Huntington's disease (HD) modifiers include mismatch-repair (MMR) genes, but their connections to neuronal pathogenesis remain unclear. Here, we genetically tested 9 HD genome-wide association study (GWAS)/MMR genes in mutant Huntingtin (mHtt) mice with 140 inherited CAG repeats (Q140). Knockout (KO) of genes encoding a distinct MMR complex either strongly (Msh3 and Pms1) or moderately (Msh2 and Mlh1) rescues phenotypes with early onset in striatal medium-spiny neurons (MSNs) and late onset in the cortical neurons: somatic CAG-repeat expansion, transcriptionopathy, and mHtt aggregation. Msh3 deficiency ameliorates open-chromatin dysregulation in Q140 neurons. Mechanistically, the fast linear rate of mHtt modal-CAG-repeat expansion in MSNs (8.8 repeats/month) is drastically reduced or stopped by MMR mutants. Msh3 or Pms1 deficiency prevents mHtt aggregation by keeping somatic MSN CAG length below 150. Importantly, Msh3 deficiency corrects synaptic, astrocytic, and locomotor defects in HD mice. Thus, Msh3 and Pms1 drive fast somatic mHtt CAG-expansion rates in HD-vulnerable neurons to elicit repeat-length/threshold-dependent, selective, and progressive pathogenesis in vivo.

Dendritic arbors are essential for neuronal computation and signal propagation, yet large-scale single-neuron morphology studies remain challenging. Here we present a systems biology approach, termed 'dendritome mapping', to profile the dendritic morphology of genetically defined single neurons in mice, unveiling striatal medium spiny neuron (MSN) morphological territories and aging-associated or disease-associated alterations. We generated 3,762 three-dimensional-reconstructed and reference-atlas-mapped striatal D1-type and D2-type MSNs, revealing distinct impacts of D1/D2 genotypes and striatal locations on MSN morphology. To analyze dendritic variation at a finer resolution than known anatomical landmarks permit, we assigned MSNs to latticed cubic boxes within the reference brain atlas, summarized a morphometric representation ('eigen-morph') for each box and clustered boxes with shared morphometry. This identified six modules with characteristic dendritic features and spanning contiguous striatal territories, each receiving distinct corticostriatal inputs. Finally, we found that aging confers dendritic atrophy in both D1-MSNs and D2-MSNs, whereas Huntington's disease mice exhibit MSN-type and regional-specific defects.

Spinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD), are motor diseases caused by CAG expansions in ATXN1 and HTT , where SCA1 shows prominent cerebellar neurodegeneration and HD shows prominent striatal neurodegeneration, particularly in the Medium Spiny Neurons (MSNs). Since human and mouse studies demonstrate progressive striatal vulnerability in SCA1, we examined age-dependent molecular, cellular and functional striatal attributes in SCA1 (f-ATXN1 146Q/2Q ) knockin mice, by assessing RNA-sequencing, immunohistochemistry and electrophysiology. Striatal mRNAs are downregulated in SCA1 mice, many in common with HD mice, and specificity in MSNs is supported by the rescue of transcriptomic dysregulation with deletion of mutant Ataxin1 from MSNs. Immunohistochemistry assessed dopamine receptor 1 (D1R) and 2 (D2R) expression in indirect and direct MSNs. In HD mice ( Htt Q175/Q7 ), expression of both D1R and D2R proteins in MSNs decreased with age in parallel with their RNA levels. In the SCA1 mouse striatum, D1R protein expression decreased with age as seen in murine HD striatum. In contrast, while D2R protein level was decreased similar to D1R protein at 5-weeks of age, by 40-weeks expression of D2R protein recovered to levels recorded in WT mice. Electrophysiological assessment showed a reduction of excitatory synaptic transmission in SCA1 mouse MSNs, indicating functional deficits early in disease. In contrast to cerebellar and many other aspects of SCA1 pathology known to depend on proper nuclear localization of ATXN1 with an expanded polyglutamine, mutating ATXN1's nuclear localization failed to correct striatal MSN RNA and protein downregulations, indicating a difference in how ATXN1 exerts its pathological effects between the cerebellum and the striatum. Together, these data provide a molecular and cellular basis of striatal pathology in SCA1.

