Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST. OBJECTIVE: To determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism. METHODS: A four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted. RESULTS: A novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration. CONCLUSION: The adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Evaluation of a Synthetic Retinoid, Ellorarxine, in the NSC-34 Cell Model of Motor Neuron Disease.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease worldwide and is characterized by progressive muscle atrophy. There are currently two approved treatments, but they only relieve symptoms briefly and do not cure the disease. The main hindrance to research is the complex cause of ALS, with its pathogenesis not yet fully elucidated. Retinoids (vitamin A derivatives) appear to be essential in neuronal cells and have been implicated in ALS pathogenesis. This study explores 4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydroquinoxalin-2-yl)ethylnyl]benzoic acid (Ellorarxine, or DC645 or NVG0645), a leading synthetic retinoic acid, discussing its pharmacological mechanisms, neuroprotective properties, and relevance to ALS. The potential therapeutic effect of Ellorarxine was analyzed in vitro using the WT and SOD1G93A NSC-34 cell model of ALS at an administered concentration of 0.3-30 nM. Histological, functional, and biochemical analyses were performed. Elorarxine significantly increased MAP2 expression and neurite length, increased AMPA receptor GluA2 expression and raised intracellular Ca2+ baseline, increased level of excitability, and reduced Ca2+ spike during depolarization in neurites. Ellorarxine also displayed both antioxidant and anti-inflammatory effects. Overall, these results suggest Ellorarxine shows relevance and promise as a novel therapeutic strategy for treatment of ALS.

Rewiring movements: A single neuronal population in the spinal cord is crucial to restore walking after paralysis.

A single neuronal population in the spinal cord is crucial to restore walking after paralysis.

Mangiferin activates the nuclear factor erythroid 2-related factor pathway to protect SOD1-G93A induced NSC-34 motor neurons from oxidative stress and apoptosis.

One of the main factors in the pathophysiology of amyotrophic lateral sclerosis is oxidative stress. Mangiferin (MF), a natural plant polyphenol, has anti-inflammatory and antioxidant effects. The aim of our study was to investigate the protective effects and mechanisms of MF in the hSOD1-G93A ALS cell model. Our result revealed that MF treatment reduced the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), decreased oxidative damage, and reduced apoptosis. Additionally, it was observed that MF significantly increased the synthesis of the antioxidant genes hemeoxygenase-1 and NAD(P)H: quinone oxidoreductase 1, which are downstream of the Nrf2 signaling pathway, and increased the expression and activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 knockdown greatly promoted apoptosis, which was reversed by MF treatment. To summarize, MF promoted the Nrf2 pathway and scavenged MDA and ROS to protect the ALS cell model.

Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

Endocytosis of Synaptic Vesicle in Motor Nerve Endings of FUS Transgenic Mice with a Model of Amyotrophic Lateral Sclerosis.

In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

Poly-GP accumulation due to C9orf72 loss of function induces motor neuron apoptosis through autophagy and mitophagy defects.

The GGGGCC hexanucleotide repeat expansion (HRE) of the C9orf72 gene is the most frequent cause of amyotrophic lateral sclerosis (ALS), a devastative neurodegenerative disease characterized by motor neuron degeneration. C9orf72 HRE is associated with lowered levels of C9orf72 expression and its translation results in the production of dipeptide-repeats (DPRs). To recapitulate C9orf72-related ALS disease in vivo, we developed a zebrafish model where we expressed glycine-proline (GP) DPR in a c9orf72 knockdown context. We report that C9orf72 gain- and loss-of-function properties act synergistically to induce motor neuron degeneration and paralysis with poly(GP) accumulating preferentially within motor neurons along with Sqstm1/p62 aggregation indicating macroautophagy/autophagy deficits. Poly(GP) levels were shown to accumulate upon c9orf72 downregulation and were comparable to levels assessed in autopsy samples of patients carrying C9orf72 HRE. Chemical boosting of autophagy using rapamycin or apilimod, is able to rescue motor deficits. Proteomics analysis of zebrafish-purified motor neurons unravels mitochondria dysfunction confirmed through a comparative analysis of previously published C9orf72 iPSC-derived motor neurons. Consistently, 3D-reconstructions of motor neuron demonstrate that poly(GP) aggregates colocalize to mitochondria, thus inducing their elongation and swelling and the failure of their processing by mitophagy, with mitophagy activation through urolithin A preventing locomotor deficits. Finally, we report apoptotic-related increased amounts of cleaved Casp3 (caspase 3, apoptosis-related cysteine peptidase) and rescue of motor neuron degeneration by constitutive inhibition of Casp9 or treatment with decylubiquinone. Here we provide evidence of key pathogenic steps in C9ALS-FTD that can be targeted through pharmacological avenues, thus raising new therapeutic perspectives for ALS patients.

