Molecular and Cellular Mechanisms of Motor Circuit Development.

Motor circuits represent the main output of the central nervous system and produce dynamic behaviors ranging from relatively simple rhythmic activities like swimming in fish and breathing in mammals to highly sophisticated dexterous movements in humans. Despite decades of research, the development and function of motor circuits remain poorly understood. Breakthroughs in the field recently provided new tools and tractable model systems that set the stage to discover the molecular mechanisms and circuit logic underlying motor control. Here, we describe recent advances from both vertebrate (mouse, frog) and invertebrate (nematode, fruit fly) systems on cellular and molecular mechanisms that enable motor circuits to develop and function and highlight conserved and divergent mechanisms necessary for motor circuit development.

Expression of Macrophage Colony-Stimulating Factor CSF-1 in Spinal Cord Neurons in Mice with Experimental Autoimmune Encephalomyelitis.

In mice with acute and chronic models of experimental autoimmune encephalomyelitis (EAE), a quantitative study of the dynamics of CSF-1 and IL-34 protein levels in the spinal cord and blood plasma was conducted by ELISA and the specificity of CSF-1 expression in spinal cord cells was examined by immunohistological methods. A significant increase in the level of CSF-1 in the spinal cord was detected and populations of motoneurons with intense CSF-1 immunoreactivity were identified in mice with acute and chronic EAE. The obtained results suggest a possible role of CSF-1 in the pathogenesis and progression of EAE/multiple sclerosis.

The relationship between the functional status of upper extremity motor neurons and motor function and prognosis in stroke patients.

Introduction: This study investigates the correlation between neuroelectrop-hysiological assessments such as motor unit number estimation (MUNE) and F-waves with upper extremity motor function and one-year prognosis in stroke patients. Methods: Neuroelectrophysiological assessments of the abductor pollicis brevis muscle, including MUNE and F-waves, were conducted. Upper extremity motor function was evaluated using the Fugl-Meyer Assessment of Upper Extremity (FMA-UE) and the Modified Ashworth Scale (MAS). Pearson correlation and multiple linear regression analyses were performed to explore the relationship between upper extremity motor function and variables such as MUNE and F-waves. ROC curve analysis assessed the predictive ability of MUNE and F-waves for upper extremity motor function, and binary logistic regression analysis examined factors related to motor function improvement 1 year post-discharge. Results: A total of 130 patients were ultimately included. Significant differences in MUNE and occupancy rate of non-repeater F-waves (non-ORF) were found between hemiplegic and unaffected sides (p < 0.001), with a significant difference in F-wave mean latency (p < 0.05). Pearson correlation analysis showed a positive correlation between FMA-UE at admission and hemiplegic side's MUNE and non-ORF (p < 0.001). Multiple linear regression indicated that hemiplegic side's MUNE (ß = 0.88, p < 0.001) and non-ORF (ß = 0.275, p = 0.005) influenced FMA-UE. ROC analysis demonstrated higher predictive ability for hemiplegic side's MUNE (AUC = 0.696, p < 0.001) than non-ORF (AUC = 0.622, p = 0.018). Binary logistic regression showed that hemiplegic side's MUNE was associated with FMA-UE improvement 1 year post-discharge. Conclusion: MUNE and F-waves are correlated with upper extremity motor function in patients, reflecting their motor function status. These indicators have good predictive value for motor function and are associated with the prognosis of upper extremity motor function to a certain extent.

Strategies for the development of in vitro models of spinal cord ischemia-reperfusion injury: Oxygen-glucose deprivation and reoxygenation.

BACKGROUND: In vitro models tailored for spinal cord ischemia-reperfusion injury are pivotal for investigation of the mechanisms underlying spinal cord injuries. We conducted a two-phased study to identify the optimal conditions for establishing an in vitro model of spinal cord ischemia-reperfusion injury using primary rat spinal motor neurons. NEW METHOD: In the first phase, cell cultures were subjected to oxygen deprivation (OD) only, glucose deprivation (GD) only, or simultaneous deprivation of oxygen and glucose [oxygen-glucose deprivation (OGD)] for different durations (1, 2, and 6 h). In the second phase, different durations of re-oxygenation (1, 12, and 24 h) were applied after 1 h of OGD to determine the optimal duration simulating reperfusion injury. RESULTS AND COMPARISON WITH EXISTING METHOD(S): GD for 6 h significantly reduced cell viability (91 % of control, P<0.001) and increase cytotoxicity (111 % of control, P<0.001). OGD for 1 h and 2 h, resulted in a significant decrease in cell viability (80 % of control P<0.001, respectively), and increase in cytotoxicity (130 % of control, P<0.001, respectively). Re-oxygenation for 1, 12, and 24 h worsened ischemic injury following 1 h of OGD (all P<0.05). CONCLUSIONS: Our results may provide a valuable guide to devise in vitro models of spinal cord ischemia-reperfusion injury using primary spinal motor neurons.

