Huntington’s disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD. Nguyen et al. identify TDP-43 and METTL3 as key regulators of disrupted RNA splicing in Huntington’s disease, offering insight into how TDP-43 mislocalization and aberrant m6A RNA modification and localization relate to disease pathogenesis.

Huntington's Disease (HD) is caused by a CAG repeat expansion in the gene encoding huntingtin (HTT). While normal HTT function appears impacted by the mutation, the specific pathways unique to CAG repeat expansion versus loss of normal function are unclear. To understand the impact of the CAG repeat expansion, we evaluated biological signatures of HTT knockout (HTT KO) versus those that occur from the CAG repeat expansion by applying multi-omics, live cell imaging, survival analysis and a novel feature-based pipeline to study cortical neurons (eCNs) derived from an isogenic human embryonic stem cell series (RUES2). HTT KO and the CAG repeat expansion influence developmental trajectories of eCNs, with opposing effects on growth. Network analyses of differentially expressed genes and proteins associated with enriched epigenetic motifs identified subnetworks common to CAG repeat expansion and HTT KO that include neuronal differentiation, cell cycle regulation, and mechanisms related to transcriptional repression, and may represent gain-of-function mechanisms that cannot be explained by HTT loss of function alone. A combination of dominant and loss-of-function mechanisms are likely involved in the aberrant neurodevelopmental and neurodegenerative features of HD that can help inform therapeutic strategies.

Huntington disease (HD) is a genetic neurodegenerative disease caused by cytosine, adenine, guanine (CAG) expansion in the Huntingtin ( HTT ) gene, translating to an expanded polyglutamine tract in the HTT protein. Age at disease onset correlates to CAG repeat length but varies by decades between individuals with identical repeat lengths. Genome-wide association studies link HD modification to DNA repair and mitochondrial health pathways. Clinical studies show elevated DNA damage in HD, even at the premanifest stage. A major DNA repair node influencing neurodegenerative disease is the PARP pathway. Accumulation of poly adenosine diphosphate (ADP)-ribose (PAR) has been implicated in Alzheimer and Parkinson diseases, as well as cerebellar ataxia. We report that HD mutation carriers have lower cerebrospinal fluid PAR levels than healthy controls, starting at the premanifest stage. Human HD induced pluripotent stem cell-derived neurons and patient-derived fibroblasts have diminished PAR response in the context of elevated DNA damage. We have defined a PAR-binding motif in HTT, detected HTT complexed with PARylated proteins in human cells during stress, and localized HTT to mitotic chromosomes upon inhibition of PAR degradation. Direct HTT PAR binding was measured by fluorescence polarization and visualized by atomic force microscopy at the single molecule level. While wild-type and mutant HTT did not differ in their PAR binding ability, purified wild-type HTT protein increased in vitro PARP1 activity while mutant HTT did not. These results provide insight into an early molecular mechanism of HD, suggesting possible targets for the design of early preventive therapies.

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene that alters cellular homeostasis, particularly in the striatum and cortex. Astrocyte signaling that establishes and maintains neuronal functions are often altered under pathological conditions. We performed single-nuclei RNA-sequencing on human HD patient-induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from R6/2 HD mice to investigate high-resolution HD astrocyte cell state transitions. We observed altered maturation and glutamate signaling in HD human and mouse astrocytes. Human HD astrocytes also showed upregulated actin-mediated signaling, suggesting that some states may be cell-autonomous and human specific. In both species, astrogliogenesis transcription factors may drive HD astrocyte maturation deficits, which are supported by rescued climbing deficits in HD drosophila with NFIA knockdown. Thus, dysregulated HD astrocyte states may induce dysfunctional astrocytic properties, in part due to maturation deficits influenced by astrogliogenesis transcription factor dysregulation.

Astrocytes and brain endothelial cells are components of the neurovascular unit that comprises the blood-brain barrier (BBB) and their dysfunction contributes to pathogenesis in Huntington's disease (HD). Defining the contribution of these cells to disease can inform cell-type-specific effects and uncover new disease-modifying therapeutic targets. These cells express integrin (ITG) adhesion receptors that anchor the cells to the extracellular matrix (ECM) to maintain the integrity of the BBB. We used HD patient-derived induced pluripotent stem cell (iPSC) modeling to study the ECM-ITG interface in astrocytes and brain microvascular endothelial cells and found ECM-ITG dysregulation in human iPSC-derived cells that may contribute to the dysfunction of the BBB in HD. This disruption has functional consequences since reducing ITG expression in glia in an HD Drosophila model suppressed disease-associated CNS dysfunction. Since ITGs can be targeted therapeutically and manipulating ITG signaling prevents neurodegeneration in other diseases, defining the role of ITGs in HD may provide a novel strategy of intervention to slow CNS pathophysiology to treat HD.

Summary Huntington’s disease (HD) is a neurodegenerative disease caused by an expanded CAG repeat within the Huntingtin ( HTT ) gene having dysregulated cellular homeostasis in the central nervous system, particularly in the striatum and cortex. Astrocytes establish and maintain neuronal functions through the secretion of soluble factors and physical interactions with other neurovascular unit cell types. Under pathological conditions, astrocytes can become reactive, causing cell state transitions that affect brain function. To investigate transitions between cellular states in unaffected and HD astrocytes at high resolution, single-nuclei RNA-sequencing (snRNA-seq) was performed on human HD patient induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from a rapidly progressing HD mouse model (R6/2). Analysis of HD human and mouse astrocytes revealed both models have alterations in morphology, glutamate uptake, and dysregulation of astrocyte identity and maturation, whereas dysregulated actin-mediated signaling was unique to human iPSC-derived astrocytes. Representative proteins showed altered levels by Western. In both species, HD transcriptional changes reveal potential astrocyte maturation deficits that were potentially driven by astrogliogenesis transcription factors, including ATF3 and NFIA. When perturbed in a drosophila model of HD, knockdown of NFIA in glia rescued the climbing deficit. These data further support the hypothesis that mutant HTT induces dysregulated astrocyte cell states resulting in dysfunctional astrocytic properties, suggests that some of these states are cell autonomous and maybe unique to human HD, and implicate ATF3 and maturation deficits in HD pathogenesis.

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1+ neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.