Decreased spinal cord motor neuron numbers in mice depleted of central nervous system copper.

Disrupted copper availability in the central nervous system (CNS) is implicated as a significant feature of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Solute carrier family 31 member 1 (Slc31a1; Ctr1) governs copper uptake in mammalian cells and mutations affecting Slc31a1 are associated with severe neurological abnormalities. Here, we examined the impact of decreased CNS copper caused by ubiquitous heterozygosity for functional Slc31a1 on spinal cord motor neurons in Slc31a1+/- mice. Congruent with the CNS being relatively susceptible to disrupted copper availability, brain and spinal cord tissue from Slc31a1+/- mice contained significantly less copper than wild-type littermates, even though copper levels in other tissues were unaffected. Slc31a1+/- mice had less spinal cord α-motor neurons compared to wild-type littermates, but they did not develop any overt physical signs of motor impairment. By contrast, ALS model SOD1G37R mice had fewer α-motor neurons than control mice and exhibited clear signs of motor function impairment. With the expression of Slc31a1 notwithstanding, spinal cord expression of genes related to copper handling revealed only minor differences between Slc31a1+/- and wild-type mice. This contrasted with SOD1G37R mice where changes in the expression of copper handling genes were pronounced. Similarly, the expression of genes related to toxic glial activation was unchanged in spinal cords from Slc31a1+/- mice but highly upregulated in SOD1G37R mice. Together, results from the Slc31a1+/- mice and SOD1G37R mice indicate that although depleted CNS copper has a significant impact on spinal cord motor neuron numbers, the manifestation of overt ALS-like motor impairment requires additional factors.

ALS-associated C21ORF2 variant disrupts DNA damage repair, mitochondrial metabolism, neuronal excitability and NEK1 levels in human motor neurons.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS.

RBM5 induces motor neuron apoptosis in hSOD1G93A-related amyotrophic lateral sclerosis by inhibiting Rac1/AKT pathways.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder distinguished by gradual depletion of motor neurons. RNA binding motif protein 5 (RBM5), an abundantly expressed RNA-binding protein, plays a critical role in the process of cellular death. However, little is known about the role of RBM5 in the pathogenesis of ALS. Here, we found that RBM5 was upregulated in ALS hSOD1G93A-NSC34 cell models and hSOD1G93A mice due to a reduction of miR-141-5p. The upregulation of RBM5 increased the apoptosis of motor neurons by inhibiting Rac1-mediated neuroprotection. In contrast, genetic knockdown of RBM5 rescued motor neurons from hSOD1G93A-induced degeneration by activating Rac1 signaling. The neuroprotective effect of RBM5-knockdown was significantly inhibited by the Rac1 inhibitor, NSC23766. These findings suggest that RBM5 could potentially serve as a therapeutic target in ALS by activating the Rac1 signalling.

ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes.

The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.

Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS.

Hyperexcitability of motor neurons and spinal cord motor circuitry has been widely reported in the early stages of Amyotrophic Lateral Sclerosis (ALS). Changes in the relative amount of excitatory to inhibitory inputs onto a neuron (E:I synaptic ratio), possibly through a developmental shift in synapse formation in favour of excitatory transmission, could underlie pathological hyperexcitability. Given that astrocytes play a major role in early synaptogenesis and are implicated in ALS pathogenesis, their potential contribution to disease mechanisms involving synaptic imbalances and subsequent hyperexcitability is also of great interest. In order to assess E:I ratios in ALS, we utilised a novel primary spinal neuron / astrocyte co-culture system, derived from neonatal mice, in which synapses are formed in vitro. Using multiple ALS mouse models we found that no combination of astrocyte or neuron genotype produced alterations in E:I synaptic ratios assessed using pre- and post-synaptic anatomical markers. Similarly, we observed that ephrin-B1, a major contact-dependent astrocytic synaptogenic protein, was not differentially expressed by ALS primary astrocytes. Further to this, analysis of E:I ratios across the entire grey matter of the lumbar spinal cord in young (post-natal day 16-19) ALS mice revealed no differences versus controls. Finally, analysis in co-cultures of human iPSC-derived motor neurons and astrocytes harbouring the pathogenic C9orf72 hexanucleotide repeat expansion showed no evidence of a bias toward excitatory versus inhibitory synapse formation. We therefore conclude, utilising multiple ALS models, that we do not observe significant changes in the relative abundance of excitatory versus inhibitory synapses as would be expected if imbalances in synaptic inputs contribute to early hyperexcitability.