Emerging targets in amyotrophic lateral sclerosis (ALS): The promise of ATP-binding cassette (ABC) transporter modulation.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative primarily affecting motor neurons, leading to disability and neuronal death, and ATP-Binding Cassette (ABC) transporter due to their role in drug efflux and modulation of various cellular pathways contributes to the pathogenesis of ALS. In this article, we extensively investigated various molecular and mechanistic pathways linking ALS transporter to the pathogenesis of ALS; this involves inflammatory pathways such as Mitogen-Activated Protein Kinase (MAPK), Phosphatidylinositol-3-Kinase/Protein Kinase B (PI3K/Akt), Toll-Like Receptor (TLR), Glycogen Synthase Kinase 3ß (GSK-3ß), Nuclear Factor Kappa-B (NF-κB), and Cyclooxygenase (COX). Oxidative pathways such as Astrocytes, Glutamate, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Sirtuin 1 (SIRT-1), Forkhead box protein O (FOXO), Extracellular signal-regulated kinase (ERK). Additionally, we delve into the role of autophagic pathways like TAR DNA-binding protein 43 (TDP-43), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and lastly, the apoptotic pathways. Furthermore, by understanding these intricate interactions, we aim to develop novel therapeutic strategies targeting ABC transporters, improving drug delivery, and ultimately offering a promising avenue for treating ALS.

The myokine FGF21 associates with enhanced survival in ALS and mitigates stress-induced cytotoxicity.

Amyotrophic lateral sclerosis (ALS) is an age-related and fatal neurodegenerative disease characterized by progressive muscle weakness. There is marked heterogeneity in clinical presentation, progression, and pathophysiology with only modest treatments to slow disease progression. Molecular markers that provide insight into this heterogeneity are crucial for clinical management and identification of new therapeutic targets. In a prior muscle miRNA sequencing investigation, we identified altered FGF pathways in ALS muscle, leading us to investigate FGF21. We analyzed human ALS muscle biopsy samples and found a large increase in FGF21 expression with localization to atrophic myofibers and surrounding endomysium. A concomitant increase in FGF21 was detected in ALS spinal cords which correlated with muscle levels. FGF21 was increased in the SOD1G93A mouse beginning in presymptomatic stages. In parallel, there was dysregulation of the co-receptor, ß-Klotho. Plasma FGF21 levels were increased and high levels correlated with slower disease progression, prolonged survival, and increased body mass index. In NSC-34 motor neurons and C2C12 muscle cells expressing SOD1G93A or exposed to oxidative stress, ectopic FGF21 mitigated loss of cell viability. In summary, FGF21 is a novel biomarker in ALS that correlates with slower disease progression and exerts trophic effects under conditions of cellular stress.

Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus.

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

West Nile virus, but not Usutu virus, can productively infect human motor neurons as a possible route of neuroinvasion.

Expanded ATXN1 alters transcription and calcium signaling in SCA1 human motor neurons differentiated from induced pluripotent stem cells.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited and lethal neurodegenerative disease caused by the abnormal expansion of CAG repeats in the ATAXIN-1 (ATXN1) gene. Pathological studies identified dysfunction and loss of motor neurons (MNs) in the brain stem and spinal cord, which are thought to contribute to premature lethality by affecting the swallowing and breathing of SCA1 patients. However, the molecular and cellular mechanisms of MN pathogenesis remain unknown. To study SCA1 pathogenesis in human MNs, we differentiated induced pluripotent stem cells (iPSCs) derived from SCA1 patients and their unaffected siblings into MNs. We examined proliferation of progenitor cells, neurite outgrowth, spontaneous and glutamate-induced calcium activity of SCA1 MNs to investigate cellular mechanisms of pathogenesis. RNA sequencing was then used to identify transcriptional alterations in iPSC-derived MN progenitors (pMNs) and MNs which could underlie functional changes in SCA1 MNs. We found significantly decreased spontaneous and evoked calcium activity and identified dysregulation of genes regulating calcium signaling in SCA1 MNs. These results indicate that expanded ATXN1 causes dysfunctional calcium signaling in human MNs.

