The ability of the nervous system to repair itself following injury is a remarkable phenomenon, but central nervous system endogenous repair mechanisms are incomplete. Cortical ischemic stroke produces a limited process of neural repair, characterized by axonal sprouting and reorganization in the surviving tissue adjacent to the infarct. Changes in gene expression patterns have been observed in this peri-infarct region, in populations of neurons that specifically sprout new axons following stroke, and in neurons that have received delivery of growth factors following stroke, providing transcriptomic profiles of neuronal growth states post-stroke. Within these transcriptomic data sets are a number of differentially regulated transcription factors that have been implicated in brain development. Developmental transcription factors present an intriguing target for study due to their inherent ability to act as master regulators of genes responsible for neuronal growth. This thesis investigates a selection of these differentially regulated transcription factors for their ability to regulate axonal sprouting and functional recovery following stroke. Genes were screened for their ability to promote axonal growth in primary cortical neurons. From these studies, Hhex and Ctip2 emerged as candidates for further exploration. Overexpression of these genes following stroke revealed an ability of both Hhex and Ctip2 to promote long-distance axonal sprouting, but only Hhex to promote axonal sprouting in the peri-infarct region. Overexpression of Hhex or Ctip2 showed an ability to promote recovery of gait function following stroke to the forelimb motor cortex, but only overexpression of Hhex was associated with recovery of forelimb reach movements and fine motor manipulation. Transcriptional profiling of neurons that have Ctip2 or Hhex overexpressed following stroke revealed that these transcription factors are driving different molecular pathways to achieve axonal growth and functional recovery. Ultimately, these studies implicate a role for these transcription factors in axonal growth in the adult brain and provide further evidence for unique intrinsic growth programs in neural repair processes.

White matter stroke (WMS) is a debilitating disorder, characterized by the formation of ischemic lesions along the white matter tracts in the brain. The accumulation of these white matter lesions leads to vascular dementia, which is the second leading cause of dementia, and accelerates the pathology of Alzheimer’s Disease [11]. Currently, there are no therapy options available to halt the progression of vascular dementia. Hence, we wanted to better understand the molecular mechanisms and behavioral outcomes that may result as a consequence of multiple strokes happening within the white matter region of the brain. We created a two-stroke protocol in which an initial large white matter stroke was induced in C57BL/6J mice models, then 1-month later, a small white matter stroke was induced contralaterally. This models the progressive nature of ischemic white matter damage, which is a core feature of the human condition. We measured the recovery of these cohorts through motor behavior tests (grid-walking and pole test) and cognitive tests (fear conditioning and novel object recognition) at baseline, 1-month, 2-month, and 3-month timepoints. Our data indicates that for the grid-walking test, the Large WMS mice have significantly more foot faults at the 1-month and 2-month timepoints, and then plateau at the 3-month timepoint, when compared to the Large WMS + Small WMS. In the pole test at the 3-month timepoint, the Large WMS mice have shorter t-turn times when compared to the Large WMS + Small WMS cohort. During the fear conditioning test, the Large WMS mice had higher freeze times across all timepoints when compared to the Large WMS + Small WMS mice. Overall, this data suggests that the Large WMS + Small WMS mice have more difficulty performing fine motor tasks, such as the pole test, at a later timepoint and have hindered memory and recall ability when compared to the Large WMS only cohort.

Stroke is a devastating neurological condition and the leading cause of lasting motor, sensory and cognitive deficits. These disabilities occur due to the central nervous system’s limited ability to repair itself after injury. Following cortical ischemic stroke, adjacent surviving tissue initiates a limited process of repair characterized in part by axonal sprouting and cortical remodeling. Neurons that undergo axonal sprouting have a unique transcriptional profile, differentially regulating genes in a time and age-specific manner. Among these genes are a number of differentially regulated extracellular matrix proteins, including those that are important in peripheral nervous system regeneration. The extracellular matrix is comprised of a network of proteins that provide support to cells throughout the body. In the central nervous system, the extracellular matrix is implicated in development, ensuring developing axons are guided to the correct destination, modulating signaling pathways that coordinate proliferating cells, and providing a general environment of support. In the mature brain, the extracellular matrix plays key roles in plasticity and support, and undergoes dramatic changes in composition and function following injury. This thesis investigates two differentially regulated extracellular matrix proteins for their ability to induce axonal sprouting and functional recovery following stroke. Matrilin-2 (Matn2) is an adapter protein important for peripheral nerve regeneration but paradoxically is one of the most down-regulated genes in sprouting neurons in young animals. Unique cartilage matrix associated protein (Ucma) is a novel extracellular matrix protein that is one of the most upregulated genes in young animals, but whose expression is almost totally abolished in aged animals following stroke, potentially contributing to the lack of recovery seen as a result of age. Both proteins increased neurite outgrowth in primary neurons, and rescued neurite outgrowth in a stroke-like growth inhibitory environment. Viral overexpression of Matn2 and Ucma revealed that both enhanced axonal sprouting in the post-stroke brain. When assessing ability to enhance functional recovery, Matn2 overexpression accelerated return of motor function to baseline, while Ucma overexpression initially accelerated recovery but only partially restored motor function. Further studies to test translatability of these proteins revealed that restriction of Matn2 adjacent to the injury site still enhanced axonal sprouting, but Ucma overexpression in this context was detrimental to axonal sprouting. In summary, these studies identify novel pro-growth extracellular matrix proteins that enhance axonal sprouting and functional recovery after stroke and identify the pro-growth extracellular matrix as a new target for neural repair after injury.