Low-intensity pulsed ultrasound modulates disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord, and there are no effective drug treatments. Low-intensity pulsed ultrasound (LIPUS) has garnered attention as a promising noninvasive neuromodulation method. In this study, we investigate its effects on the motor cortex and underlying mechanisms using the SOD1G93A mouse model of ALS. Our results show that LIPUS treatment delays disease onset and prolongs lifespan in ALS mice. LIPUS significantly increases cerebral blood flow in the motor cortex by preserving vascular endothelial cell integrity and increasing microvascular density, which may be mediated via the ion channel TRPV4. RNA sequencing analysis reveals that LIPUS substantially reduces the expression of genes associated with neuroinflammation. These findings suggest that LIPUS applied to the motor cortex may represent a potentially effective therapeutic tool for the treatment of ALS.

Heterozygous knockout of Synaptotagmin13 phenocopies ALS features and TP53 activation in human motor neurons.

Spinal motor neurons (MNs) represent a highly vulnerable cellular population, which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study, we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/- hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover, analysis of the SYT13+/- transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures, displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes, which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family, SYT13, is sufficient to trigger a series of abnormal alterations leading to MN sufferance, thus revealing novel insights into the selective vulnerability of this cell population.

Astrocyte-Neuron Interactions Contributing to Amyotrophic Lateral Sclerosis Progression.

Amyotrophic lateral sclerosis (ALS) is a complex disease impacting motor neurons of the brain, brainstem, and spinal cord. Disease etiology is quite heterogeneous with over 40 genes causing the disease and a vast ~90% of patients having no prior family history. Astrocytes are major contributors to ALS, particularly through involvement in accelerating disease progression. Through study of genetic forms of disease including SOD1, TDP43, FUS, C9orf72, VCP, TBK1, and more recently patient-derived cells from sporadic individuals, many biological mechanisms have been identified to cause intrinsic or glial-mediated neurotoxicity to motor neurons. Overall, many of the normally supportive and beneficial roles that astrocytes contribute to neuronal health and survival instead switch to become deleterious and neurotoxic. While the exact pathways may differ based on disease-origin, altered astrocyte-neuron communication is a common feature of ALS. Within this chapter, distinct genetic forms are examined in detail, along with what is known from sporadic patient-derived cells. Overall, this chapter highlights the interplay between astrocytes and neurons in this complex disease and describes the key features underlying: astrocyte-mediated motor neuron toxicity, excitotoxicity, oxidative/nitrosative stress, protein dyshomeostasis, metabolic imbalance, inflammation, trophic factor withdrawal, blood-brain/blood-spinal cord barrier involvement, disease spreading, and the extracellular matrix/cell adhesion/TGF-ß signaling pathways.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Morphometric analysis of spinal motor neuron degeneration in sporadic amyotrophic lateral sclerosis.

OBJECTIVES: This study aimed to clarify the relationship between 43-kDa TAR DNA-binding protein (TDP-43) pathology and spinal cord anterior horn motor neuron (AHMN) atrophy in sporadic amyotrophic lateral sclerosis (SALS). METHODS: Eight patients with SALS and 12 controls were included in this study. Formalin-fixed specimens of lumbar spinal cord samples were paraffin-embedded and sectioned at the level of the fourth lumbar spinal cord with a 4 µm thickness. Using a microscope, the long diameters of the neurons with nucleoli were measured in spinal AHMNs stained with an anti-SMI-32 antibody. AHMNs were divided into medial and lateral nuclei for statistical analysis. We also used previously reported data to measure the long diameter of AHMNs with initial TDP-43 pathology, in which TDP-43 was present both in the nucleus and cytoplasm. RESULTS: The long diameter of the lumbar spinal AHMNs in patients with SALS was smaller in the medial nucleus (42.54 ± 9.33 µm, n = 24) and the lateral nucleus (49.41 ± 13.86 µm, n = 129) than in controls (medial nucleus: 55.84 ± 13.49 µm, n = 85, p < 0.001; lateral nucleus: 62.39 ± 13.29 µm, n = 756, p < 0.001, Mann-Whitney U test). All 21 motor neurons with initial TDP-43 pathology were in the lateral nucleus, and their long diameter (67.60 ± 18.3 µm, p = 0.352) was not significantly different from that of controls. CONCLUSION: Motor neuron atrophy in SALS does not occur during the initial stages of TDP-43 pathology, and TDP-43 pathology is already advanced in the atrophied motor neurons.

The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons.

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

Nuclear Localization of Human SOD1 in Motor Neurons in Mouse Model and Patient Amyotrophic Lateral Sclerosis: Possible Links to Cholinergic Phenotype, NADPH Oxidase, Oxidative Stress, and DNA Damage.

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6-14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.