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Fragile X Syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder, is caused by expansion of a CGG trinucleotide repeat in the 5ʹ-UTR of the Fragile X Mental Retardation-1 (FMR1) gene. Epigenetic silencing of FMR1 results in loss of the Fragile X Mental Retardation Protein (FMRP). Although most studies to date have focused on excitatory neurons, recent evidence suggests that GABAergic inhibitory networks are also affected. To investigate human GABAergic neurogenesis, we established a method to reproducibly derive inhibitory neurons from multiple FXS and control human pluripotent stem cell (hPSC) lines. Electrophysiological analyses suggested that the developing FXS neurons had a delay in the GABA functional switch, a transition in fetal development that converts the GABAA channel’s function from depolarization to hyperpolarization, with profound effects on the developing brain. To investigate the cause of this delay, we analyzed 14 400 single-cell transcriptomes from FXS and control cells at 2 stages of GABAergic neurogenesis. While control and FXS cells were similar at the earlier time point, the later-stage FXS cells retained expression of neuroblast proliferation-associated genes and had lower levels of genes associated with action potential regulation, synapses, and mitochondria compared with controls. Our analysis suggests that loss of FMRP prolongs the proliferative stage of progenitors, which may result in more neurons remaining immature during the later stages of neurogenesis. This could have profound implications for homeostatic excitatory-inhibitory circuit development in FXS, and suggests a novel direction for understanding disease mechanisms that may help to guide therapeutic interventions.

In Huntington’s disease (HD), a CAG repeat expansion mutation in theHTTgene drives a gain-of-function toxicity that disrupts mRNA processing. Although widespread dysregulation of gene splicing in the striatum has been shown in human HD post-mortem brain tissue, post-mortem analyses are likely confounded by cell type composition changes due to neuronal loss and astrogliosis in late stage HD. This limits the ability to identify dysregulation related to early pathogenesis. To study alternative splicing changes in early HD, we performed RNA-sequencing analysis in an established isogenic HD neuronal cell model. We report cell type-associated and CAG length-dependent splicing changes, and find an enrichment of RNA processing genes coupled with neuronal function-related genes showing mutantHTT-associated splicing changes. Comparison with post-mortem data also identified splicing events associated with early pathogenesis that persist to later stages of disease. In summary, our results highlight splicing dysregulation in RNA processing genes in early and late-stage HD, which may lead to disrupted neuronal function and neuropathology.

Background Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained. Results Analyzing single-cell RNA sequencing from Alzheimer’s disease (AD) and Huntington’s disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/− cells, a model for autism spectrum disorder. Conclusions Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.

Huntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in HTT. The length of this repeat, however, only explains a proportion of the variability in age of onset in patients. Genome-wide association studies have identified modifiers that contribute toward a proportion of the observed variance. By incorporating tissue-specific transcriptomic information with these results, additional modifiers can be identified. We performed a transcriptome-wide association study assessing heritable differences in genetically determined expression in diverse tissues, with genome-wide data from over 4000 patients. Functional validation of prioritized genes was undertaken in isogenic HD stem cells and patient brains. Enrichment analyses were performed with biologically relevant gene sets to identify the core pathways. HD-associated gene coexpression modules were assessed for associations with neurological phenotypes in an independent cohort and to guide drug repurposing analyses. Transcriptomic analyses identified genes that were associated with age of HD onset and displayed colocalization with gene expression signals in brain tissue (FAN1, GPR161, PMS2, SUMF2), with supporting evidence from functional experiments. This included genes involved in DNA repair, as well as novel-candidate modifier genes that have been associated with other neurological conditions. Further, cortical coexpression modules were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder. Further, these analyses expanded the pathways potentially involved in modifying HD onset and prioritized candidate therapeutics for future study., Graphical Abstract Graphical Abstract Schematic representation of methodology employed to identify transcriptomic modifiers of Huntington disease (HD). Transcriptome-wide association analyses facilitated (A) the identification of top transcriptomic HD onset modifier genes, (B) relevant biologically diverse gene sets that are enriched for this trait and (C) human cortical coexpression modules that are relevant for HD onset and related phenotypes in an aging cohort, along with signature matching of HD signals for drug repurposing.

