The pathogenesis of Alzheimer's disease (AD) depends on environmental and heritable factors, with remarkable differences evident between individuals at the molecular level. Here we present a transcriptomic survey of AD using spatial transcriptomics (ST) and single-nucleus RNA-seq in cortical samples from early-stage AD, late-stage AD, and AD in Down Syndrome (AD in DS) donors. Studying AD in DS provides an opportunity to enhance our understanding of the AD transcriptome, potentially bridging the gap between genetic mouse models and sporadic AD. Our analysis revealed spatial and cell-type specific changes in disease, with broad similarities in these changes between sAD and AD in DS. We performed additional ST experiments in a disease timecourse of 5xFAD and wildtype mice to facilitate cross-species comparisons. Finally, amyloid plaque and fibril imaging in the same tissue samples used for ST enabled us to directly link changes in gene expression with accumulation and spread of pathology.

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.

The complexity of affected brain regions and cell types is a challenge for Huntington’s 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.

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.

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.

Transcriptomic studies of Alzheimer's disease (AD) have identified both tissue-level and cell-type specific gene expression changes. However, with both "bulk"-tissue and single-cell approaches, we lose pertinent spatial information, such as cell-to-cell proximity or proximity to pathological features. Recently several techniques have emerged, aiming to profile gene expression while preserving the spatial architecture, and we have leveraged one of these techniques—spatial transcriptomics—to interrogate AD gene expression changes in the 5XFAD, an amyloid mouse model, in a spatial and temporal manner. We generated spatial transcriptomic (10x Genomics Visium) data from 5XFAD and wildtype mice at the ages of 4, 6, 8, and 12 months (n = 80 total samples, sex-balanced). Prior to generating libraries, we stained our tissue sections with Amylo-glo and the conformation-specific antibody OC to analyze gene expression changes in spatial relativity to amyloid pathology. We profiled 18,000-20,000 total genes per sample with 1,800-2,700 genes per spatial spot and identified brain region-specific transcriptionally distinct clusters. We also examined the spatial distribution of AD risk genes, identified by GWAS, throughout the disease progression. Further, we identified gene expression changes spatially related to amyloid pathology localization. We have characterized the 5XFAD transcriptome, identifying spatiotemporal AD gene expression changes and providing further insight into the role of amyloid pathology in the modulation of gene expression in AD.

The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating a menagerie of biological processes across distinct cell-types. Understanding these regulatory programs requires a holistic experimental and analytical approach, especially in understanding neurodegenerative disorders such as Alzheimer’s Disease (AD). In this study, we used single-nucleus ATAC-seq (snATAC-seq) to profile the epigenome in all major cell types of the human brain in AD, and we single-nucleus RNA-seq (snRNA-seq) in the same biological samples, together painting a more complete portrait of the changes brought on by disease. We constructed an oligodendrocyte trajectory using 58,221 single-nuclei chromatin profiles and 36,773 single-nuclei transcriptomes. We developed a new analysis approach, scWGCNA, a method for interrogation of cell population-specific co-expression networks. We investigated the dynamics of transcription factor (TF) binding motifs throughout the oligodendrocyte trajectory, and evaluated chromatin accessibility, TF motif enrichment, gene expression, and cis-regulatory elements to identify SREBF1, a master regulator of cholesterol biosynthesis and myelination, downregulated in late-stage AD in oligodendrocytes. We extend our analysis of SREBF1 in oligodendrocytes by examining its downstream regulatory targets in the context of gene co-expression networks. Notably, we found several oligodendrocyte modules significantly enriched for targets of SREBF1, indicating the importance of SREBF1 in regulating gene expression in these modules. Using a multi-scale dataset of bulk-tissue RNA-seq, high-throughput proteomics, and SREBF1 ChIP-seq data (ENCODE), we defined a protein-protein interaction (PPI) network of SREBF1 target genes. Additionally, we found module eigengene expression of SREBF1 targets downregulated in early- and late-pathology AD cases at the level of proteins and RNA, corroborated by downregulation of SREBF1 activity in snATAC-seq data. Altogether, our data and analyses highlight key biological role of SREBF1 in AD biology. While the causative molecular mechanisms of sporadic AD remain unknown, our work is an important stepping stone in fully unraveling the nature of gene regulation in AD, especially in regards to genomic loci with well-described heritable disease risk. Additional work is needed to spatially resolve the complexity of single-cell gene expression and epigenomics in AD and neurodegeneration, in general.

