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Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex. Single-cell transcriptomics of more than 20,000 cells from two functionally distinct areas of the mouse neocortex identifies 133 transcriptomic types, and provides a foundation for understanding the diversity of cortical cell types.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain. RNA-sequencing analysis of cells in the human cortex enabled identification of diverse cell types, revealing well-conserved architecture and homologous cell types as well as extensive differences when compared with datasets covering the analogous region of the mouse brain.

As more people live longer, age-related neurodegenerative diseases are an increasingly important societal health issue. Treatments targeting specific pathologies such as amyloid beta in Alzheimer’s disease (AD) have not led to effective treatments, and there is increasing evidence of a disconnect between traditional pathology and cognitive abilities with advancing age, indicative of individual variation in resilience to pathology. Here, we generated a comprehensive neuropathological, molecular, and transcriptomic characterization of hippocampus and two regions cortex in 107 aged donors (median = 90) from the Adult Changes in Thought (ACT) study as a freely-available resource (http://aging.brain-map.org/). We confirm established associations between AD pathology and dementia, albeit with increased, presumably aging-related variability, and identify sets of co-expressed genes correlated with pathological tau and inflammation markers. Finally, we demonstrate a relationship between dementia and RNA quality, and find common gene signatures, highlighting the importance of properly controlling for RNA quality when studying dementia.

Background Dysfunction of the Basal Ganglia is implicated in several neurodegenerative diseases such as Parkinson’s and Huntington’s. A substructure of the Basal Ganglia, the caudate nucleus, is observed to have diffuse amyloid plaques in Alzheimer’s disease (AD), in Thal phase III. Additionally, literature suggests the presence of AD ‐related tangles. Functionally, the caudate is known to be involved in cognitive functions impacted by AD such as memory. The caudate also receives signals and has efferent projections to significantly affected regions in AD such as cortex and hippocampus respectively. Despite these connections, caudate nucleus remains understudied in AD. Method AT8 (pTau) and 6e10 (Aβ) immunohistochemical staining was performed on the caudate from 42 donors with only canonical proteionopathies and no comorbities. Single nucleus RNA and ATAC‐seq (multiome or singleome) was collected for all donors in the cohort. Spatial transcriptomics was performed on a subset of 5 Thal I‐III and 5 Thal IV‐V donors, with post‐hoc immunostaining of AT8 and 6e10. Cells were labeled using deep learning with a reference caudate dataset from healthy BRAIN Initiative donors. Changes in expression and cell type abundance were modeled in terms of levels of AT8 and 6e10 using Bayesian and general linear mixed effects models respectively. Result We identified caudate specific pTau associated abundance increases in astrocyte and microglia types. These microglia types were not the stereotypical disease associated types described in cortex. We also identified pTau associated abundance decreases in oliogodendrocyte subtypes consistent with cortex. Almost all neuronal populations in the caudate show little change in their cellular abundances. Most effects in cellular composition or differential expression were observed specifically with respect to level of pTau and not Aβ. Conclusion AD’s impact in caudate head contrasts with established effects on the cortex. Regionally unique increases in certain non‐neuronal populations suggest a caudate specific response to AD. Additionally, little neuronal loss, even with respect to significant pTau pathology suggests either environmental or cellular factors that confer resilience, or distinct pTau conditions in the caudate. Finally, our data suggests that the predominantly diffuse plaques in caudate are not sufficient for a plaque induced response in microglia.

