HNRNPK alleviates RNA toxicity by counteracting DNA damage in C9orf72 ALS.

A 'GGGGCC' repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.

Mitochondrial bioenergetic deficits in C9orf72 amyotrophic lateral sclerosis motor neurons cause dysfunctional axonal homeostasis.

Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), where the key pathological cell-type, the motor neuron (MN), has an axon extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. To address this, we undertook cellular and molecular interrogation of multiple patient-derived induced pluripotent stem cell lines and patient autopsy samples harbouring the most common ALS causing mutation, C9orf72. Using paired mutant and isogenic expansion-corrected controls, we show that C9orf72 MNs have shorter axons, impaired fast axonal transport of mitochondrial cargo, and altered mitochondrial bioenergetic function. RNAseq revealed reduced gene expression of mitochondrially encoded electron transport chain transcripts, with neuropathological analysis of C9orf72-ALS post-mortem tissue importantly confirming selective dysregulation of the mitochondrially encoded transcripts in ventral horn spinal MNs, but not in corresponding dorsal horn sensory neurons, with findings reflected at the protein level. Mitochondrial DNA copy number was unaltered, both in vitro and in human post-mortem tissue. Genetic manipulation of mitochondrial biogenesis in C9orf72 MNs corrected the bioenergetic deficit and also rescued the axonal length and transport phenotypes. Collectively, our data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function.

Dysregulation of AMPA receptor subunit expression in sporadic ALS post-mortem brain.

Amyotrophic lateral sclerosis (ALS) is characterised by progressive motor neuron degeneration. Although there are over 40 genes associated with causal monogenetic mutations, the majority of ALS patients are not genetically determined. Causal ALS mutations are being increasingly mechanistically studied, though how these mechanisms converge and diverge between the multiple known familial causes of ALS (fALS) and sporadic forms of ALS (sALS) and furthermore between different neuron types, is poorly understood. One common pathway that is implicated in selective motor neuron death is enhanced α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPAR)-mediated excitoxicity. Specifically, human in vitro and pathological evidence has linked the C9orf72 repeat expansion mutation to a relative increase in the Ca2+ -permeable AMPAR population due to AMPAR subunit dysregulation. Here, we provide the first comparative quantitative assessment of the expression profile of AMPAR subunit transcripts, using BaseScope, in post-mortem lower motor neurons (spinal cord, anterior horn), upper motor neurons (motor cortex) and neurons of the pre-frontal cortex in sALS and fALS due to mutations in SOD1 and C9orf72. Our data indicated that AMPAR dysregulation is prominent in lower motor neurons in all ALS cases. However, sALS and mutant C9orf72 cases exhibited GluA1 upregulation whereas mutant SOD1 cases displayed GluA2 down regulation. We also showed that sALS cases exhibited widespread AMPAR dysregulation in the motor and pre-frontal cortex, though the exact identity of the AMPAR subunit being dysregulated was dependent on brain region. In contrast, AMPAR dysregulation in mutant SOD1 and C9orf72 cases was restricted to lower motor neurons only. Our data highlight the complex dysregulation of AMPAR subunit expression that reflects both converging and diverging mechanisms at play between different brain regions and between ALS cohorts. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology.

Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.

Familial t(1;11) translocation is associated with disruption of white matter structural integrity and oligodendrocyte-myelin dysfunction.

Although the underlying neurobiology of major mental illness (MMI) remains unknown, emerging evidence implicates a role for oligodendrocyte-myelin abnormalities. Here, we took advantage of a large family carrying a balanced t(1;11) translocation, which substantially increases risk of MMI, to undertake both diffusion tensor imaging and cellular studies to evaluate the consequences of the t(1;11) translocation on white matter structural integrity and oligodendrocyte-myelin biology. This translocation disrupts among others the DISC1 gene which plays a crucial role in brain development. We show that translocation-carrying patients display significant disruption of  white matter integrity compared with familial controls. At a cellular level, we observe dysregulation of key pathways controlling oligodendrocyte development and morphogenesis in induced pluripotent stem cell (iPSC) derived case oligodendrocytes. This is associated with reduced proliferation and a stunted morphology in vitro. Further, myelin internodes in a humanized mouse model that recapitulates the human translocation as well as after transplantation of t(1;11) oligodendrocyte progenitors were significantly reduced when  compared with controls. Thus we provide evidence that the t(1;11) translocation has biological effects at both the systems and cellular level that together suggest oligodendrocyte-myelin dysfunction.

