Biology subjects

Thompson, L. M.

Publications and source records attributed to Thompson, L. M..

At least 19 recordsLinked to original sources

bioRxiv2026

iPSC Neurodegenerative Disease Initiative isogenic CAG repeat iPSC line for Huntingtons disease

STRUCTURED ABSTRACTO_ST_ABSPurpose of ResearchC_ST_ABSThe generation of iPSC lines expressing 21, 56 and 79 glutamine repeats within the HTT protein and homozygous KO of HTT in the KOLF2.1J background as an additional disease series within the iPSC Neurodegenerative Disease Initiative (iNDI) collection. Major FindingsAll iPSCs, even those expressing long repeats of 79Q or HTT KO, were capable of differentiating to striatal and cortical neurons, astrocytes and microglia using established protocols. General quality control stains and morphological analyses are described for each differentiation. A selected set of assays were carried out on differentiated cells; expanded repeat expressing astrocytes showed altered expression of astrocyte protein markers and morphological characteristics, and striatal neurons showed altered DARPP-32/CTIP2 colocalization. mRNAseq carried out for striatal neurons showed high similarities in gene expression changes between 79Q and KO lines compared to the unexpanded repeat. ConclusionsThe KOLF2.1J isogenic CAG repeat series serves as a community resource to study HD mechanisms with the potential for direct comparison across other neurodegenerative diseases through the iNDI collection.

neuroscience↗

bioRxiv2026

Mouse models with human antibody repertoires for inducing multiple lineages of HIV-1 broadly neutralizing antibodies

The complementarity determining region (CDR) 1, 2 and 3 of antibodies are the principal antigen contact sites. The heavy chain CDR3 (CDR H3) is highly variable, because it includes random nucleotide additions by the terminal deoxynucleotidyl transferase (TdT). Some broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1) rely heavily on CDR H3 to recognize conserved epitopes on HIV-1 Envelope (Env) protein. Elicitation of comparable bnAbs is a prime goal of HIV-1 vaccine development, but the shortage of precursor antibodies with suitable CDR H3s in human repertoire is a major challenge for immunogen design. To aid this effort, we generated six mouse models for inducing bnAbs against major HIV-1 Env epitopes. In each mouse model, the immunoglobulin heavy and light chain loci were engineered to predominantly rearrange the germline V, D and J segments of a bnAb lineage. Owing to CDR3 diversity, only a small subset of the V(D)J recombination products may encode variable regions that can engage bnAb epitopes with sufficient affinity to initiate immune response. Therefore, these mouse models can be used to test and optimize immunization strategies to induce bnAbs from rare and diverse precursors in complex antibody repertoires.

immunology↗

bioRxiv2026

Maturation of HIV-1 neutralizing antibodies in a germinal center conditional expression mouse model

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway. Author summaryIn response to antigenic stimulation, cognate B cells become activated and form germinal centers in lymphoid tissues. Germinal center B cells modify their antigen receptors through somatic hypermutation (SHM) of immunoglobulin variable region gene exons, with antigen selecting for high affinity B cells by providing survival advantage. This mechanism accounts for antibody affinity maturaton over the gradual course of an immune response. Affinity maturation is critical for generating potent, neutralizing antibodies against diverse strains of the human immunodeficiency virus-1 (HIV-1). These broadly neutralizing antibodies (bnAbs) are heavily mutated, reflecting lengthy affinity maturation over years of chronic infection. Recapitulating the affinity maturation process is a major challenge for bnAb induction by vaccination. In immunization experiments, bnAb development often stalls at rate limiting steps that involve infrequent, but functionally important, mutational events. Overcoming such obstacles requires boost immunogens that can stimulate the stalled B cells to acquire the requisite mutations. To this end, we recapitulated the maturation arrest of a bnAb lineage by expressing a stalled antibody in mouse germinal center B cells. Using this mouse model, we developed boost immunization conditions that advanced the antibody maturation beyond a roadblock to attain neutralizing activities against heterogenous viruses.

immunology↗

bioRxiv2026

Multimodal analysis of cell-free DNA identifies epigenetic biomarkers for amyotrophic lateral sclerosis diagnosis and progression

The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid-biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 non-disease controls. Following targeted enzymatic methyl-sequencing (EM-seq) of [~]4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 {+/-} 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of [~]70% of ALS patients with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.