The study of disease modifiers is a powerful way to identify patho-mechanisms associated with disease. Using the strong genetic traits of Huntington's disease (HD), we identified a rare, single-nucleotide polymorphism (SNP) in WDFY3 associated with a delayed age of onset of up to 23 years. Remarkably, the introduction of the orthologous SNP into mice recapitulates this neuroprotection, significantly delaying neuropathological and behavioral dysfunction in two models of HD. The SNP increases expression of the protein autophagy-linked Fab1, YOTB, Vac1, and EEA1 (FYVE) protein (Alfy), an autophagy adaptor protein for the clearance of aggregated proteins, whose ectopic overexpression is sufficient to capture the neuroprotective effects of the variant. Increasing Alfy expression protects not only against HD but also against the toxicity due to phospho-α-synuclein and AT8-positive accumulation. By combining human and mouse genetics, we have uncovered a pathway that protects against multiple proteinopathies, revealing a much-sought-after, shared therapeutic target across a broad range of neurodegenerative diseases.

Morphology is a cardinal feature of a neuron that mediates its functions, but profiling neuronal morphologies at scale remains a formidable challenge. Here we describe a generalizable pipeline for large-scale brainwide study of dendritic morphology of genetically-defined single neurons in the mouse brain. We generated a dataset of 3,762 3D-reconstructed and reference-atlas mapped striatal D1- and D2- medium spiny neurons (MSNs). Integrative morphometric analyses reveal distinct impacts of anatomical locations and D1/D2 genetic types on MSN morphologies. To analyze striatal regional features of MSN dendrites without prior anatomical constraints, we assigned MSNs to a lattice of cubic boxes in the reference brain atlas, and summarized morphometric representation ("eigen-morph") for each box and clustered boxes with shared morphometry. This analysis reveals 6 modules with characteristic dendritic features and spanning contiguous striatal territories, each receiving distinct corticostriatal inputs. Finally, we found aging confers robust dendritic length and branching defects in MSNs, while Huntington's disease (HD) mice exhibit selective length-related defects. Together, our study demonstrates a systems-biology approach to profile dendritic morphology of genetically-defined single-neurons; and defines novel striatal D1/D2-MSN morphological territories and aging- or HD-associated pathologies.

Modifiers of Huntington's disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly ( Msh3 and Pms1 ) or moderately ( Msh2 and Mlh1 ) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3 -dependent. Remarkably, Q140/homozygous Msh3-KO lacks visible mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3 -deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R 2 =0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no detectable mHtt nuclear or neuropil aggregates. Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions. One Sentence Summary:Msh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.

Despite its monogenic etiology, the pathogenic mechanisms of Huntington's disease (HD) have been challenging to disentangle. Mouse models of HD recapitulate several disease features, including striatum-selective, mutant Huntingtin (mHTT) CAG-repeat length–dependent and age-dependent disease processes, offering tools to study HD pathogenesis in vivo. Systems biology integrates large-scale molecular profiling approaches with unbiased bioinformatic tools (e.g., coexpression network analysis) to provide high-dimensional, information-rich, and reproducible findings that can inform disease mechanisms. Recent transcriptomic, epigenomic, and proteomic studies have uncovered key roles for expanded and uninterrupted mHTT CAG-repeats and other molecular factors in striatum-selective pathogenesis including transcriptional dysregulation in HD. Cell-type-specific or single-cell profiling reveals distinct molecular defects in striatal and cortical neurons and glial cells. Finally, novel systems biology pipelines, toolsets, and databases (HDinHD) provide rich and scalable readouts to evaluate genetic or pharmacological perturbations to validate disease mechanisms and facilitate HD therapeutic discovery.