Spinal neuron diversity scales exponentially with swim-to-limb transformation during frog metamorphosis.

Vertebrates exhibit a wide range of motor behaviors, ranging from swimming to complex limb-based movements. Here we take advantage of frog metamorphosis, which captures a swim-to-limb-based movement transformation during the development of a single organism, to explore changes in the underlying spinal circuits. We find that the tadpole spinal cord contains small and largely homogeneous populations of motor neurons (MNs) and V1 interneurons (V1s) at early escape swimming stages. These neuronal populations only modestly increase in number and subtype heterogeneity with the emergence of free swimming. In contrast, during frog metamorphosis and the emergence of limb movement, there is a dramatic expansion of MN and V1 interneuron number and transcriptional heterogeneity, culminating in cohorts of neurons that exhibit striking molecular similarity to mammalian motor circuits. CRISPR/Cas9-mediated gene disruption of the limb MN and V1 determinants FoxP1 and Engrailed-1, respectively, results in severe but selective deficits in tail and limb function. Our work thus demonstrates that neural diversity scales exponentially with increasing behavioral complexity and illustrates striking evolutionary conservation in the molecular organization and function of motor circuits across species.

Lowered GnT-I Activity Decreases Complex-Type N-Glycan Amounts and Results in an Aberrant Primary Motor Neuron Structure in the Spinal Cord.

The attachment of sugar to proteins and lipids is a basic modification needed for organismal survival, and perturbations in glycosylation cause severe developmental and neurological difficulties. Here, we investigated the neurological consequences of N-glycan populations in the spinal cord of Wt AB and mgat1b mutant zebrafish. Mutant fish have reduced N-acetylglucosaminyltransferase-I (GnT-I) activity as mgat1a remains intact. GnT-I converts oligomannose N-glycans to hybrid N-glycans, which is needed for complex N-glycan production. MALDI-TOF MS profiles identified N-glycans in the spinal cord for the first time and revealed reduced amounts of complex N-glycans in mutant fish, supporting a lesion in mgat1b. Further lectin blotting showed that oligomannose N-glycans were more prevalent in the spinal cord, skeletal muscle, heart, swim bladder, skin, and testis in mutant fish relative to WT AB, supporting lowered GnT- I activity in a global manner. Developmental delays were noted in hatching and in the swim bladder. Microscopic images of caudal primary (CaP) motor neurons of the spinal cord transiently expressing EGFP in mutant fish were abnormal with significant reductions in collateral branches. Further motor coordination skills were impaired in mutant fish. We conclude that identifying the neurological consequences of aberrant N-glycan processing will enhance our understanding of the role of complex N-glycans in development and nervous system health.

Potential contribution of spinal interneurons to the etiopathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) consists of a group of adult-onset fatal and incurable neurodegenerative disorders characterized by the progressive death of motor neurons (MNs) throughout the central nervous system (CNS). At first, ALS was considered to be an MN disease, caused by cell-autonomous mechanisms acting specifically in MNs. Accordingly, data from ALS patients and ALS animal models revealed alterations in excitability in multiple neuronal populations, including MNs, which were associated with a variety of cellular perturbations such as protein aggregation, ribonucleic acid (RNA) metabolism defects, calcium dyshomeostasis, modified electrophysiological properties, and autophagy malfunctions. However, experimental evidence rapidly demonstrated the involvement of other types of cells, including glial cells, in the etiopathogenesis of ALS through non-cell autonomous mechanisms. Surprisingly, the contribution of pre-motor interneurons (INs), which regulate MN activity and could therefore critically modulate their excitability at the onset or during the progression of the disease, has to date been severely underestimated. In this article, we review in detail how spinal pre-motor INs are affected in ALS and their possible involvement in the etiopathogenesis of the disease.

Live Imaging Analysis of Axonal Regeneration in Human iPSC-Derived Motor Neurons Using a Microfluidic System.

Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.

Timeline of hypoglossal motor neuron death and intrinsic tongue muscle denervation in high-copy number SOD1G93A mice.