The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating a menagerie of biological processes across distinct cell types. Here, we present a multi-omic single-nucleus study of 191,890 nuclei in late-stage Alzheimer's disease (AD), accessible through our web portal, profiling chromatin accessibility and gene expression in the same biological samples and uncovering vast cellular heterogeneity. We identified cell-type-specific, disease-associated candidate cis-regulatory elements and their candidate target genes, including an oligodendrocyte-associated regulatory module containing links to APOE and CLU. We describe cis-regulatory relationships in specific cell types at a subset of AD risk loci defined by genome-wide association studies, demonstrating the utility of this multi-omic single-nucleus approach. Trajectory analysis of glial populations identified disease-relevant transcription factors, such as SREBF1, and their regulatory targets. Finally, we introduce single-nucleus consensus weighted gene coexpression analysis, a coexpression network analysis strategy robust to sparse single-cell data, and perform a systems-level analysis of the AD transcriptome.

AbstractBackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative disease, characterized by changes in cell‐type proportions and marked alterations of the epigenome and transcriptome. Recent “bulk” tissue RNA‐seq on thousands of postmortem AD brain samples by AMP‐AD consortia has shed light on new AD biology. Unfortunately, cell‐type specific disease biology is completely missed or averaged out using conventional “bulk” tissue RNA‐seq approaches. Single‐cell transcriptomics using single‐nuclei RNA‐seq (snRNA‐seq) and epigenomics (single‐nuclei ATAC‐seq) promises to capture cell‐type heterogeneity and unravel relevant biology related to cell‐to‐cell variability. Such single‐cell approaches have been recently used in psychiatric diseases through PsychENCODE consortia and integrated with bulk tissue RNA‐seq to understand cellular heterogeneity in these diseases. Our work is the first to define single‐nuclear clusters based on open‐chromatin (snATAC‐seq) and gene‐expression (snRNA‐seq) that are dysregulated during the progression of the disease.MethodUsing snATAC‐seq, we have profiled more than 150,000 single‐nuclei open chromatin profiles from normal human (n = 10) and late‐stage AD frontal cortex (n = 11) samples. We have also used same samples to further profile gene‐expression changes at single‐cell resolution using snRNA‐seq from more than 70,000 nuclei. In addition, we have performed bulk‐tissue RNA‐seq from control and AD frontal cortex (n = 50/group) samples and developed gene co‐expression methods to integrate bulk‐tissue and snRNA‐seq to unravel cell‐type specific co‐expression changes altered in AD.ResultOur analysis has identified distinct cell‐clusters based on both open‐chromatin (snATAC‐seq) and transcriptomics (snRNA‐seq) which are altered in late‐stage AD. We have identified several oligodendrocyte cell‐clusters which are increased only during disease progression. Pseudo‐time analysis of data indicates that AD‐specific oligodendrocyte clusters have a distinct developmental trajectory that define the terminal fate of these clusters. In addition, co‐expression analysis with bulk‐tissue RNA‐seq and snRNA‐seq data has identified functional modules (groups of genes) that are dysregulated in these AD‐specific oligodendrocyte clusters. These data highlight the potential role of myelination deficits that occur during the progression of disease.ConclusionOur tour‐de‐force work has identified distinct oligodendrocyte cell‐clusters that are altered in late‐stage AD. We have also identified core regulatory networks in these oligodendroglia cells that promises to provide novel therapeutic targets for AD.

Alzheimer's disease (AD) is a devastating neurological disorder characterized by changes in cell-type proportions and consequently marked alterations of the transcriptome. Here we use a data-driven systems biology meta-analytical approach across three human AD cohorts, encompassing six cortical brain regions, and integrate with multi-scale datasets comprising of DNA methylation, histone acetylation, transcriptome- and genome-wide association studies and quantitative trait loci to further characterize the genetic architecture of AD. We perform co-expression network analysis across more than 1200 human brain samples, identifying robust AD-associated dysregulation of the transcriptome, unaltered in normal human aging. We assess the cell-type specificity of AD gene co-expression changes and estimate cell-type proportion changes in human AD by integrating co-expression modules with single-cell transcriptome data generated from 27 321 nuclei from human postmortem prefrontal cortical tissue. We also show that genetic variants of AD are enriched in a microglial AD-associated module and identify key transcription factors regulating co-expressed modules. Additionally, we validate our results in multiple published human AD gene expression datasets, which can be easily accessed using our online resource (https://swaruplab.bio.uci.edu/consensusAD).

