7 research outputs found
Deep Molecular Characterization of Human Cortical Cell-Types
Human cortical cytoarchitecture is as beautiful as it is complex. Harboring dozens of morphologically, functionally, and molecularly distinct cell-types, the neocortex is organized into cytoarchitectonically and functionally diverse regions. In concert, the diverse cell-types that are found within the diverse cortical regions build the center of all higher cognitive functions, including but not limited to sensory processing, instruction and execution of motor movements, abstract thinking, perception, as well as speech processing and production. Over the past decades, detailed knowledge on the molecular characteristics of cortical cell-types has been obtained from genetically engineered animal models. These deep insights into the molecular underpinnings of cortical cell-types, paired with recent advances in cellular nuclei isolation technologies allow for the isolation and deep molecular profiling of human neocortical cell-types. We developed a novel serial fluorescence activated nuclei sorting strategy, which we will henceforth refer to as sFANS. sFANS stands for serial fluorescence activated nuclei sorting and allows for the isolation of up to sixteen cell-types from the human cerebral cortex. In this thesis, we present data from fourteen routinely isolated cell-types, which include layer 2/3, layer 4, region specific populations of layer 5, termed layer 5 region-specific (layer 5rs), layer 5a, as well as layer 6a, and layer 6b excitatory pyramidal neurons, VIP, LAMP5, RELN, and PVALB expressing interneurons, astrocytes, microglia, oligodendrocytes, and OPCs. We show that our isolation strategy is highly cell-type specific and reproducible, permitting deep molecular profiling of the cell-types across several regions of post-mortem human cortex. Moreover, we show that isolated and cell-type specific populations can be passed on to a number of different downstream assays. Specifically, in the course of this work, we conducted RNAseq, snRNAseq, ATACseq, as well as CAG expansion assays on hundreds of cell-type specific populations and these data, obtained from several different regions of the human neocortex, will be presented in this work. As many neurological diseases are known to affect the neocortex, cell-type specific studies are instrumental for our understanding of the underlying pathophysiological and molecular alterations that underly these conditions. It has been widely recognized that cell-type specific knowledge is critically needed to bring us closer to more effective treatment strategies. Here, we focused our efforts on the investigations of the neuronal neocortical alterations observed in Huntington’s disease. Our precise and reproducible methodology revealed that an HTR2C expressing layer 5a excitatory neuron population is selectively vulnerable and drastically diminished across all stages of Huntington’s disease. In collaborative studies, in which we used samples from an AAV2.retro, striatal injected nonhuman primate model, we show that the vulnerable HTR2C expressing neurons constitute a cortico-striatal projecting cell-type. Moreover, we quantified the relative number of nuclei per cell-type using a population enriched snRNAseq strategy and, while our cell-type specific CAG expansion assays confirmed dramatic expansions in all deep layer excitatory neurons, only those nuclei of the layer 5a type were diminished in Huntington’s disease. Our data therefore suggest that CAG expansion is necessary but not sufficient to drive the loss of HTR2C expressing layer 5a excitatory neurons and that potential alterations of the cortico-striatal connections that are formed by this particular cell-type may be a major driver for the vulnerability of HTR2C expressing neurons in Huntington’s disease. Chapter one of this thesis offers an introduction into the neuroanatomical aspects of the human neocortex. The major cell-types that constitute the cortex, their morphological, functional, and molecular hallmarks will be introduced. Functionally and cytoarchitectonically diverse regions of cortex that are of particular importance to this work will be discussed and several aspects of regional and cellular connectivity will be highlighted. Chapter two provides background and rationale for the development of our sFANSeq isolation strategy. Detailed information on our methods, including nuclei isolation, labeling, and gating strategies are provided. Results and confirmation of reproducibility and cell-type specificity will be presented. In chapter three, we introduce current knowledge on the neuropathological alterations of Huntington’s disease with a focus on the neocortex and expand on this knowledge through presentation and discussion of the results and findings from our sFANS experiments, RNAseq, snRNAseq, ATAC, and CAG expansion data. This work is intended to provide the ground stone for the application of our sFANS strategy for the isolation and deep characterization of cortical cell-types in human health and disease. Our aim is to introduce the sFANS strategy as a tool that provides highly robust, reproducible, and celltype specific results for all major human neocortical cell-types
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Layer 5a Corticostriatal Projection Neurons are Selectively Vulnerable in Huntington's Disease
The properties of the cell types that are most vulnerable in the Huntington's disease (HD) cortex, the nature of somatic CAG expansion of
in these cells, and their importance in CNS circuitry have not been delineated. Here we have employed serial fluorescence activated nuclear sorting (sFANS), deep molecular profiling, and single nucleus RNA sequencing (snRNAseq) to demonstrate that layer 5a pyramidal neurons are selectively vulnerable in primary motor cortex and other cortical areas. Extensive somatic
-CAG expansion occurs in vulnerable layer 5a pyramidal cells, and in Betz cells, layer 6a, layer 6b neurons that are not lost in HD. Retrograde tracing experiments in the macaque brain identify the vulnerable layer 5a neurons as corticostriatal pyramidal cells. Our data establish that
-CAG expansion is not sufficient for cell loss in the cerebral cortex of HD, and suggest that cortico-striatal disconnection in early-stage HD patients may play an important role in neurodegeneration
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Selective vulnerability of layer 5a corticostriatal neurons in Huntington's disease
The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here, we employed serial fluorescence-activated nuclear sorting (sFANS), deep molecular profiling, and single-nucleus RNA sequencing (snRNA-seq) of motor-cortex samples from thirteen predominantly early stage, clinically diagnosed HD donors and selected samples from cingulate, visual, insular, and prefrontal cortices to demonstrate loss of layer 5a pyramidal neurons in HD. Extensive mHTT CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layers 6a and 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in HD cerebral cortex