Background The contribution of grey matter (GM) and white matter (WM) degeneration to the progressive brain atrophy in Huntington Disease (HD) has been well studied. The pathology of the spinal cord in HD is comparatively less well documented. Aim Here we sought to investigate spinal cord pathology in a mouse model of HD, and in particular WM and myelination abnormalities. Method To determine whether GM and WM regions are atrophied in the spinal cord of 12 months old BACHD mice, we measured the area of GM and WM in the spinal cord by histological assessment. Electron microscopy was used to analyze myelin fibers in the cervical area of the spinal cord, where g-ratio of myelinated axons was used as a measure of myelin thickness. To investigate the impact of inactivation of mutant huntingtin (mHTT) in oligodendroglia on these measures, we used the previously described BACHDxNG2Cre (BN) mouse line where mHTT is specifically reduced in oligodendrocyte progenitor cells (OPC). Results We show that spinal GM and WM areas are significantly atrophied in BACHD mice compared to wild-type (WT) controls. We further demonstrate that specific reduction of mHTT in oligodendroglial cells rescues the atrophy of spinal cord WM, but not GM, as observed in BACHD mice. Inactivation of mHTT in oligodendroglia had no effect on the density of oligodendroglial cells but enhanced the expression of myelin-related proteins in the spinal cord. Conclusions Overall, our findings firstly demonstrate that the myelination abnormalities observed in brain WM structures in HD extend to the spinal cord, and secondly, they suggest that specific expression of mHTT in oligodendrocytes contributes to such abnormalities.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

Background: The relative contribution of grey matter (GM) and white matter (WM) degeneration to the progressive brain atrophy in Huntington’s disease (HD) has been well studied. The pathology of the spinal cord in HD is comparatively less well documented. Objective: We aim to characterize spinal cord WM abnormalities in a mouse model of HD and evaluate whether selective removal of mutant huntingtin (mHTT) from oligodendroglia rescues these deficits. Methods: Histological assessments were used to determine the area of GM and WM in the spinal cord of 12-month-old BACHD mice, while electron microscopy was used to analyze myelin fibers in the cervical area of the spinal cord. To investigate the impact of inactivation of mHTT in oligodendroglia on these measures, we used the previously described BACHDxNG2Cre mouse line where mHTT is specifically reduced in oligodendrocyte progenitor cells. Results: We show that spinal GM and WM areas are significantly atrophied in HD mice compared to wild-type controls. We further demonstrate that specific reduction of mHTT in oligodendroglial cells rescues the atrophy of spinal cord WM, but not GM, observed in HD mice. Inactivation of mHTT in oligodendroglia had no effect on the density of oligodendroglial cells but enhanced the expression of myelin-related proteins in the spinal cord. Conclusion: Our findings demonstrate that the myelination abnormalities observed in brain WM structures in HD extend to the spinal cord and suggest that specific expression of mHTT in oligodendrocytes contributes to such abnormalities.

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation. Agency for Science, Technology and Research (A*STAR) Nanyang Technological University Published version M.A.P. was supported by funds from theAgency for Science Technology and Research and the NationalUniversity of Singapore. S.P. was supported by funds from LKC Schoolof Medicine and SCELSE.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5′ untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulate. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that aging further reduces HTT palmitoylation. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons.HighlightsPalmitoylation of mHTT is reduced in multiple transgenic HD mouse modelsHTT palmitoylation decreases with increasing polyQ length in HD patient cellsmHTT-lowering in mouse brains does not rescue aberrant palmitoylationmHTT palmitoylation in HD patient-derived cells can be rescued via APT inhibitionPromoting palmitoylation reduces mHTT aggregation and cytotoxicity in vitro

The generation of structurally standardized human pluripotent stem cell (hPSC)‐derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio‐temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly‐organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination‐like folding, where primitive ectodermal cells undergo E‐to‐N‐cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio‐temporal patterning of the mesoendoderm, which guides the development and self‐organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFβ signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient‐derived hPSCs as well as differentiating between different classes of neural tube defect‐inducing drugs., This paper reports the generation of a reproducible and highly organized neuroepithelial (NE) tissue by combining human pluripotent stem cell (hPSC) micropatterning and a temporally sequenced induction protocol to specify NE cells in spatial juxtaposition to mesoendoderm cells. By evaluating NE tissue structural dysmorphia in the micropatterned NE model, we can successfully model gene‐ and drug‐induced neurodevelopmental defects.