Maturation and electrophysiological properties of human pluripotent stem cell-derived oligodendrocytes.

Rodent-based studies have shown that the membrane properties of oligodendrocytes play prominent roles in their physiology and shift markedly during their maturation from the oligodendrocyte precursor cell (OPC) stage. However, the conservation of these properties and maturation processes in human oligodendrocytes remains unknown, despite their dysfunction being implicated in human neurodegenerative diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Here, we have defined the membrane properties of human oligodendrocytes derived from pluripotent stem cells as they mature from the OPC stage, and have identified strong conservation of maturation-specific physiological characteristics reported in rodent systems. We find that as human oligodendrocytes develop and express maturation markers, they exhibit a progressive decrease in voltage-gated sodium and potassium channels and a loss of tetrodotoxin-sensitive spiking activity. Concomitant with this is an increase in inwardly rectifying potassium channel activity, as well as a characteristic switch in AMPA receptor composition. All these steps mirror the developmental trajectory observed in rodent systems. Oligodendrocytes derived from mutant C9ORF72-carryng ALS patient induced pluripotent stem cells did not exhibit impairment to maturation and maintain viability with respect to control lines despite the presence of RNA foci, suggesting that maturation defects may not be a primary feature of this mutation. Thus, we have established that the development of human oligodendroglia membrane properties closely resemble those found in rodent cells and have generated a platform to enable the impact of human neurodegenerative disease-causing mutations on oligodendrocyte maturation to be studied.

Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

Neuronal Circuit Dysfunction in Amyotrophic Lateral Sclerosis.

The primary neural circuit affected in Amyotrophic Lateral Sclerosis (ALS) patients is the corticospinal motor circuit, originating in upper motor neurons (UMNs) in the cerebral motor cortex which descend to synapse with the lower motor neurons (LMNs) in the spinal cord to ultimately innervate the skeletal muscle. Perturbation of these neural circuits and consequent loss of both UMNs and LMNs, leading to muscle wastage and impaired movement, is the key pathophysiology observed. Despite decades of research, we are still lacking in ALS disease-modifying treatments. In this review, we document the current research from patient studies, rodent models, and human stem cell models in understanding the mechanisms of corticomotor circuit dysfunction and its implication in ALS. We summarize the current knowledge about cortical UMN dysfunction and degeneration, altered excitability in LMNs, neuromuscular junction degeneration, and the non-cell autonomous role of glial cells in motor circuit dysfunction in relation to ALS. We further highlight the advances in human stem cell technology to model the complex neural circuitry and how these can aid in future studies to better understand the mechanisms of neural circuit dysfunction underpinning ALS.

Plekhg5 controls the unconventional secretion of Sod1 by presynaptic secretory autophagy.

Increasing evidence suggests an essential function for autophagy in unconventional protein secretion (UPS). However, despite its relevance for the secretion of aggregate-prone proteins, the mechanisms of secretory autophagy in neurons have remained elusive. Here we show that the lower motoneuron disease-associated guanine exchange factor Plekhg5 drives the UPS of Sod1. Mechanistically, Sod1 is sequestered into autophagosomal carriers, which subsequently fuse with secretory lysosomal-related organelles (LROs). Exocytosis of LROs to release Sod1 into the extracellular milieu requires the activation of the small GTPase Rab26 by Plekhg5. Deletion of Plekhg5 in mice leads to the accumulation of Sod1 in LROs at swollen presynaptic sites. A reduced secretion of toxic ALS-linked SOD1G93A following deletion of Plekhg5 in SOD1G93A mice accelerated disease onset while prolonging survival due to an attenuated microglia activation. Using human iPSC-derived motoneurons we show that reduced levels of PLEKHG5 cause an impaired secretion of ALS-linked SOD1. Our findings highlight an unexpected pathophysiological mechanism that converges two motoneuron disease-associated proteins into a common pathway.

