Autism spectrum disorder (ASD) is associated with functional abnormalities of the anterior cingulate cortex (ACC), a brain area that mediates social behavior. Given evidence of a role of inflammation in ASD, markers of pro-inflammatory and anti-inflammatory microglia were studied using postmortem ACC tissues from ASD and age-matched typically developed control donors. Gene expression levels of pro-inflammatory (CD68, HLA-DRA, IL1B, NOS2, PTGS2) and anti-inflammatory (ARG1, IGF1, MRC1, PPARG) microglial genes were measured using quantitative real-time PCR. Additionally, brain sections were immunohistochemically stained for a microglial marker. Expression levels of IGF1 were modestly higher, while the expression of MRC1 was modestly lower in ASD donors when compared to control donors. No other differences in gene expression levels between the two groups of donors were observed. Statistical significance for changes in expression levels IGF1 and MRC1 did not survive correction for multiple comparisons. Further research on anti-inflammatory microglial involvement in ASD is warranted.

Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a short, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of experiments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines. Stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with microglial activation. Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of sigma receptor activation and HUCBC therapy in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to oxygen glucose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain over time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brain in appreciable numbers at any timepoint. IL10 and IFN?; levels were found to significantly increase by 96hours post MCAO. This increase in IL10 and IFNγ expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the relative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatments to modulate the peripheral immune systems response to injury.

Schizophrenia is a devastating mental illness characterized by a broad range of clinical manifestations including hallucinations, social cognitive impairments, and disordered thinking and behavior, all of which impair daily functioning. The immune molecule complement component 4 (C4), located in the major histocompatibility locus (MHC) on chromosome 6 in humans, is highly associated with schizophrenia such that specific structural variants and regulatory regions increase the expression of C4 and confer greater risk for this brain disorder. Besides their established role in brain immune defense, complement proteins play a role in various stages of brain development including neurogenesis, migration and synaptic development. However, C4 has never been experimentally upregulated to determine the impact of increased expression of this immune gene on brain development. Here, I study the role of C4 in layer 2/3 pyramidal neurons in the medial prefrontal cortex of mice to study the hypothesis that C4 overexpression causes circuit dysfunction by leading to the pathological elimination of synapses. Specifically, neuronal connectivity was assayed by measuring dendritic spine density using confocal microscopy and functional connectivity through whole-cell electrophysiology recordings. Additionally, the role of microglia in altering the developmental wiring of the brain was examined by quantifying microglia engulfment in the medial prefrontal cortex. Lastly, complement-induced changes to the prefrontal cortex were accompanied by deficits in social behavior in both juvenile and adult mice. Overall, these studies show that C4 affects brain connectivity by reducing dendritic spine density and excitatory drive through enhanced microglia-engulfment of synaptic material which was sufficient to cause lasting deficits in mouse social behavior.

Olfactory sensory neurons (OSNs) are contained within the olfactory epithelium (OE) and are responsible for detecting odorant molecules in the air. The exposure of OSNs to the external environment is necessary for their function, but it also leaves them exposed to potentially harmful elements and thus results in a high turnover rate. Despite the high turnover, the olfactory sense is maintained throughout life through the division of a population of stem cells that produce new OSNs both during normal turnover and after an injury occurs in the OE. When new OSNs are born, they must extend axons from the OE to the olfactory bulb (OB) where they make specific synaptic contacts. To determine the timeline of axon extension in normal turnover and after a methimazole-induced injury, we used fate-tracing utilizing an inducible Cre-LoxP model in which a fluorescent reporter was expressed by neuronal precursors and subsequently used to track axonal growth as the OSNs matured. Our results show that axon extension in both conditions follow the same timeline. However, markers of synaptic connectivity in the OB were delayed after injury. The delay in synaptic connectivity was also corroborated with delays in olfactory behavior after injury, which took 40 days to recover to control levels. Additionally, we investigated the process of removal of axonal debris created after an injury. Immunohistochemical analysis after injury indicated upregulation of IBA1+ cells within the 3 olfactory nerve layer of the OB, suggesting a role of microglia in this process. These microglia also showed an activated morphology and some had very large cell bodies with multiple nuclei. Furthermore, qPCR analysis of post-injury OB tissue shows upregulation of the CD11b receptor that is expressed on microglia. Our results have also shown upregulation of components of the complement pathway after injury, which is suggestive of a mechanism that underlies axonal debris removal after injury in the OB. Taken together, these results shed light on the process by which the olfactory system is able to recover after injury and could lead to discovery of mechanisms that could translate to treatments for injuries in other areas of the nervous system.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases predominantly affecting infants and children, which in some cases can present into adulthood. There are fourteen genes comprising the 13 known subtypes of NCLs (CLN1-CLN8, CLN10-CLN14; CLN9 has been reclassified as CLN4). The NCL diseases share common molecular and clinical features, including cellular accumulation of autofluorescent storage material, characteristic histological findings (curvilinear inclusions, fingerprint profiles, and granular osmophilic deposits), markedly low brain weight, seizures, blindness, motor dysfunction and behavioral disabilities. Though the functions of the CLN proteins are not fully understood, they are mainly localized to the lysosomal compartment and autophagic pathway. Previous works have focused on understanding the individual functions of the CLN proteins. However, there is little research examining the interactions between CLN proteins and their involvement in neurogenesis. The CLN proteins also show involvement in various other signaling pathways, notably the mTOR and p53 pathways, and may therefore have implication as important signaling molecules during development and aging. In this thesis, I outline a variety of interactions between CLN proteins, as well as their role in lysosome formation and autophagy. I further examine the involvement of these proteins in lysosomes of microglia, and potential functions of microglia during neurogenesis in childhood and adulthood. I hypothesize that the CLN proteins are likely involved in a common pathway which is highly regulated during neurogenesis through microglial release of pro-inflammatory molecules. Though these diseases are incurable, enzyme replacement shows promise as a treatment for NCL; cerliponase alpha (BioMarin Pharmaceuticals) is the first and only FDA-approved enzyme replacement treatment for CLN2 disease. Future in-depth investigation of protein-protein interactions as well as their involvement in signaling pathways during development is necessary in order to find a cure for these devastating diseases.