IntroductionPersons living with HIV (PLWH) experience the early onset of age-related illnesses, even in the setting of successful human immunodeficiency virus (HIV) suppression with highly active antiretroviral therapy (HAART). HIV infection is associated with accelerated epigenetic aging as measured using DNA methylation (DNAm)-based estimates of biological age and of telomere length (TL).MethodsDNAm levels (Infinium MethylationEPIC BeadChip) from peripheral blood mononuclear cells from 200 PLWH and 199 HIV-seronegative (SN) participants matched on chronologic age, hepatitis C virus, and time intervals were used to calculate epigenetic age acceleration, expressed as age-adjusted acceleration residuals from 4 epigenetic clocks [Horvath’s pan-tissue age acceleration residual (AAR), extrinsic epigenetic age acceleration (EEAA), phenotypic epigenetic age acceleration (PEAA), and grim epigenetic age acceleration (GEAA)] plus age-adjusted DNAm-based TL (aaDNAmTL). Epigenetic age acceleration was compared for PLWH and SN participants at two visits: up to 1.5 years prior and 2–3 years after HAART (or equivalent visits). Flow cytometry was performed in PLWH and SN participants at both visits to evaluate T-cell subsets.ResultsEpigenetic age acceleration in PLWH decreased after the initiation of HAART but remained greater post-HAART than that in age-matched SN participants, with differences in medians of 6.6, 9.1, and 7.7 years for AAR, EEAA, and PEAA, respectively, and 0.39 units of aaDNAmTL shortening (all p < 0.001). Cumulative HIV viral load after HAART initiation was associated with some epigenetic acceleration (EEAA, PEAA, and aaDNAmTL), but even PLWH with undetectable HIV post-HAART showed persistent epigenetic age acceleration compared to SN participants (p < 0.001). AAR, EEAA, and aaDNAmTL showed significant associations with total, naïve, and senescent CD8 T-cell counts; the total CD4 T-cell counts were associated with AAR, EEAA, and PEAA (p = 0.04 to <0.001). In an epigenome-wide analysis using weighted gene co-methylation network analyses, 11 modules demonstrated significant DNAm differences pre- to post-HAART initiation. Of these, nine were previously identified as significantly different from pre- to post-HIV infection but in the opposite direction.DiscussionIn this large longitudinal study, we demonstrated that, although the magnitude of the difference decreases with HAART is associated with the cumulative viral load, PLWH are persistently epigenetically older than age-matched SN participants even after the successful initiation of HAART, and these changes are associated with changes in T-cell subsets.

Background:Genetic study of late-onset Alzheimer's disease (AD) reveals that a rare Arginine-to-Histamine mutation at amino acid residue 47 (R47H) in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) results in increased disease risk. TREM2 plays critical roles in regulating microglial response to amyloid plaques in AD, leading to their clustering and activation surrounding the plaques. We previously showed that increasing human TREM2 gene dosage exerts neuroprotective effects against AD-related deficits in amyloid depositing mouse models of AD. However, the in vivo effects of the R47H mutation on human TREM2-mediated microglial reprogramming and neuroprotection remains poorly understood.Method:Here we created a BAC transgenic mouse model expressing human TREM2 with the R47H mutation in its cognate genomic context (BAC-TREM2-R47H). Importantly, the BAC used in this study was engineered to delete critical exons of other TREM-like genes on the BAC to prevent confounding effects of overexpressing multiple TREM-like genes. We crossed BAC-TREM2- R47H mice with 5xFAD [1], an amyloid depositing mouse model of AD, to evaluate amyloid pathologies and microglial phenotypes, transcriptomics and in situ expression of key TREM2 -dosage dependent genes. We also compared the key findings in 5xFAD/BAC-TREM2-R47H to those observed in 5xFAD/BAC-TREM2 mice.Result:Both BAC-TREM2 and BAC-TREM2-R47H showed proper expression of three splicing isoforms of TREM2 that are normally found in human. In 5xFAD background, elevated TREM2-R47H gene dosages significantly reduced the plaque burden, especially the filamentous type. The results were consistent with enhanced phagocytosis and altered NLRP3 inflammasome activation in BAC- TREM2-R47H microglia in vitro. However, unlike TREM2 overexpression, elevated TREM2- R47H in 5xFAD failed to ameliorate cognitive and transcriptomic deficits. In situ analysis of key TREM2 -dosage dependent genes and microglial morphology uncovered that TREM2-R47H showed a loss-of-function phenotype in reprogramming of plaque-associated microglial reactivity and gene expression in 5xFAD.Conclusion:Our study demonstrated that the AD-risk variant has a previously unknown, mixture of partial and full loss of TREM2 functions in modulating microglial response in AD mouse brains. Together, our new BAC-TREM2-R47H model and prior BAC-TREM2 mice are invaluable resource to facilitate the therapeutic discovery that target human TREM2 and its R47H variant to ameliorate AD and other neurodegenerative disorders.

