Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Heintz Laboratory

Abstract

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.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Download

Included in

Life Sciences Commons

Share

COinS