In amyotrophic lateral sclerosis (ALS) postmortem tissue and the SOD1 mouse model at mid-disease, death of hypoglossal motor neurons (XII MNs) is evident. These XII MNs innervate the intrinsic and extrinsic tongue muscles, and despite their importance in many oral and lingual motor behaviours that are affected by ALS (e.g., swallowing, speech, and respiratory functions), little is known about the timing and extent of tongue muscle denervation. Here in the well-characterised SOD1G93A (high-copy) mouse model, we evaluated XII MN numbers and intrinsic tongue muscle innervation using standard histopathological approaches, which included stereological evaluation of Nissl-stained brainstem, and the presynaptic and postsynaptic evaluation of neuromuscular junctions (NMJs), using synapsin, neurofilament, and α-bungarotoxin immunolabelling, at presymptomatic, onset, mid-disease, and endstage timepoints. We found that reduction in XII MN size at onset preceded reduced XII MN survival, while the denervation of tongue muscle did not appear until the endstage. Our study suggests that denervation-induced weakness may not be the most pertinent feature of orolingual deficits in ALS. Efforts to preserve oral and respiratory functions of XII MNs are incredibly important if we are to influence patient outcomes.

Members of an array of zinc-finger proteins specify distinct Hox chromatin boundaries.

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

α-Cyclodextrin/Moringin Induces an Antioxidant Transcriptional Response Activating Nrf2 in Differentiated NSC-34 Motor Neurons.

Oxidative stress is a common feature of neurodegenerative diseases. Different natural compounds mediate neuroprotective effects by activating the Nrf2 antioxidant response. Some isothiocyanates are Nrf2 activators, including Moringin (MOR). In this study, the transcriptional profile of differentiated NSC-34 motor neurons was evaluated after treatment for 48 h and 96 h with concentrations of 0.5, 5, and 10 µM of a new MOR formulation obtained with α-cyclodextrin (α-CD). All the concentrations increased gene expression and cytoplasmic protein levels of Nrf2 at 96 h. However, the highest dose also increased nuclear Nrf2 levels at 96 h. Then, Nrf2 interactors were selected using STRING, and common biological process (BP) terms between the groups were evaluated. α-CD/MOR was able to modulate BP related to responses to oxidative stress, proteostasis, and autophagy. Specifically, the treatment with 10 µM of α-CD/MOR for 96 h induced genes involved in glutathione synthesis and proteasome subunits and reduced the expression of genes related to endoplasmic reticulum stress. Moreover, this group showed the lowest levels of the apoptotic markers Bax, cleaved caspase 9, and cleaved caspase 3. These results indicate the beneficial effects of prolonged α-CD/MOR supplementation that are mediated, at least in part, by Nrf2 activation. Then, α-CD/MOR could be a valuable treatment against neurodegenerative diseases, in particular motor neuron degeneration.

Enterovirus-A71 preferentially infects and replicates in human motor neurons, inducing neurodegeneration by ferroptosis.

Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe2+ and peroxidated lipids. Notably, the Fe2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.

Proteomic insights into extracellular vesicles in ALS for therapeutic potential of Ropinirole and biomarker discovery.

BACKGROUND: Extracellular vesicles (EVs) hold the potential for elucidating the pathogenesis of amyotrophic lateral sclerosis (ALS) and serve as biomarkers. Notably, the comparative and longitudinal alterations in the protein profiles of EVs in serum (sEVs) and cerebrospinal fluid (CSF; cEVs) of sporadic ALS (SALS) patients remain uncharted. Ropinirole hydrochloride (ROPI; dopamine D2 receptor [D2R] agonist), a new anti-ALS drug candidate identified through induced pluripotent stem cell (iPSC)-based drug discovery, has been suggested to inhibit ALS disease progression in the Ropinirole Hydrochloride Remedy for Amyotrophic Lateral Sclerosis (ROPALS) trial, but its mechanism of action is not well understood. Therefore, we tried to reveal longitudinal changes with disease progression and the effects of ROPI on protein profiles of EVs. METHODS: We collected serum and CSF at fixed intervals from ten controls and from 20 SALS patients participating in the ROPALS trial. Comprehensive proteomic analysis of EVs, extracted from these samples, was conducted using liquid chromatography/mass spectrometer (LC/MS). Furthermore, we generated iPSC-derived astrocytes (iPasts) and performed RNA sequencing on astrocytes with or without ROPI treatment. RESULTS: The findings revealed notable disparities yet high congruity in sEVs and cEVs protein profiles concerning disease status, time and ROPI administration. In SALS, both sEVs and cEVs presented elevated levels of inflammation-related proteins but reduced levels associated with unfolded protein response (UPR). These results mirrored the longitudinal changes after disease onset and correlated with the revised ALS Functional Rating Scale (ALSFRS-R) at sampling time, suggesting a link to the onset and progression of SALS. ROPI appeared to counteract these changes, attenuating inflammation-related protein levels and boosting those tied to UPR in SALS, proposing an anti-ALS impact on EV protein profiles. Reverse translational research using iPasts indicated that these changes may partly reflect the DRD2-dependent neuroinflammatory inhibitory effects of ROPI. We have also identified biomarkers that predict diagnosis and disease progression by machine learning-driven biomarker search. CONCLUSIONS: Despite the limited sample size, this study pioneers in reporting time-series proteomic alterations in serum and CSF EVs from SALS patients, offering comprehensive insights into SALS pathogenesis, ROPI-induced changes, and potential prognostic and diagnostic biomarkers.

Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model.

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including SOD1. So far, clinical records, rodent studies, and in vitro models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC, the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings, we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model, myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers, lack of sarcomeres, morphologically different nAChR clusters, and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably, trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary, a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS.

Assembling a Coculture System to Prepare Highly Pure Induced Pluripotent Stem Cell-Derived Neurons at Late Maturation Stages.

Generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) offers an unprecedented approach to modeling movement disorders such as dystonia and amyotrophic lateral sclerosis. However, achieving survival poses a significant challenge when culturing induced MNs, especially when aiming to reach late maturation stages. Utilizing hiPSC-derived motor neurons and primary mouse astrocytes, we assembled two types of coculture systems: direct coculturing of neurons with astrocytes and indirect coculture using culture inserts that physically separate neurons and astrocytes. Both systems significantly enhance neuron survival. Compared with these two systems, no significant differences in neurodevelopment, maturation, and survival within 3 weeks, allowing to prepare neurons at maturation stages. Using the indirect coculture system, we obtained highly pure MNs at the late mature stage from hiPSCs. Transcriptomic studies of hiPSC-derived MNs showed a typical neurodevelopmental switch in gene expression from the early immature stage to late maturation stages. Mature genes associated with neurodevelopment and synaptogenesis are highly enriched in MNs at late stages, demonstrating that these neurons achieve maturation. This study introduces a novel tool for the preparation of highly pure hiPSC-derived neurons, enabling the determination of neurological disease pathogenesis in neurons at late disease onset stages through biochemical approaches, which typically necessitate highly pure neurons. This advancement is particularly significant in modeling age-related neurodegeneration.

Negative features of sporadic amyotrophic lateral sclerosis: Motor neurons of Onuf's nucleus survive in ADAR2-conditional knockout mice.

Patients with amyotrophic lateral sclerosis (ALS) do not develop oculomotor disturbances and vesicorectal dysfunction until end-stage disease owing to the survival of certain motor neurons (MNs), including oculomotor neurons and MNs within Onuf's nucleus. In sporadic ALS, adenosine deaminase acting on RNA 2 (ADAR2)-mediated editing of GluA2 mRNA at the Q/R site is compromised in lower MNs. We previously developed genetically modified mice with a conditional knockout of ADAR2 in cholinergic neurons (ADAR2flox/flox/VAChT-Cre, Fast; AR2). These mice displayed slow and progressive lower motor neuron death with TAR DNA-binding protein 43 (TDP-43) pathology, attributable to insufficient editing at the GluA2 Q/R site due to ADAR2 deficiency. MN death was more common in fast-fatigable MNs owing to differential vulnerability under conditions of ADAR2 deficiency. Although facial and hypoglossal nerves were impaired in AR2 mice, cell death did not occur within the oculomotor nerve nucleus, as observed in patients with sporadic ALS. Since the basis for avoiding cystorectal damage in ALS is unknown, we compared the features of Onuf's nucleus MNs in 12-month-old AR2 mice with those in age-matched wild-type mice. Although the number of MNs was not significantly lower in AR2 mice, the neurons exhibited a shrunken morphology and TDP-43 pathology. Onuf's nucleus MNs could survive in an ADAR2-deficient state and mainly included fast fatigue-resistant (FR) and slow (S) MNs. In summary, FR and S MNs show increased resilience to ADAR2 deficiency, potentially participating in an important neuronal death avoidance mechanism in ALS.