BackgroundMany neurodegenerative diseases (NDs) develop only later in life, when cells in the nervous system lose their structure or function. In genetic forms of NDs, this late onset phenomenon remains largely unexplained.ResultsAnalyzing single cell RNA sequencing (scRNA-seq) from Alzheimer’s disease (AD) patients, we find increased transcriptional heterogeneity in AD excitatory neurons. We hypothesized that transcriptional heterogeneity precedes ND pathologies. To test this idea experimentally, we used juvenile forms (72Q; 180Q) of Huntington’s disease (HD) iPSCs, differentiated them into committed neuronal progenitors, and obtained single cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking-down several differentially-variable genes resulted in increased aggregate formation, a pathology associated with HD. We further validated the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder.ConclusionsOverall, our results suggest that although NDs develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy., German Research Foundation (DFG); European Union (European Union); NEUROMICS Network; International Coordination Action (ICA); Fund for Scientific Research Flanders (FWO); Netherlands Organization for Scientific Research (ZONMW VIDI); National Medical Research Council, Singapore; A Strategic Positioning Fund on Genetic Orphan Diseases (GODAFIT); Industry Alignment Fund on Singapore Childhood Undiagnosed Diseases Program (SUREKids); Biomedical Research Council, A*STAR; Diana and Steve Marienhoff Fashion Industries Guild Endowed Fellowship in Pediatric Neuromuscular Diseases; Fondazione Bambino Gesù (Vite Coraggiose); Canadian Institutes of Health Research; Natural Sciences and Engineering Research Council of Canada; Eurocores Program EuroEPINOMICS; University of Antwerp Research Fund; FRAXA Foundation; Brain & Behavior Research Foundation, NARSAD Young Investigator Grant

Background Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking. Methods Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology. Results We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder–associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient–derived human-induced pluripotent stem cell neural cells. Conclusions Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.

Ermin is an actin-binding protein found almost exclusively in the central nervous system (CNS) as a component of myelin sheaths. Although Ermin has been predicted to play a role in the formation and stability of myelin sheaths, this has not been directly examined in vivo. Here we show that Ermin is essential for myelin sheath integrity and normal saltatory conduction. Loss of Ermin in mice caused de-compacted and fragmented myelin sheaths and led to slower conduction along with progressive neurological deficits. RNA sequencing of the corpus callosum, the largest white matter structure in the CNS, pointed to inflammatory activation in aged Ermin-deficient mice, which was corroborated by increased levels of microgliosis and astrogliosis. The inflammatory milieu and myelin abnormalities were further associated with increased susceptibility to immune-mediated demyelination insult in Ermin knockout mice. Supporting a possible role of Ermin deficiency in inflammatory white matter disorders, a rare inactivating mutation in the ERMN gene was identified in multiple sclerosis patients. Our findings demonstrate a critical role for Ermin in maintaining myelin integrity. Given its near exclusive expression in myelinating oligodendrocytes, Ermin deficiency represents a compelling “inside-out” model of inflammatory dysmyelination and may offer a new paradigm for the development of myelin stability-targeted therapies.