Oral anticoagulants are a critical component of stroke prevention, but carry a risk of brain hemorrhage. These hemorrhagic complications tend to occur in elderly individuals, especially those with predisposing conditions such as cerebral amyloid angiopathy (CAA). Clinical evidence suggests that non-vitamin K antagonist oral anticoagulants are safer than traditional oral anticoagulants. We analyzed whether the anticoagulant dabigatran produces cerebral microhemorrhage (the pathological substrate of MRI-demonstrable cerebral microbleeds) or intracerebral hemorrhage in aged mice with and without hemorrhage-predisposing angiopathy. We studied aged (22 months old) Tg2576 (a model of CAA) and wild-type (WT) littermate mice. Mice received either dabigatran etexilate (DE) (Tg N = 7; WT N = 10) or vehicle (Tg N = 9; WT N = 7) by gavage for 4 weeks. Anticoagulation effects of DE were confirmed using thrombin time assay. No mice experienced intracerebral hemorrhage. Cerebral microhemorrhage analysis, performed using Prussian-blue and H&E staining, showed no significant change in either number or size of cerebral microhemorrhage in DE-treated animals. Analysis of biochemical parameters for endothelial activation (ICAM-1), blood-brain barrier disruption (IgG, claudin-5, fibrinogen), microglial activation (Iba-1), or astrocyte activation (GFAP) showed neither exacerbation nor protective effects of DE in either Tg2576 or WT mice. Our study provides histological and biochemical evidence that aged mice, with or without predisposing factors for brain hemorrhage, tolerate anticoagulation with dabigatran. The absence of dabigatran-induced intracerebral hemorrhage or increased frequency of acute microhemorrhage may provide some reassurance for its use in high-risk patient populations.

Intracerebral hemorrhage (ICH) accounts for 10–15% of all strokes and is a major cause of disability and mortality. Introduction of blood components (e.g., thrombin, heme, and platelets) following ICH initiates neuroinflammatory responses mainly mediated by microglia, which are the resident immune cells in the central nervous system. Microglia have been shown to have dual roles in ICH, both beneficial and detrimental. The beneficial role involves phagocytosis of cellular debris and red blood cells after the hemorrhagic incident, while the detrimental role involves the production of pro-inflammatory cytokines and chemokines resulting in neuroinflammation. These dual and contradictory roles of microglia are thought to be implemented by two distinct phenotypes: classically-activated microglia and alternatively-activated microglia. We discuss herein the role of microglia in ICH with particular emphasis on its role in brain injury and recovery after ICH.

The visual wulst is the telencephalic target of the avian thalamofugal visual system. It contains several retinotopically organised representations of the contralateral visual field. We used optical imaging of intrinsic signals, electrophysiological recordings, and retrograde tracing with two fluorescent tracers to evaluate properties of these representations in the zebra finch, a songbird with laterally placed eyes. Our experiments revealed that there is some variability of the neuronal maps between individuals and also concerning the number of detectable maps. It was nonetheless possible to identify three different maps, a posterolateral, a posteromedial, and an anterior one, which were quite constant in their relation to each other. The posterolateral map was in contrast to the two others constantly visible in each successful experiment. The topography of the two other maps was mirrored against that map. Electrophysiological recordings in the anterior and the posterolateral map revealed that all units responded to flashes and to moving bars. Mean directional preferences as well as latencies were different between neurons of the two maps. Tracing experiments confirmed previous reports on the thalamo-wulst connections and showed that the anterior and the posterolateral map receive projections from separate clusters within the thalamic nuclei. Maps are connected to each other by wulst intrinsic projections. Our experiments confirm that the avian visual wulst contains several separate retinotopic maps with both different physiological properties and different thalamo-wulst afferents. This confirms that the functional organization of the visual wulst is very similar to its mammalian equivalent, the visual cortex.

Large-scale brain activity patterns can be visualized by optical imaging of intrinsic signals (OIS) based on activity-dependent changes in the blood oxygenation level. Another method, flavoprotein autofluorescence imaging (AFI), exploits the mitochondrial flavoprotein autofluorescence, which is enhanced during neuronal activity. In birds, topographic mapping of visual space has been shown in the visual wulst, the avian homologue of the mammalian visual cortex by using OIS. We here applied the AFI method to visualize topographic maps in the visual wulst because with OIS, which depends on blood flow changes, blood vessel artifacts often obscure brain activity maps. We then compared both techniques quantitatively in zebra finches and in C57Bl/6J mice using the same setup and stimulation conditions. In addition to experiments with craniotomized animals, we also examined mice with intact skull (in zebra finches, intact skull imaging is not feasible probably due to the skull construction). In craniotomized animals, retinotopic maps were obtained by both methods in both species. Using AFI, artifacts caused by blood vessels were generally reduced, the magnitude of neuronal activity significantly higher and the retinotopic map quality better than that obtained by OIS in both zebra finches and mice. In contrast, our measurements in non-craniotomized mice did not reveal any quantitative differences between the two methods. Our results thus suggest that AFI is the method of choice for investigations of visual processing in zebra finches. In mice, however, if researchers decide to use the advantages of imaging through the intact skull, they will not be able to exploit the higher signals obtainable by the AFI-method.