genetics↗

bioRxiv2025

Multimodal spatial transcriptomics determines repeat expansion, huntingtin aggregation, and selective cortical neuron loss in Huntington Disease

Huntingtons disease (HD) is caused by CAG expansion in HTT, yet how somatic repeat instability and huntingtin aggregation relate to selective cell loss in the human brain remains unclear. We have developed a multimodal spatial transcriptomics approach that enables defining transcriptional programs with subcellular resolution, somatic CAG repeat lengths, and six other pathology marks including huntingtin aggregates in every cell of intact brain sections. Imaging 428,173 cells in HD cortex revealed selective vulnerability: L5-6 NP and L6b deep-layer excitatory neurons undergo >50% loss, closely linked to very large (>380{+/-}55) somatic expansions. Intranuclear aggregation was most prevalent at intermediate somatic repeat expansion (220-300 CAGs) and was accompanied by broader transcriptional changes. In contrast, chandelier and somatostatin+ inhibitory interneurons are lost despite only modest repeat expansion or aggregation. These data provide a comprehensive resource and establish a broadly applicable framework for connecting repeat expansion and protein pathology across diverse cell types. Short Bullet pointsO_LIDevelopment of a novel, multimodal spatial transcriptomics platform enables definition of RNA transcriptomes with subcellular resolution, somatic repeat expansions, and protein accumulation in cells within tissue sections C_LIO_LIGeneration of two complementary datasets for HD and control cortex: 428,173 cells to quantify comprehensively cell-type vulnerability, and a deeper multimodal dataset in which CAG repeat expansion, huntingtin aggregation, six additional pathological readouts, and expression of 1,128 genes were measured in 185,721 cells, providing a comprehensive resource for the neurodegeneration community. C_LIO_LIDeep layer excitatory neuron loss (L5-6 NP and L6b) was associated with very large somatic CAG expansions (>380{+/-}55), while selective inhibitory neuron loss (chandelier and somatostatin interneurons) occurred with modest CAG repeat expansion or huntingtin intranuclear aggregation C_LIO_LIIntranuclear aggregation, not somatic repeat expansion, was more predictive of transcriptional changes, including chromatin remodeling and RNA export factors C_LI

neuroscience↗

bioRxiv2025

PIAS1/PIAS4-Mediated SUMOylation of TDP-43 is Induced by Oxidative Stress

Abstract/SummaryTAR DNA-binding protein 43 (TDP-43) is a conserved RNA and DNA binding protein that functions in transcriptional repression, pre-mRNA splicing and mRNA stabilization. Under pathological conditions found in multiple neurodegenerative diseases, TDP-43 shows aberrant mislocalization from the nucleus and cytoplasmic accumulation and aggregation. TDP-43 also appears to play a role in DNA damage repair, specifically in non-homologous end-joining (NHEJ), suggesting that nuclear depletion of TDP-43 may contribute towards the accumulation of DNA damage observed in TDP-43 proteinopathies. These TDP-43 pathological inclusions are decorated with post-translational modifications, most notably phosphorylation. SUMOylation (Small Ubiquitin-like Modifier) is a dynamic post-translational modification that regulates many protein properties and is implicated in neurodegenerative disease pathology. DNA damage repair proteins are commonly regulated through SUMOylation, and the SUMO E3 ligases PIAS1 and PIAS4 are required for efficient DNA repair of double-strand DNA breaks. Given these findings, we investigated TDP-43 SUMOylation and whether SUMO modification impacts TDP-43s DNA damage repair function. We show that TDP-43 can be modified by SUMO1 and SUMO2/3 and confirm SUMOylation in response to oxidative stress. We also determine which regions of TDP-43 are SUMOylated and show that this modification is facilitated by the SUMO E3 ligases PIAS1 and PIAS4. Etoposide-induced DNA damage did not promote SUMOylation of TDP-43; studies are ongoing to determine the impact of TDP-43 SUMOylation on DNA repair.

neuroscience↗

bioRxiv2025

Huntingtin preserves mitochondrial genome integrity in neurons, which is impaired in Huntingtons disease