Historically, the role of immune cells in the nervous system was predominantly examined throughthe lens of disease. In recent years, studies have shown that the complex, orchestrated events of immune activity throughout embryonic and postnatal critical periods are crucial for proper nervous system development. While previous studies have suggested limited immune heterogeneity in the adult brain, the diverse roles of the hippocampus in cognition and pathological development would suggest variation of immune cells in this region. Specifically, the hippocampus is known to be a site of adult neurogenesis. However, fundamental traits of immune cells in this region have not been well characterized. In chapter one, I present a summary of literature that discusses what was previously known of immune regulation of adult neurogenesis during health and disease. In chapter two, I compare different reporter lines and marker genes to evaluate responses in various cell types in the neurogenic niche and in other regions of the brain in the context of injury and pharmacological modulation. I discuss preliminary evidence suggesting microglial depletion may result in phenotypic changes in astrocytes throughout the hippocampus. In chapter three, I provide evidence of heterogeneity in myeloid-lineage cells in the hippocampus. I leveraged the highthroughput nature of cell suspension based single cell RNA-sequencing to collect transcriptomes of over 20,000 myeloid lineage cells from murine hippocampi. Using a series of bioinformatic techniques, I was able to computationally dissect different populations within this system and found spatial mapping of one distinct subset specifically localized to the neurogenic niche of the hippocampus. The transcriptomic signature of these cells alongside immunoreactivity to candidate genes, and morphological properties of this population resemble those of reactive microglia associated with the restriction of neurodegenerative diseases. In chapter four, I discuss how the immune landscape of the hippocampus responds to perturbation using a model of Focused Ultrasound mediated Blood-Brain Barrier opening. Subtypes of myeloid lineage cells change in composition and in transcriptomic response. We find distinct, temporally defined transcriptional responses in microglial and macrophage populations, indicating discrete roles for microglia and macrophages in immune activity during the transition from acute to chronic response. Together, these findings point towards diverse properties of microglia in the adult hippocampus.

No description available.

No description available.

Synaptic connectivity is highly plastic in early development and undergoes extensive remodeling in response to changes in neuronal activity and sensory experience. Microglia, the resident central nervous system macrophages, participate in shaping mature neuronal circuits by dynamically surveying the brain parenchyma and pruning away less active synaptic connections. However, it is unknown how changes in neuronal activity regulates microglial pruning within circuits and whether this activity-dependent pruning is necessary to achieve plasticity. Using the rodent somatosensory circuit, I identified that microglia engulf and eliminate synapses in the cortex following early postnatal (P4) unilateral removal of mouse whiskers. I found this early life microglial synaptic remodeling requires specific chemokine signaling between neurons and microglia. Mice that lack expression of either the neuronal chemokine CX3CL1 (fractalkine), or its microglial receptor CX3CR1, have significantly reduced microglial synapse engulfment and fail to eliminate synapses following whisker removal. To gain more insight into how this signaling is regulated, I performed both single-cell RNA sequencing of the primary somatosensory cortex as well as microglia-specific Translating Ribosome Affinity Purification (TRAP) sequencing. I identified that the majority of central nervous system (CNS) cell populations in the somatosensory cortex, including microglia, undergo transcriptional changes following whisker removal. Further, the transcriptional changes in microglia after whisker cauterization require expression of the receptor CX3CR1. Importantly, I also found that Adam10, a gene encoding the metalloprotease known to post-translationally cleave CX3CL1 into a soluble chemokine, is upregulated in the deprived cortex after whisker ablation. Pharmacological inhibition of ADAM10 inhibits microglia-mediated removal of synapses in the deprived cortex. These data support a mechanism by which cleavage of membrane-bound CX3CL1 by ADAM10 is necessary for neuronal signaling to microglia via CX3CR1 to induce transcriptional changes within microglia upstream of synaptic engulfment and elimination following sensory deprivation.