FXS is the most common genetic cause of intellectual (ID) and autism spectrum disorders (ASD). FXS is caused by loss of FMRP, an RNA-binding protein involved in the translational regulation of a large number of neuronal mRNAs. Absence of FMRP has been shown to lead to elevated protein synthesis and is thought to be a major cause of the synaptic plasticity and behavioural deficits in FXS. The increase in protein synthesis results in part from abnormal activation of key protein translation pathways downstream of ERK1/2 and mTOR signalling. Pharmacological and genetic interventions that attenuate hyperactivation of these pathways can normalize levels of protein synthesis and improve phenotypic outcomes in animal models of FXS. Several efforts are currently underway to trial this strategy in patients with FXS. To date, elevated global protein synthesis as a result of FMRP loss has not been validated in the context of human neurons. Here, using an isogenic human stem cell-based model, we show that de novo protein synthesis is elevated in FMRP-deficient neural cells. We further show that this increase is associated with elevated ERK1/2 and Akt signalling and can be rescued by metformin treatment. Finally, we examined the effect of normalizing protein synthesis on phenotypic abnormalities in FMRP-deficient neural cells. We find that treatment with metformin attenuates the increase in proliferation of FMRP-deficient neural progenitor cells but not the neuronal deficits in neurite outgrowth. The elevated level of protein synthesis and the normalization of neural progenitor proliferation by metformin treatment were validated in additional control and FXS patient-derived hiPSC lines. Overall, our results validate that loss of FMRP results in elevated de novo protein synthesis in human neurons and suggest that approaches targeting this abnormality are likely to be of partial therapeutic benefit in FXS.

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by mutant huntingtin (mHTT). Phosphorylation at serine-421 (pS421) of mHTT has been shown to be neuroprotective in cellular and rodent models. However, the genetic context of these models differs from that of HD patients. Here we employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, we generated isogenic hiPSC lines in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phospho-resistant alanine (S421A). We observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although we observed no effect of S421D on mHTT clearance or axonal transport, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, our analysis revealed modulation of several aspects of mitochondrial form and function. These include mitochondrial surface area, volume, and counts, as well as improved mitochondrial membrane potential and oxidative phosphorylation. Our study validates the protective role of pS421 on mHTT and highlights a facet of the relationship between mHTT and mitochondrial changes in the context of human physiology with potential relevance to the pathogenesis of HD.

Olfactory dysfunction and altered neurogenesis are observed in several neurodegenerative disorders including Huntington disease (HD). These deficits occur early and correlate with a decline in global cognitive performance, depression and structural abnormalities of the olfactory system including the olfactory epithelium, bulb and cortices. However, the role of olfactory system dysfunction in the pathogenesis of HD remains poorly understood and the mechanisms underlying this dysfunction are unknown. We show that deficits in odour identification, discrimination and memory occur in HD individuals. Assessment of the olfactory system in an HD murine model demonstrates structural abnormalities in the olfactory bulb (OB) and piriform cortex, the primary cortical recipient of OB projections. Furthermore, a decrease in piriform neuronal counts and altered expression levels of neuronal nuclei and tyrosine hydroxylase in the OB are observed in the YAC128 HD model. Similar to the human HD condition, olfactory dysfunction is an early phenotype in the YAC128 mice and concurrent with caspase activation in the murine HD OB. These data provide a link between the structural olfactory brain region atrophy and olfactory dysfunction in HD and suggest that cell proliferation and cell death pathways are compromised and may contribute to the olfactory deficits in HD.

Laquinimod, an immunomodulatory agent under clinical development for Huntington disease (HD), has recently been shown to confer behavioural improvements that are coupled with prevention of atrophy of the white matter (WM)-rich corpus callosum (CC) in the YAC128 HD mice. However, the nature of the WM improvements is not known yet. Here we investigated the effects of laquinimod on HD-related myelination deficits at the cellular, molecular and ultrastructural levels. We showed that laquinimod treatment improves motor learning and motor function deficits in YAC128 HD mice, and confirmed its antidepressant effect even at the lowest dose used. In addition, we demonstrated for the first time the beneficial effects of laquinimod on myelination in the posterior region of the CC where it reversed changes in myelin sheath thickness and rescued Mbp mRNA and protein deficits. Furthermore, the effect of laquinimod on myelin-related gene expression was not region-specific since the levels of the Mbp and Plp1 transcripts were also increased in the striatum. Also, we did not detect changes in immune cell densities or levels of inflammatory genes in 3-month-old YAC128 HD mice, and these were not altered with laquinimod treatment. Thus, the beneficial effects of laquinimod on HD-related myelination abnormalities in YAC128 HD mice do not appear to be dependent on its immunomodulatory activity. Altogether, our findings describe the beneficial effects of laquinimod treatment on HD-related myelination abnormalities and highlight its therapeutic potential for the treatment of WM pathology in HD patients.

