such as tetrodotoxin, to block action potential firing, or inhibitors of ionotropic glutamate receptors. The tool can also offer information about local homeostatic calcium activity events in neurites.

Hot-spot KIF5A mutations cause familial ALS.

Heterozygous missense mutations in the N-terminal motor or coiled-coil domains of the kinesin family member 5A (KIF5A) gene cause monogenic spastic paraplegia (HSP10) and Charcot-Marie-Tooth disease type 2 (CMT2). Moreover, heterozygous de novo frame-shift mutations in the C-terminal domain of KIF5A are associated with neonatal intractable myoclonus, a neurodevelopmental syndrome. These findings, together with the observation that many of the disease genes associated with amyotrophic lateral sclerosis disrupt cytoskeletal function and intracellular transport, led us to hypothesize that mutations in KIF5A are also a cause of amyotrophic lateral sclerosis. Using whole exome sequencing followed by rare variant analysis of 426 patients with familial amyotrophic lateral sclerosis and 6137 control subjects, we detected an enrichment of KIF5A splice-site mutations in amyotrophic lateral sclerosis (2/426 compared to 0/6137 in controls; P = 4.2 × 10-3), both located in a hot-spot in the C-terminus of the protein and predicted to affect splicing exon 27. We additionally show co-segregation with amyotrophic lateral sclerosis of two canonical splice-site mutations in two families. Investigation of lymphoblast cell lines from patients with KIF5A splice-site mutations revealed the loss of mutant RNA expression and suggested haploinsufficiency as the most probable underlying molecular mechanism. Furthermore, mRNA sequencing of a rare non-synonymous missense mutation (predicting p.Arg1007Gly) located in the C-terminus of the protein shortly upstream of the splice donor of exon 27 revealed defective KIF5A pre-mRNA splicing in respective patient-derived cell lines owing to abrogation of the donor site. Finally, the non-synonymous single nucleotide variant rs113247976 (minor allele frequency = 1.00% in controls, n = 6137), also located in the C-terminal region [p.(Pro986Leu) in exon 26], was significantly enriched in familial amyotrophic lateral sclerosis patients (minor allele frequency = 3.40%; P = 1.28 × 10-7). Our study demonstrates that mutations located specifically in a C-terminal hotspot of KIF5A can cause a classical amyotrophic lateral sclerosis phenotype, and underline the involvement of intracellular transport processes in amyotrophic lateral sclerosis pathogenesis.

Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.