<p>Evidence from experimental models of epilepsy support that prolonged seizures (status epilepticus, SE) promote pathological hippocampal synaptodendritic remodeling which contributes to the development of seizures and cognitive decline. One potential mechanism underlying the SE-induced sequelae is microgliosis. </p> <p>Evidence from models of experimental epilepsy supports a significant spatiotemporal correlation between SE-induced decreases in the microtubule associated protein 2 (Map2) loss and microgliosis in the hippocampus. In addition, pharmacological suppression of microgliosis after SE with the drug rapamycin attenuated the losses of Map2 and the dendritic ion channels Kv.4.2 and HCN1 in the hippocampus. This microglia suppression paralleled a recovery of the SE-induced recognition and spatial memory deficits. Based on these studies, we hypothesized that the inhibition of microgliosis during epileptogenesis will attenuate the SE-induced hippocampal dendritic and cognitive pathology. To further investigate the role of microgliosis in the SE-induced dendritic pathology, we tested the efficacy of a more selective inhibitor <a>of the survival and proliferation of </a>microglia, PLX3397, using the pilocarpine model of SE and acquired epilepsy. PLX3397 binds to colony stimulating factor 1 receptor (CSF1R) on microglia and inhibits the downstream signaling responsible for survival and proliferation of these cells. </p> <p>To test this hypothesis, we induced SE in male rats with pilocarpine (280-300mg/kg) <a>while and controls (Ctrl) received saline. </a>Rats were randomly assigned to a diet of either chow alone (vehicle; Veh) or chow with PLX3397 (50mg/kg) for 20 days post-SE. At two weeks post-SE, rats were subjected to novel object recognition (NOR) and Barnes maze (BM) to evaluate hippocampal-dependent recognition memory, and spatial learning and memory, respectively. Following the behavioral assessments, rats were sacrificed for brain analysis at 20 days post-SE. We used histological analysis to determine the amount of microgliosis with IBA1 and dendritic stability with Map2. We used western blotting to measure the protein levels of molecules involved in the crosstalk between microglia and astrocytes: GFAP, IL-6, C3, and iC3b. We also measured the protein levels of the dendritic ion channels Kv4.2 and HCN1, and the synaptic marker PSD95.</p> <p>NOR showed that the Ctrl+Veh and Ctrl+PLX3397 groups spent significantly more time exploring the novel object (<i>p</i> < .05), while the SE+Veh and SE+PLX3397 did not. Similar results were observed in the BM test, Ctrl+Veh and Ctrl+PLX3397 groups had a faster latency to find the target compared to the SE+Veh and SE+PLX3397 groups (<i>p</i> < .05). These data suggest that recognition and spatial memory deficits induced by SE were not attenuated by treatment with PLX3397. We found that the PLX3397 treatment significantly decreased microgliosis in Ctrl+PLX3397 rats compared to Ctrl+Veh rats (<i>p</i> < .05). As expected, we found a significant increase in the number of microglial cells in hippocampi of SE+Veh rats compared to Ctrl+Veh rats (<i>p</i> < .05). Interestingly, in the PLX3397-treated SE group, we observed two distinctive groups which we categorized as responders and non-responders when compared to the SE+Veh group. The SE+PLX responders had significantly decreased microgliosis compared to the SE+Veh group (<i>p</i> < .05). The SE+PLX non-responders had higher levels of microgliosis compared to the SE+Veh group (<i>p</i> < .05). We found levels of GFAP were increased in the SE+Veh group compared to the Ctrl+Veh group (<i>p</i> < .05). Treatment with PLX3397 in the SE group reduced these levels compared to the vehicle treated SE group (<i>p</i> < .05). We also found increases in C3 and iC3b following the induction of SE compared to Ctrl+Veh group (<i>p</i> < .05), and these levels remained similar in the SE+PLX3397 group compare to the SE+Veh group (<i>p</i> > .05). There was a reduction in Map2 immunoreactivity as well as the protein levels of Kv4.2 and PSD95 in the SE+Veh group compared to the Ctrl+Veh group (<i>p</i> < .05). We found that treatment with PLX3397 recovered the SE-induced loss of Map2 labeled dendrites compared to SE+Veh group (<i>p</i> < .05). However, treatment with PLX3397 did not recover the SE-induced reduction Kv4.2 and PSD95 (<i>p</i> > .05). <a>In parallel, we found that a group of SE+PLX3397 animals did not have reduced microgliosis compared to the SE+Veh group (<i>p </i>< .05), and therefore was categorized as a non-responder group. </a></p> Our findings are the first to show that blocking CSF1R signaling with PLX3397 suppressed microgliosis in the hippocampus, partially recovered the SE-induced decline of Map2 immunoreactivity in the hippocampal CA1 region but had no effect in the recognition or spatial memory deficits. These data suggest that while hippocampal microgliosis may play a role in the disruption of dendritic structural stability in the hippocampus it does not seem to critically contribute to the memory decline that occurs during epileptogenesis.