The Neurolipid Atlas: a lipidomics resource for neurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4.

Lipid changes in the brain have been implicated in many neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's disease and Amyotrophic Lateral Sclerosis. To facilitate comparative lipidomic research across brain-diseases we established a data commons named the Neurolipid Atlas, that we have pre-populated with novel human, mouse and isogenic induced pluripotent stem cell (iPSC)-derived lipidomics data for different brain diseases. We show that iPSC-derived neurons, microglia and astrocytes display distinct lipid profiles that recapitulate in vivo lipotypes. Leveraging multiple datasets, we show that the AD risk gene ApoE4 drives cholesterol ester (CE) accumulation in human astrocytes recapitulating CE accumulation measured in the human AD brain. Multi-omic interrogation of iPSC-derived astrocytes revealed that cholesterol plays a major role in astrocyte interferon-dependent pathways such as the immunoproteasome and major histocompatibility complex (MHC) class I antigen presentation. We show that through enhanced cholesterol esterification ApoE4 suppresses immune activation of astrocytes. Our novel data commons, available at neurolipidatlas.com, provides a user-friendly tool and knowledge base for a better understanding of lipid dyshomeostasis in neurodegenerative diseases.

Towards Standardizing Nomenclature in Huntington's Disease Research.

The field of Huntington's disease research covers many different scientific disciplines, from molecular biology all the way through to clinical practice, and as our understanding of the disease has progressed over the decades, a great deal of different terminology has accrued. The field is also renowned for its collaborative spirit and use of standardized reagents, assays, datasets, models, and clinical measures, so the use of standardized terms is especially important. We have set out to determine, through a consensus exercise involving basic and clinical scientists working in the field, the most appropriate language to use across disciplines. Nominally, this article will serve as the style guide for the Journal of Huntington's Disease (JHD), the only journal devoted exclusively to HD, and we lay out the preferred and standardized terminology and nomenclature for use in JHD publications. However, we hope that this article will also serve as a useful resource to the HD research community at large and that these recommended naming conventions will be adopted widely.

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Identifying dysregulated regions in amyotrophic lateral sclerosis through chromatin accessibility outliers.

The high heritability of amyotrophic lateral sclerosis (ALS) contrasts with its low molecular diagnosis rate post-genetic testing, pointing to potential undiscovered genetic factors. To aid the exploration of these factors, we introduced EpiOut, an algorithm to identify chromatin accessibility outliers that are regions exhibiting divergent accessibility from the population baseline in a single or few samples. Annotation of accessible regions with histone chromatin immunoprecipitation sequencing and Hi-C indicates that outliers are concentrated in functional loci, especially among promoters interacting with active enhancers. Across different omics levels, outliers are robustly replicated, and chromatin accessibility outliers are reliable predictors of gene expression outliers and aberrant protein levels. When promoter accessibility does not align with gene expression, our results indicate that molecular aberrations are more likely to be linked to post-transcriptional regulation rather than transcriptional regulation. Our findings demonstrate that the outlier detection paradigm can uncover dysregulated regions in rare diseases. EpiOut is available at github.com/uci-cbcl/EpiOut.

Disease related changes in ATAC-seq of iPSC-derived motor neuron lines from ALS patients and controls.

Amyotrophic Lateral Sclerosis (ALS), like many other neurodegenerative diseases, is highly heritable, but with only a small fraction of cases explained by monogenic disease alleles. To better understand sporadic ALS, we report epigenomic profiles, as measured by ATAC-seq, of motor neuron cultures derived from a diverse group of 380 ALS patients and 80 healthy controls. We find that chromatin accessibility is heavily influenced by sex, the iPSC cell type of origin, ancestry, and the inherent variance arising from sequencing. Once these covariates are corrected for, we are able to identify ALS-specific signals in the data. Additionally, we find that the ATAC-seq data is able to predict ALS disease progression rates with similar accuracy to methods based on biomarkers and clinical status. These results suggest that iPSC-derived motor neurons recapitulate important disease-relevant epigenomic changes.

BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease.

Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

Aberrant splicing in Huntington's disease via disrupted TDP-43 activity accompanied by altered m6A RNA modification.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene encoding huntingtin. Prior reports have established a correlation between CAG expanded HTT and altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. 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 striatum from 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 novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.

Transplanted human neural stem cells rescue phenotypes in zQ175 Huntington's disease mice and innervate the striatum.

Huntington's disease (HD), a genetic neurodegenerative disorder, primarily affects the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that, with short-term hNSC transplantation into the striatum of HD R6/2 mice, human cells differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here, we show that long-term (8 months) implantation of hNSCs into the striatum of HD zQ175 mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) levels, and reduces mutant huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry, single-nucleus RNA sequencing (RNA-seq), and electron microscopy demonstrate that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells, and form connections. Single-nucleus RNA-seq analysis also shows restoration of several mHTT-mediated transcriptional changes of endogenous striatal HD mouse cells. Remarkably, engrafted cells receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.