Huntingtin (HTT) function is enigmatic, as the native protein plays critical roles in neuronal health, while mutant HTT (mHTT), carrying an expanded polyglutamine stretch, triggers neurotoxicity and contributes to the pathogenesis of Huntingtons disease (HD). We recently found that HTT is part of a nuclear transcription-coupled DNA repair (TCR) complex with DNA repair enzymes including polynucleotide-kinase-3-phosphatase (PNKP). This complex resolves DNA lesions during transcription to maintain genome integrity, while in HD, mHTT impairs the activity of this complex, resulting in accumulation of DNA lesions. Using molecular, cellular biology and computational methods, we find that HTT has a role in assembling a functional DNA repair complex in mitochondria. Together with mitochondrial RNA polymerase and transcription factors, HTT resolves mitochondrial DNA lesions to preserve mitochondrial genome integrity and function. Pathogenic mHTT impairs this activity, resulting in persistent DNA lesions and reduced mitochondrial function in HD. Importantly, restoring activity of this complex in a Drosophila HD model through ectopic HTT or PNKP expression significantly improves mitochondrial genome integrity and ameliorates motor deficits. HIGHLIGHTSO_LIHTT organizes a functional, multifactorial mitochondrial DNA repair complex C_LIO_LIMutant HTT impairs the mitochondrial DNA repair complex causing DNA damage accumulation C_LIO_LIHTT-associated repair complex resolves mitochondrial DNA lesions and DNA integrity C_LIO_LIRestoring repair activity in HD flies rescues mitochondrial DNA integrity and motor defects C_LI

neuroscience↗

bioRxiv2025

Prevention of Transgene Silencing During Human PluripotentStem Cell Differentiation

While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.

neuroscience↗

bioRxiv2025

Intersecting impact of CAG repeat and Huntingtin knockout in stem cell-derived cortical neurons

Huntingtons 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 the 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.

cell biology↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗

bioRxiv2024

Cognitive effects of early life exposure to PCBs: Sex-specific behavioral, hormonal and neuromolecular mechanisms involving the brain dopamine system.

Endocrine-disrupting chemicals (EDCs) are environmental toxicants that disrupt hormonal and neurodevelopmental processes. Among these chemicals, polychlorinated biphenyls (PCBs) are particularly concerning due to their resistance to biodegradation and tendency to bioaccumulate. PCBs affect neurodevelopmental function and disrupt the brains dopamine (DA) system, which is crucial for attentional, affective, and reward processing. These disruptions may contribute to the rising prevalence of DA-mediated neuropsychiatric disorders such as ADHD, depression, and substance use disorders. Notably, these behaviors are sexually dimorphic, in part due to differences in sex hormones and their receptors, which are targets of estrogenic PCBs. Therefore, this study determined effects of early life PCB exposure on behaviors and neurochemistry related to potential disruption of dopaminergic signaling. Male and female Sprague Dawley rats were exposed to PCBs or vehicle perinatally and then underwent a series of behavioral tests, including the sucrose preference test to measure affect, conditioned orienting to assess incentive-motivational phenotype, and attentional set-shifting to evaluate cognitive flexibility and response latency. Following these tests, rats were euthanized, and we measured serum estradiol (E2), midbrain DA cells, and gene expression in the midbrain. Female rats exposed perinatally to A1221 exhibited decreased sucrose preference, and both male and female A1221 rats had reduced response latency in the attentional set-shifting task compared to vehicle counterparts. Conditioned orienting, serum estradiol (E2), and midbrain DA cell numbers were not affected in either sex; however, A1221-exposed male rats displayed higher expression of estrogen receptor alpha (Esr1) in the midbrain and non-significant effects on other DA-signaling genes. Additionally, E2 uniquely predicted behavioral outcomes and DAergic cell numbers in A1221-exposed female rats, whereas DA signaling genes were predictive of behavioral outcomes in males. These data highlight sex-specific effects of A1221 on neuromolecular and behavioral phenotypes.

animal behavior and cognition↗

bioRxiv2024

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 Alzheimers Disease (AD), Parkinsons 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.

neuroscience↗

bioRxiv2023

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

Huntingtons 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.

neuroscience↗

bioRxiv2023

Identifying dysregulated regions in amyotrophic lateral sclerosis through DNA accessibility outliers