Dysfunction of the Basal Ganglia is implicated in several neurodegenerative diseases such as Parkinson's and Huntington's. A substructure of the Basal Ganglia, the caudate nucleus, is observed to have diffuse amyloid plaques in Alzheimer's disease (AD), in Thal phase III. Additionally, literature suggests the presence of AD -related tangles. Functionally, the caudate is known to be involved in cognitive functions impacted by AD such as memory. The caudate also receives signals and has efferent projections to significantly affected regions in AD such as cortex and hippocampus respectively. Despite these connections, caudate nucleus remains understudied in AD. AT8 (pTau) and 6e10 (Aβ) immunohistochemical staining was performed on the caudate from 42 donors with only canonical proteionopathies and no comorbities. Single nucleus RNA and ATAC-seq (multiome or singleome) was collected for all donors in the cohort. Spatial transcriptomics was performed on a subset of 5 Thal I-III and 5 Thal IV-V donors, with post-hoc immunostaining of AT8 and 6e10. Cells were labeled using deep learning with a reference caudate dataset from healthy BRAIN Initiative donors. Changes in expression and cell type abundance were modeled in terms of levels of AT8 and 6e10 using Bayesian and general linear mixed effects models respectively. We identified caudate specific pTau associated abundance increases in astrocyte and microglia types. These microglia types were not the stereotypical disease associated types described in cortex. We also identified pTau associated abundance decreases in oliogodendrocyte subtypes consistent with cortex. Almost all neuronal populations in the caudate show little change in their cellular abundances. Most effects in cellular composition or differential expression were observed specifically with respect to level of pTau and not Aβ. AD's impact in caudate head contrasts with established effects on the cortex. Regionally unique increases in certain non-neuronal populations suggest a caudate specific response to AD. Additionally, little neuronal loss, even with respect to significant pTau pathology suggests either environmental or cellular factors that confer resilience, or distinct pTau conditions in the caudate. Finally, our data suggests that the predominantly diffuse plaques in caudate are not sufficient for a plaque induced response in microglia.

The scientific mission of the Project MindScope is to understand neocortex, the part of the mammalian brain that gives rise to perception, memory, intelligence, and consciousness. We seek to quantitatively evaluate the hypothesis that neocortex is a relatively homogeneous tissue, with smaller functional modules that perform a common computational function replicated across regions. We here focus on the mouse as a mammalian model organism with genetics, physiology, and behavior that can be readily studied and manipulated in the laboratory. We seek to describe the operation of cortical circuitry at the computational level by comprehensively cataloging and characterizing its cellular building blocks along with their dynamics and their cell type-specific connectivities. The project is also building large-scale experimental platforms (i.e., brain observatories) to record the activity of large populations of cortical neurons in behaving mice subject to visual stimuli. A primary goal is to understand the series of operations from visual input in the retina to behavior by observing and modeling the physical transformations of signals in the corticothalamic system. We here focus on the contribution that computer modeling and theory make to this long-term effort.

The CEST properties of EuDOTA-tetraamide complexes bearing pendant carboxylate and carboxyl ethyl esters were measured as a function of pH. The CEST signal from the Eu³⁺-bound water molecule decreased in intensity between pH 8.5 and 4.5 while the proton exchange rates (kex) increased over this same pH range. In comparison, the CEST signal in the corresponding carboxyl ester derivatives was nearly constant. Both observations are consistent with stepwise protonation of the four carboxylic acid groups over this same pH range. This indicates that negative charges on the carboxyl groups above pH 6 facilitate the formation of a strong hydrogen-bonding network in the coordination second sphere above the single Eu³⁺-bound water molecule, thereby decreasing prototropic exchange of protons on the bound water molecule with bulk water protons. The percentage of square antiprismatic versus twisted square antiprism coordination isomers also decreased as the appended carboxylic acid groups were positioned further away from the amide. The net effect of lowering the pH was an overall increase in kex and a quenching of the CEST signal. This article is part ot the themed issue 'Challenges for chemistry in molecular imaging'.

Alzheimer's disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. We applied single nucleus RNA sequencing (RNAseq), ATAC-seq, and Multiome profiling to over 7 million high-quality nuclei from 10 brain regions-spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex-sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co-morbidities). We predicted the cell-type for each nucleus by mapping to an expanded BRAIN initiative cell-type taxonomy, which included AD-associated non-neuronal states and ∼70 brain region-specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell-type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Our multimodal, multi-region single-cell atlas reveals common and region-specific patterns of cellular vulnerability in AD. These cell-types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.

Background Alzheimer’s disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid‐beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. Method We applied single nucleus RNA sequencing (RNAseq), ATAC‐seq, and Multiome profiling to over 7 million high‐quality nuclei from 10 brain regions—spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex—sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co‐morbidities). We predicted the cell‐type for each nucleus by mapping to an expanded BRAIN initiative cell‐type taxonomy, which included AD‐associated non‐neuronal states and ∼70 brain region‐specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. Result We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell‐type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Conclusion Our multimodal, multi‐region single‐cell atlas reveals common and region‐specific patterns of cellular vulnerability in AD. These cell‐types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.