HD and SCA1: Tales from two 30-year journeys since gene discovery.

One of the more transformative findings in human genetics was the discovery that the expansion of unstable nucleotide repeats underlies a group of inherited neurological diseases. A subset of these unstable repeat neurodegenerative diseases is due to the expansion of a CAG trinucleotide repeat encoding a stretch of glutamines, i.e., the polyglutamine (polyQ) repeat neurodegenerative diseases. Among the CAG/polyQ repeat diseases are Huntington's disease (HD) and spinocerebellar ataxia type 1 (SCA1), in which the expansions are within widely expressed proteins. Although both HD and SCA1 are autosomal dominantly inherited, and both typically cause mid- to late-life-onset movement disorders with cognitive decline, they each are characterized by distinct clinical characteristics and predominant sites of neuropathology. Importantly, the respective affected proteins, Huntingtin (HTT, HD) and Ataxin 1 (ATXN1, SCA1), have unique functions and biological properties. Here, we review HD and SCA1 with a focus on how their disease-specific and shared features may provide informative insights.

CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons.

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.

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects.

Using induced pluripotent stem cells (iPSCs) to understand the mechanisms of neurological disease holds great promise; however, there is a lack of well-curated lines from a large array of participants. Answer ALS has generated over 1,000 iPSC lines from control and amyotrophic lateral sclerosis (ALS) patients along with clinical and whole-genome sequencing data. The current report summarizes cell marker and gene expression in motor neuron cultures derived from 92 healthy control and 341 ALS participants using a 32-day differentiation protocol. This is the largest set of iPSCs to be differentiated into motor neurons, and characterization suggests that cell composition and sex are significant sources of variability that need to be carefully controlled for in future studies. These data are reported as a resource for the scientific community that will utilize Answer ALS data for disease modeling using a wider array of omics being made available for these samples.

Single-nuclei transcriptome analysis of Huntington disease iPSC and mouse astrocytes implicates maturation and functional deficits.

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.

An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells.

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.

An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models.

Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d-CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d-CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR-Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation.

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

Diminished LC3-Associated Phagocytosis by Huntington's Disease Striatal Astrocytes.

BACKGROUND: In recent years the functions of astrocytes have shifted from conventional supportive roles to also include active roles in altering synapses and engulfment of cellular debris. Recent studies have implicated astrocytes in both protective and pathogenic roles impacting Huntington's disease (HD) progression. OBJECTIVE: The goal of this study is to determine if phagocytosis of cellular debris is compromised in HD striatal astrocytes. METHODS: Primary adult astrocytes were derived from two HD mouse models; the fast-progressing R6/2 and slower progressing Q175. With the use of laser nanosurgery, a single astrocyte was lysed within an astrocyte network. The phagocytic response of astrocytes was observed with phase contrast and by fluorescence microscopy for GFP-LC3 transiently transfected cells. RESULTS: Astrocyte phagocytosis was significantly diminished in primary astrocytes, consistent with the progression of HD in R6/2 and Q175 mouse models. This was defined by the number of astrocytes responding via phagocytosis and by the average number of vesicles formed per cell. GFP-LC3 was found to increasingly localize to phagocytic vesicles over a 20-min imaging period, but not in HD mice, suggesting the involvement of LC3 in astrocyte phagocytosis. CONCLUSION: We demonstrate a progressive decrease in LC3-associated phagocytosis in HD mouse striatal astrocytes.

Translating cell therapies for neurodegenerative diseases: Huntington's disease as a model disorder.

There has been substantial progress in the development of regenerative medicine strategies for CNS disorders over the last decade, with progression to early clinical studies for some conditions. However, there are multiple challenges along the translational pipeline, many of which are common across diseases and pertinent to multiple donor cell types. These include defining the point at which the preclinical data are sufficiently compelling to permit progression to the first clinical studies; scaling-up, characterization, quality control and validation of the cell product; design, validation and approval of the surgical device; and operative procedures for safe and effective delivery of cell product to the brain. Furthermore, clinical trials that incorporate principles of efficient design and disease-specific outcomes are urgently needed (particularly for those undertaken in rare diseases, where relatively small cohorts are an additional limiting factor), and all processes must be adaptable in a dynamic regulatory environment. Here we set out the challenges associated with the clinical translation of cell therapy, using Huntington's disease as a specific example, and suggest potential strategies to address these challenges. Huntington's disease presents a clear unmet need, but, importantly, it is an autosomal dominant condition with a readily available gene test, full genetic penetrance and a wide range of associated animal models, which together mean that it is a powerful condition in which to develop principles and test experimental therapeutics. We propose that solving these challenges in Huntington's disease would provide a road map for many other neurological conditions. This white paper represents a consensus opinion emerging from a series of meetings of the international translational platforms Stem Cells for Huntington's Disease and the European Huntington's Disease Network Advanced Therapies Working Group, established to identify the challenges of cell therapy, share experience, develop guidance and highlight future directions, with the aim to expedite progress towards therapies for clinical benefit in Huntington's disease.

Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins.

The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.