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease., Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

Summary Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells, including impaired neural rosette formation, increased susceptibility to growth factor withdrawal, and deficits in mitochondrial respiration, are rescued in isogenic controls. Importantly, using genome-wide expression analysis, we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines, suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities, and the importance of isogenic controls for disease modeling using hiPSCs., Graphical Abstract, Highlights • A CRISPR-Cas9 and PiggyBac-based approach allows efficient correction of HD hiPSCs • The corrected hiPSCs can be differentiated into synaptically active neurons • Correction of HD gene mutation reverses a number of phenotypic abnormalities • Isogenic hiPSCs help distinguish mutation from genetic background-related effects, Pouladi and colleagues show that genetic correction of HD human induced pluripotent stem cells using a CRISPR-Cas9 and piggyBac transposon-based approach rescues a number of phenotypic abnormalities in derived neural cells, including increased susceptibility to growth factor withdrawal and deficits in mitochondrial respiration, and helps distinguish HD-specific from genetic background-related molecular and cellular phenotypes.

Huntington disease (HD) manifests in both adult and juvenile forms. Mutant HTT gene carriers are thought to undergo normal brain development followed by a degenerative phase, resulting in progressive clinical manifestations. However, recent studies in children and prodromal individuals at risk for HD have raised the possibility of abnormal neurodevelopment. Although key findings in rodent models support this notion, direct evidence in the context of human physiology remains lacking. Using a panel of isogenic HD human embryonic pluripotent stem cells and cerebral organoids, we investigated the impact of mutant HTT on early neurodevelopment. We find that ventricular zone-like neuroepithelial progenitor layer expansion is blunted in an HTT CAG repeat length-dependent manner due to premature neurogenesis in HD cerebral organoids, driven by cell intrinsic processes. Transcriptional profiling and imaging analysis revealed impaired cell cycle regulatory processes, increased G1 length, and increased asymmetric division of apical progenitors, collectively contributing to premature neuronal differentiation. We demonstrate increased activity of the ATM-p53 pathway, an up-stream regulator of cell cycle processes, and show that treatment with ATM antagonists partially rescues the blunted neuroepithelial progenitor expansion in HD organoids. Our findings suggest that CAG repeat length regulates the balance between neural progenitor expansion and differentiation during early neurodevelopment. Our results further support the view that HD, at least in its early-onset forms, may not be a purely neurodegenerative disorder, and that abnormal neurodevelopment may be a component of HD pathophysiology.

Huntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in the HTT gene. In an attempt to identify genomic modifiers that contribute towards the age of onset of HD, we performed a transcriptome wide association study assessing heritable differences in genetically determined expression in diverse tissues, employing genome wide data from over 4,000 patients. This identified genes that showed evidence for colocalization and replication, with downstream functional validation being performed in isogenic HD stem cells and patient brains. Enrichment analyses detected associations with various biologically-relevant gene sets and striatal coexpression modules that are mediated by CAG length. Further, cortical coexpression modules that are relevant for HD onset were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder.

White matter abnormalities are a nearly universal pathological feature of neurodegenerative disorders including Huntington disease (HD). A long-held assumption is that this white matter pathology is simply a secondary outcome of the progressive neuronal loss that manifests with advancing disease. Using a mouse model of HD, here we show that white matter and myelination abnormalities are an early disease feature appearing before the manifestation of any behavioral abnormalities or neuronal loss. We further show that selective inactivation of mutant huntingtin (mHTT) in the NG2+ oligodendrocyte progenitor cell population prevented myelin abnormalities and certain behavioral deficits in HD mice. Strikingly, the improvements in behavioral outcomes were seen despite the continued expression of mHTT in nonoligodendroglial cells including neurons, astrocytes, and microglia. Using RNA-seq and ChIP-seq analyses, we implicate a pathogenic mechanism that involves enhancement of polycomb repressive complex 2 (PRC2) activity by mHTT in the intrinsic oligodendroglial dysfunction and myelination deficits observed in HD. Our findings challenge the long-held dogma regarding the etiology of white matter pathology in HD and highlight the contribution of epigenetic mechanisms to the observed intrinsic oligodendroglial dysfunction. Our results further suggest that ameliorating white matter pathology and oligodendroglial dysfunction may be beneficial for HD. Agency for Science, Technology and Research (A*STAR) Accepted version We thank members of the M.A.P. laboratory for helpful discussions and comments. C.F.B. is supported by a Singapore International Graduate Award from the Agency for Science, Technology and Research (A*STAR). M.A.P. is supported by grants from A*STAR and the National University of Singapore.