Stroke is a leading cause of disability and there is a need for therapies which enhance the spontaneous amount of brain recovery that occurs naturally. PDE2a is a promising molecular target which may limit recovery. Here, two-photon calcium imaging is employed to study the primary motor cortex (M1) and the premotor cortex (M2) of mice before and after inducing a PT stroke to M1. Mice received daily PDE2a-inhibitor as a recovery agent after stroke, and were imaged weekly for 5 weeks. Head-fixed behavior was concurrently assessed during spontaneous and forced-use contexts. Calcium recordings were processed and analyzed, and functional connectivity (FC) was assessed based on the correlatedness of the neurons in the recording. Stroke was found to have significant negative effects on FC, especially in M1. Stroke had differential effects in M2 during Spontaneous behavior vs forced use behavior. Finally, PDE2a-inhibition shows a strong trend for improving head-fixed recovery after stroke and it is a significantly positive enhancer of FC in M1 and M2. The results suggest that calcium imaging can act as a biomarker for stroke and that PDE2a-inhibition is an ideal molecular target.

Stroke is the number one leading cause of adult disability in the US. While medical intervention has significantly decreased mortality rate; stroke survivors still face devastating disability that affect their quality of life. With limited therapeutic interventions, promoting the brain repair and recovery remains an unmet need in medical sciences. While the brain has limited regeneration capability, its endogenous reactive and repair mechanism is not well-understood. Pericytes, a less-well-understood member of brain, are a heterogenous population of perivascular cell that regulate development and maintenance of structural elements of the blood brain barrier, vascular stability, and angiogenesis. Recent studies have confirmed their remarkable contractile ability, their control of cerebral blood flow and crosstalk with other cell types after injury. Due to its lack of exclusive markers, its overlapping markers with other cell types in the brain, including a newly identified population of perivascular fibroblasts and its heterogeneity, findings of brain pericyte after injury have been controversial. Through development of an array of novel molecular tools, coupled with the use of single cell RNA sequencing and novel pharmacological agents, this dissertation characterizes the post-stroke pericyte heterogeneity and its contribution to the brain repair processes. Pericyte population was characterized after stroke on the tissue level to understand their proliferative capacity and their association with the regenerative neurovascular niche. Using two novel molecular lineage-tracing models, subpopulations of pericyte were labeled and lineage-traced to delineate its contribution to the fibrotic scar. Using single cell RNA seq, the finding was confirmed and further characterized the molecular mechanism of pericyte contribution to (1) the brain fibrotic scar and (2) its role in establishing the regenerative neurovascular niche. Using the RNAseq dataseq as a guiding foundation, three specific manipulation studies were carried out to further delineate the heterogenous role of pericyte after stroke: Stat3 knockout in brain-specific pericyte, Loxl2/3 pathway inhibition by small molecule drugs and brain-specific pericyte ablation. These findings from these studies revealed the highly conserved nature of the brain’s fibrotic scar formation processes and the essential contribution of pericyte to the scar formation and maintenance. Pericyte heterogeneity, especially the fibrotic-scar-formation population and the peri-infarct population that associate with the neurovascular niche, needs to be taken into consideration when developing therapeutic treatment. Ultimately, these studies implicate the multi-faceted roles of pericyte in the brain repair and recovery processes, and provide further evidence to the growing body of research being done on the biology of pericyte in homeostasis and after injury

Stroke is the leading cause of adult disability and leaves millions of patients worldwide with chronic deficits in sensory, motor, and cognitive function. Although there are currently no pharmacological treatments for brain recovery after stroke, clinical trials have shown that a limb overuse paradigm, known as constraint-induced movement therapy (CIMT), can lead to significant and lasting motor improvements. This functional improvement is strongly associated with increased activity in the ipsilesional premotor cortex, but the exact neuronal connections and molecular processes that underlie this plasticity process are unknown.

The body of work within this dissertation identifies one potential mechanism of cortical axonal sprouting after limb overuse in a mouse model of stroke and forelimb overuse that approximates human CIMT. Via quantitative neuroanatomical mapping, we find that limb overuse drives the novel formation of a connection from retrosplenial cortex to premotor cortex (RSC-PMC). We then isolate the very neurons that comprise this connection by FACS (Fluorescence-Activated Cell Sorting) purification for RNA-Seq transcriptional profiling. The RSC-PMC transcriptome is characterized by expression of activity-induced and growth-related genes, several of which are new to the context of CNS injury and plasticity. Pathway analyses indicate that limb overuse induces signaling in cell growth and tissue development, calcium signaling, and Ephrin A pathways. Upstream transcriptional regulators of the differentially expressed genes within RSC-PMC include NeuroD1, NeuroG3, YY1, and Otx2. Subsequently, top candidates from the 160 differentially regulated genes by limb overuse were functionally screened using CRISPR/cas9 in an in-vitro neuronal outgrowth assay. Two molecular hits were Otx2 and NeuroG3, genes previously associated with critical period onset/closure and neurogenesis, respectively. Finally, whole transcriptome comparison studies of the RSC-PMC across various plasticity paradigms indicate relationship to activity-dependent/learning transcriptomes and divergence from developmental growth programs. In parallel, we find that GDF10 (growth and differentiation factor 10) is an early trigger of axonal sprouting after stroke. Even in the absence of limb overuse, we find that in-vivo delivery of GDF10 increases axonal peri-infarct sprouting, forms synaptic contacts in premotor cortex, and results in improved behavioral recovery. Together, these studies identify potentially synergistic mechanisms to target in future therapeutic strategies for stroke.