The high heritability of ALS and similar rare diseases contrasts with their low molecular diagnosis rate post-genetic testing, pointing to potential undiscovered genetic factors. Chromatin accessibility assays quantify the activity of functional elements genome-wide, offering invaluable insights into dysregulated regions. In this research, we introduced EpiOut, a computational toolbox to identify outliers in chromatin accessibility. These outliers represent dysregulated regions where chromatin accessibility uniquely diverges from the population baseline in a single or few samples. Annotation of accessible regions with histone ChIP-seq 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. For example, 59% of gene expression outliers can be linked to aberration in chromatin accessibility. 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 open-sourced and freely available at github.com/uci-cbcl/EpiOut.

bioinformatics↗

bioRxiv2022

BDNF and TRiC-inspired Reagents Rescue Cortical Synaptic Deficits in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) results from a CAG repeat expansion in the gene for Huntington (HTT) resulting in expansion of the polyglutamine (Q) tract in the mutant protein (mHTT). Synaptic changes are early manifestations of neuronal dysfunction in HD. However, the mechanism(s) by which mHTT impacts synapse formation and function is not well defined. Herein we explored HD pathogenesis in the BACHD and the {Delta}N17-BACHD mouse models of HD by examining cortical synapse formation and function in primary cultures maintained for up to 35 days (DIV35). We identified synapses by immunostaining with antibodies against pre-synaptic (Synapsin 1) and a post-synaptic (PSD95) marker. Consistent with earlier studies, cortical neurons from both WT and the HD models began to form synapses at DIV14; at this age there were no genotypic differences in synapse numbers. However, from DIV21 through DIV35 BACHD neurons showed progressively smaller numbers of synapses relative to WT neurons. Remarkably, BACHD synaptic deficits were completely rescued by treating cultures with BDNF. Building on earlier studies using reagents inspired by the chaperonin TRiC, we found that addition of the recombinant apical domain of CCT1 partially rescued synapse number. Unexpectedly, unlike BACHD cultures, synapses in {Delta}N17-BACHD cultures showed a progressive increase in number as compared to WT neurons, thus distinguishing synaptic changes in these HD models. Using multielectrode arrays, we discovered age-related functional deficits in BACHD cortical cultures with significant differences present by DIV28. As for synapse number, BDNF treatment prevented most synaptic deficits, including mean firing rate, spikes per burst, inter-burst interval, and synchrony. The apical domain of CCT1 showed similar, albeit less potent effects. These data are evidence that deficits in HD synapse number and function can be replicated in vitro and that treatment with either BDNF or a TRiC-inspired reagent can prevent them. Our findings support the use of cellular models to further explicate HD pathogenesis and its treatments.

neuroscience↗

bioRxiv2022

Integrated transcriptome analysis of Huntingtons disease iPSC-derived and mouse astrocytes implicates dysregulated synaptogenesis, actin, and astrocyte maturation

Huntingtons 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.

neuroscience↗

bioRxiv2022

Single nuclei RNAseq analysis of HD mouse models and human brain reveals impaired oligodendrocyte maturation and potential role for thiamine metabolism

The complexity of affected brain regions and cell types is a challenge for Huntingtons disease (HD) treatment. Here we used single nucleus RNA sequencing (snRNAseq) to investigate mechanism of pathology in the cortex and striatum from R6/2 mice at 8 and 12w and in three regions of human HD post-mortem tissue. We identified cell type-specific and cell agnostic signatures and found changes suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) were arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 were negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells showed decreased accessibility of sites regulated by OL maturation genes. Glucose and lipid metabolism were implicated in abnormal cell maturation and PRKCE and Thiamine Pyrophosphokinase 1 were identified as central genes. High dose thiamine/biotin treatment of R6/1 HD mice to target thiamine metabolism not only restored OL maturation, but also rescued pathology in neurons. These findings reveal insights into HD OL pathology that spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

neuroscience↗

bioRxiv2022

CryoET Reveals Organelle Phenotypes in Huntington Disease Patient iPSC-Derived and Mouse Primary Neurons

Huntingtons Disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. Patient-derived induced pluripotent stem cells (iPSCs) can help understand disease; however, defining pathological biomarkers is 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 mitochondria with enlarged granules and distorted cristae, and thin sheet aggregates in double membrane-bound organelles. We used artificial intelligence to quantify mitochondrial granules, and proteomics to show differential protein content in HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC-neurons and reduced phenotypes in 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.

neuroscience↗