Attempts to constitutively knockout HTT in rodents resulted in embryonic lethality, curtailing efforts to study HTT function later in development. Here we show that HTT is dispensable for early zebrafish development, contrasting published zebrafish morpholino experiment results. Homozygous HTT knockouts were embryonically viable and appeared developmentally normal through juvenile stages. Comparison of adult fish revealed significant reduction in body size and fitness in knockouts compared to hemizygotes and wildtype fish, indicating an important role for wildtype HTT in postnatal development. Our zebrafish model provides an opportunity to examine the function of wildtype HTT later in development.

White matter (WM) abnormalities are a well-established feature of Huntington disease (HD), although their nature is not fully understood. Here, we asked whether remyelination as a measure of WM plasticity is impaired in a model of HD. Using the cuprizone assay, we examined demyelination and remyelination responses in YAC128 HD mice. Treatment with 0.2% cuprizone (CPZ) for 6 weeks resulted in significant reduction in mature (GSTπ-positive) oligodendrocyte counts and FluoroMyelin staining in the corpus callosum, leading to similar demyelination states in YAC128 and wild-type (WT) mice. Six weeks following cessation of CPZ, we observed robust remyelination in WT mice as indicated by an increase in mature oligodendrocyte counts and FluoroMyelin staining. In contrast, YAC128 mice exhibited an impaired remyelination response. The increase in mature oligodendrocyte counts in YAC128 HD mice following CPZ cessation was lower than that of WT. Furthermore, there was no increase in FluoroMyelin staining compared to the demyelinated state in YAC128 mice. We confirmed these findings using electron microscopy where the CPZ-induced reduction in myelinated axons was reversed following CPZ cessation in WT but not YAC128 mice. Our findings demonstrate that remyelination is impaired in YAC128 mice and suggest that WM plasticity may be compromised in HD.

ObjectiveTo investigate the possible involvement of germline mutations in a neurologic condition involving diffuse white matter lesions.MethodsThe patients were 3 siblings born to healthy parents. We performed homozygosity mapping, whole-exome sequencing, site-directed mutagenesis, and immunoblotting.ResultsAll 3 patients showed clinical manifestations of ataxia, behavioral and mood changes, premature hair loss, memory loss, and lower back pain. In addition, they presented with inflammatory-like features and recurrent rhinitis. MRI showed abnormal diffuse demyelination lesions in the brain and myelitis in the spinal cord. We identified an insertion in high-temperature requirement A (HTRA1), which showed complete segregation in the pedigree. Functional analysis showed the mutation to affect stability and secretion of truncated protein.ConclusionsThe patients' clinical manifestations are consistent with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL; OMIM #600142), which is known to be caused by HTRA1 mutations. Because some aspects of the clinical presentation deviate from those reported for CARASIL, our study expands the spectrum of clinical consequences of loss-of-function mutations in HTRA1.

Rett syndrome (RTT) is a childhood neurodevelopmental disorder caused by mutations in MECP2. To study the molecular mechanisms underlying RTT, four sublines of H1 hESCs were generated, carrying a hemizygous knockout or mutant allele of MECP2. Exons 3 and 4 of MECP2 were targeted using the CRISPR/Cas9 nuclease system., Strategic Positioning Fund for Genetic Orphan Diseases; SUREKids from the Agency for Science Technology and Research (Singapore); TLGM-NDD from Agency for Science Technology and Research (Singapore); the National University of Singapore

In article number 2001100, Yi‐Chin Toh and co‐workers combine hPSC micropatterning and a temporally‐sequenced induction protocol to specify neuroepithelial (NE) cells in spatial juxtaposition to mesoendoderm cells. This results in the formation of a reproducible and highly‐organized NE tissue. By evaluating NE tissue structural dysmorphia in the micropatterned NE model, gene‐ and drug‐induced neurodevelopmental defects can be successfully modelled. [Image: see text]