The mammalian brain is an enormously complex organ with myriad cell types cohesively working together to carry out a host of intricate tasks, from motor functions, to the storing and execution of consciousness. These cell types broadly fall into neuronal and non-neuronal classifications, the latter of which substantially outnumber the former and provide the support system and maintenance for the electrically active neuronal component. With the advent of single-cell platforms, we now have the capability to deeply assess and characterize all cell types in the brain; however, the majority of studies to date have specifically targeted neurons, largely neglecting their non-neuronal counterpart. In our proposed plan of research, we will directly address the dearth of single-cell omics data on non-neuronal cell types by the deployment of innovative technologies we have developed to assay epigenetic properties at the single-cell level in high throughput. We will focus specifically on non-neuronal cells in the human and rodent brain by enriching for the NeuN(-) population, the reciprocal of numerous neuron-focused studies. Assays will include the assessment of regulatory element usage by deploying chromatin accessibility assays, genome-wide profiling of DNA methylation, and assessing the three-dimensional folding of chromatin in the nucleus. In addition to profiling single cells at the regional level, we will adapt our assay platform to include the tracking of spatial information by high-density regional subsampling. We will deploy the spatially aware assay variant in the context of ischemic injury, which results in a gradient of glial reactivity radiating out from the injury site. Lastly, our assessment of regulatory networks with cell type and reactive-state specificity is ideally suited for the design of highly specific transgenic reporter mice. We will produce these lines using our identified regulatory modules to drive standard and split recombinase constructs that activate the INTACT reporter system to enable rapid and efficient isolation of target cells with high purity. The resources we propose to develop will broadly enable the interpretation of data and the development of studies that target the non- neuronal component of the mammalian brain.
We have recently entered a new era in our capacity to characterize the mammalian brain at the resolution of single cells. These capabilities have predominantly been used to specifically assess transcription in neuronal cell types, largely neglecting non-neuronal cells, or the underlying epigenetic landscape that dictates transcription. Here, we provide a plan to deeply catalogue non-neuronal cell types from an epigenomic perspective as well as incorporate spatial information, and utilize these profiles to produce highly cell type specific transgenic reporter mice.