Safety and efficacy of memantine and trazodone versus placebo for motor neuron disease (MND SMART): stage two interim analysis from the first cycle of a phase 3, multiarm, multistage, randomised, adaptive platform trial.

BACKGROUND: Motor neuron disease represents a group of progressive and incurable diseases that are characterised by selective loss of motor neurons, resulting in an urgent need for rapid identification of effective disease-modifying therapies. The MND SMART trial aims to test the safety and efficacy of promising interventions efficiently and definitively against a single contemporaneous placebo control group. We now report results of the stage two interim analysis for memantine and trazodone. METHODS: MND SMART is an investigator-led, phase 3, double-blind, placebo-controlled, multiarm, multistage, randomised, adaptive platform trial recruiting at 20 hospital centres in the UK. Individuals older than 18 years with a confirmed diagnosis of either amyotrophic lateral sclerosis classified by the revised El Escorial criteria, primary lateral sclerosis, progressive muscular atrophy, or progressive bulbar palsy, regardless of disease duration, were eligible for screening. Participants were randomised (1:1:1) to receive oral trazodone 200 mg once a day, oral memantine 20 mg once a day, or matched placebo using a computer-generated minimisation algorithm delivered via a secure web-based system. Co-primary outcome measures were clinical functioning, measured by rate of change in the Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R), and survival. Comparisons were conducted in four stages, with predefined criteria for stopping at the end of stages one and two. We report interim analysis from the stage two results, which was done when 100 participants per group (excluding long survivors, defined as >8 years since diagnosis at baseline) completed a minimum of 12 months of follow-up for the candidate investigational medicinal products. The trial is registered on the European Clinical Trials Registry, 2019-000099-41, and ClinicalTrials.gov, NCT04302870, and is ongoing. FINDINGS: Between Feb 27, 2020, and July 24, 2023 (database lock for interim analysis two), 554 people with a motor neuron disease were randomly allocated to memantine (183 [33%]), trazodone (185 [33%]), or placebo (186 [34%]). The primary interim analysis population comprised 530 participants, of whom 175 (33%) had been allocated memantine, 175 (33%) had been allocated trazodone, and 180 (34%) had been allocated placebo. Over 12 months of follow-up, the mean rate of change per month in ALSFRS-R was -0·650 for memantine, -0·625 for trazodone, and -0·655 for placebo (memantine versus placebo estimated mean difference 0·033, one-sided 90% CI lower level -0·085; one-sided p=0·36; trazodone vs placebo: 0·065, -0·051; one-sided p=0·24). The one-sided p values were both above the significance threshold of 10%, indicating that neither memantine nor trazodone groups met the criteria for continuation. There were 483 participants with at least one adverse event (145 [77%] on placebo, 170 [91%] on memantine, and 168 [90%] on trazodone). There were 88 participants with at least one serious adverse event (37 [20%] on memantine, 27 [14%] on trazodone, and 24 [13%] on placebo). A total of 11 serious adverse event led to treatment discontinuation. There was no survival difference between comparisons, with 49 deaths in the memantine group, 52 deaths in the trazodone group, and 48 deaths in the placebo group. INTERPRETATION: Neither memantine nor trazodone improved efficacy outcomes compared with placebo. This result is sufficiently powered to warrant no further testing of trazodone or memantine in motor neuron disease at the doses evaluated in this study. The multiarm multistage design shows important benefits in reducing the time, cost, and participant numbers to reach a definitive result. FUNDING: The Euan MacDonald Centre, MND Scotland, My Name'5 Doddie Foundation, and Baillie Gifford.

Blood-CNS barrier dysfunction in amyotrophic lateral sclerosis: Proposed mechanisms and clinical implications.