[This corrects the article DOI: 10.1016/j.isci.2022.104488.].

In fatty liver disease, systemic homeostasis is perturbed. While pre-clinical models are used to understand its pathogenesis, translating this knowledge to patients is difficult. However, by focusing on the most preserved homeostasis systems between species and models, novel disease dimensions can be unearthed. We interrogated core liver gene co-expression networks in a mouse model of liver cancer following dietary challenge. The behaviour of immunometabolic networks in tumours mirrored their counterparts in non-tumour liver. These modules showed temporal changes under the influence of diet duration and aging. A high immune response network was associated with a lower tumour burden in mice and humans. This module in mice was enriched for genes related to haematopoietic cell differentiation. Consistently, the bone marrow haematopoietic stem and progenitor cells response was reflective of the liver immune response. Linking haematopoiesis to hepatic homeostasis uncovers a hitherto unexplored dimension of tissue crosstalk that can inform pathogenesis.

Brain tissue transcriptomes may be organized into gene coexpression networks, but their underlying biological drivers remain incompletely understood. Here, we undertook a large-scale transcriptomic study using 508 wild-type mouse striatal tissue samples dissected exclusively in the afternoons to define 38 highly reproducible gene coexpression modules. We found that 13 and 11 modules are enriched in cell-type and molecular complex markers, respectively. Importantly, 18 modules are highly enriched in daily rhythmically expressed genes that peak or trough with distinct temporal kinetics, revealing the underlying biology of striatal diurnal gene networks. Moreover, the diurnal coexpression networks are a dominant feature of daytime transcriptomes in the mouse cortex. We next employed the striatal coexpression modules to decipher the striatal transcriptomic signatures from Huntington’s disease models and heterozygous null mice for 52 genes, uncovering novel functions for Prkcq and Kdm4b in oligodendrocyte differentiation and bipolar disorder-associated Trank1 in regulating anxiety-like behaviors and nocturnal locomotion.

In Huntington's disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

Findings about chronic complex diseases are difficult to extrapolate from animal models to humans. We reason that organs may have core network modules that are preserved between species and are predictably altered when homeostasis is disrupted. To test this idea, we perturbed hepatic homeostasis in mice by dietary challenge and compared the liver transcriptome with that in human fatty liver disease and liver cancer. Co-expression module preservation analysis pointed to alterations in immune responses and metabolism (core modules) in both human and mouse datasets. The extent of derailment in core modules was predictive of survival in the cancer genome atlas (TCGA) liver cancer dataset. We identified module eigengene quantitative trait loci (module-eQTL) for these predictive co-expression modules, targeting of which may resolve homeostatic perturbations and improve patient outcomes. The framework presented can be used to understand homeostasis at systems levels in pre-clinical models and in humans. A record of this paper's transparent peer review process is included in the supplemental information.

Additional file 6: Table of pairwise correlations between preserved CTX modules. Each preserved CTX module (yellow/CTX, blue/CTX and green/CTX) is represented by its eigengene. High positive or negative correlations between eigengenes suggest that the modules are indistinguishable. Correlations between the yellow/CTX, blue/CTX and green/CTX are reported. (XLS 25 KB)

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.

In Huntington’s disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cell clusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.

Although Huntington’s disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly ( p < 10 −7 ) associated with 33 CpG sites, including the HTT gene ( p = 6.5 × 10 −26 ). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model ( p = 6.0 × 10 −8 ) and in the transgenic sheep model ( p = 2.4 × 10 −88 ). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant ( p < 10 −7 ) associations with methylation levels at three loci: near PEX14 ( p = 9.3 × 10 −9 ), GRIK4 ( p = 3.0 × 10 −8 ), and COX4I2 ( p = 6.5 × 10 −8 ). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.