There is strong evidence for blood-brain and blood-spinal cord barrier dysfunction at the early stages of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Since impairment of the blood-central nervous system barrier (BCNSB) occurs during the pre-symptomatic stages of ALS, the mechanisms underlying this pathology are likely also involved in the ALS disease process. In this review, we explore how drivers of ALS disease, particularly mitochondrial dysfunction, astrocyte pathology and neuroinflammation, may contribute to BCNSB impairment. Mitochondria are highly abundant in BCNSB tissue and mitochondrial dysfunction in ALS contributes to motor neuron death. Likewise, astrocytes adopt key physical, transport and metabolic functions at the barrier, many of which are impaired in ALS. Astrocytes also show raised expression of inflammatory markers in ALS and ablating ALS-causing transgenes in astrocytes slows disease progression. In addition, key drivers of neuroinflammation, including TAR DNA-binding protein 43 (TDP-43) pathology, matrix metalloproteinase activation and systemic inflammation, affect BCNSB integrity in ALS. Finally, we discuss the translational implications of BCNSB dysfunction in ALS, including the development of biomarkers for disease onset and progression, approaches aimed at restoring BCNSB integrity and in vitro modelling of the neurogliovascular system.

A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS.

The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.

Astrocytes mediate cell non-autonomous correction of aberrant firing in human FXS neurons.

Pre-clinical studies of fragile X syndrome (FXS) have focused on neurons, with the role of glia remaining largely underexplored. We examined the astrocytic regulation of aberrant firing of FXS neurons derived from human pluripotent stem cells. Human FXS cortical neurons, co-cultured with human FXS astrocytes, fired frequent short-duration spontaneous bursts of action potentials compared with less frequent, longer-duration bursts of control neurons co-cultured with control astrocytes. Intriguingly, bursts fired by FXS neurons co-cultured with control astrocytes are indistinguishable from control neurons. Conversely, control neurons exhibit aberrant firing in the presence of FXS astrocytes. Thus, the astrocyte genotype determines the neuronal firing phenotype. Strikingly, astrocytic-conditioned medium, and not the physical presence of astrocytes, is capable of determining the firing phenotype. The mechanistic basis of this effect indicates that the astroglial-derived protein, S100ß, restores normal firing by reversing the suppression of a persistent sodium current in FXS neurons.

Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration.

Although microglial activation is widely found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the underlying mechanism(s) are poorly understood. Here, using human-induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harboring the most common ALS/FTD mutation (C9orf72, mC9-MG), gene-corrected isogenic controls (isoC9-MG), and C9orf72 knockout hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated immune response upon stimulation with lipopolysaccharide. Analysis of the C9ORF72 interactome revealed that C9ORF72 interacts with regulators of autophagy and functional studies showed impaired initiation of autophagy in mC9-MG and C9KO-MG. Coculture studies with motor neurons (MNs) demonstrated that the autophagy deficit in mC9-MG drives increased vulnerability of mC9-MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated both cell-autonomous functional deficits in hiPSC-MG and MN death in MG-MN coculture. Together, these findings reveal an important role for C9ORF72 in regulating immune homeostasis and identify dysregulation in myeloid cells as a contributor to neurodegeneration in ALS/FTD.

Systematic, comprehensive, evidence-based approach to identify neuroprotective interventions for motor neuron disease: using systematic reviews to inform expert consensus.

OBJECTIVES: Motor neuron disease (MND) is an incurable progressive neurodegenerative disease with limited treatment options. There is a pressing need for innovation in identifying therapies to take to clinical trial. Here, we detail a systematic and structured evidence-based approach to inform consensus decision making to select the first two drugs for evaluation in Motor Neuron Disease-Systematic Multi-arm Adaptive Randomised Trial (MND-SMART: NCT04302870), an adaptive platform trial. We aim to identify and prioritise candidate drugs which have the best available evidence for efficacy, acceptable safety profiles and are feasible for evaluation within the trial protocol. METHODS: We conducted a two-stage systematic review to identify potential neuroprotective interventions. First, we reviewed clinical studies in MND, Alzheimer's disease, Huntington's disease, Parkinson's disease and multiple sclerosis, identifying drugs described in at least one MND publication or publications in two or more other diseases. We scored and ranked drugs using a metric evaluating safety, efficacy, study size and study quality. In stage two, we reviewed efficacy of drugs in MND animal models, multicellular eukaryotic models and human induced pluripotent stem cell (iPSC) studies. An expert panel reviewed candidate drugs over two shortlisting rounds and a final selection round, considering the systematic review findings, late breaking evidence, mechanistic plausibility, safety, tolerability and feasibility of evaluation in MND-SMART. RESULTS: From the clinical review, we identified 595 interventions. 66 drugs met our drug/disease logic. Of these, 22 drugs with supportive clinical and preclinical evidence were shortlisted at round 1. Seven drugs proceeded to round 2. The panel reached a consensus to evaluate memantine and trazodone as the first two arms of MND-SMART. DISCUSSION: For future drug selection, we will incorporate automation tools, text-mining and machine learning techniques to the systematic reviews and consider data generated from other domains, including high-throughput phenotypic screening of human iPSCs.

Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons.

Axonal transport is essential for the development, function, and survival of the nervous system. In an energy-demanding process, motor proteins act in concert with microtubules to deliver cargoes, such as organelles, from one end of the axon to the other. Perturbations in axonal transport are a prominent phenotype of many neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe a simple method to fluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell-derived motor neurons. This method enables the sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.

The ciliary gene INPP5E confers dorsal telencephalic identity to human cortical organoids by negatively regulating Sonic hedgehog signaling.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

40 Years of CSF Toxicity Studies in ALS: What Have We Learnt About ALS Pathophysiology?

Based on early evidence of in vitro neurotoxicity following exposure to serum derived from patients with amyotrophic lateral sclerosis (ALS), several studies have attempted to explore whether cerebrospinal fluid (CSF) obtained from people with ALS could possess similar properties. Although initial findings proved inconclusive, it is now increasingly recognized that ALS-CSF may exert toxicity both in vitro and in vivo. Nevertheless, the mechanism underlying CSF-induced neurodegeneration remains unclear. This review aims to summarize the 40-year long history of CSF toxicity studies in ALS, while discussing the various mechanisms that have been proposed, including glutamate excitotoxicity, proteotoxicity and oxidative stress. Furthermore, we consider the potential implications of a toxic CSF circulatory system in the pathophysiology of ALS, and also assess its significance in the context of current ALS research.

Dysregulation in Subcellular Localization of Myelin Basic Protein mRNA Does Not Result in Altered Myelination in Amyotrophic Lateral Sclerosis.

Pathological hallmarks of amyotrophic lateral sclerosis (ALS), including protein misfolding, are well established in oligodendrocytes. More recently, an RNA trafficking deficit of key myelin proteins has been suggested in oligodendrocytes in ALS but the extent to which this affects myelination and the relative contribution of this to disease pathogenesis is unclear. ALS autopsy research findings showing demyelination contrasts with the routine clinical-pathological workup of ALS cases where it is rare to see white matter abnormalities other than simple Wallerian degeneration secondary to widespread neuronal loss. To begin to address this apparent variance, we undertook a comprehensive evaluation of myelination at an RNA, protein and structural level using human pathological material from sporadic ALS patients, genetic ALS patients (harboring C9orf72 mutation) and age- and sex-matched non-neurological controls. We performed (i) quantitative spatial profiling of the mRNA transcript encoding myelin basic protein (MBP), (ii) quantification of MBP protein and (iii) the first quantitative structural assessment of myelination in ALS post-mortem specimens by electron microscopy. We show no differences in MBP protein levels or ultrastructural myelination, despite a significant dysregulation in the subcellular trafficking of MBP mRNA in ALS patients compared to controls. We therefore confirm that whilst there are cell autonomous mRNA trafficking deficits affecting oligodendrocytes in ALS, this has no effect on myelin structure.