Traumatic brain injury (TBI) is a major cause of disability, with approximately 1.7 million incidents reported annually. Following a TBI, patients are likely to sustain sensorimotor and cognitive impairments and are at an increased risk of developing neurodegenerative diseases later in life. Despite this, robust therapies that treat TBI neuropathology are not available in the clinic. One emerging therapeutic approach is to target epigenetic mediators that modulate a variety of molecular regulatory events acutely following injury. Specifically, previous studies demonstrated that histone deacetylase inhibitor (HDACi) administration following TBI reduced inflammation, enhanced functional outcomes, and was neuroprotective. Here, we evaluated a novel quisinostat-loaded PLA-PEG nanoparticle (QNP) therapy in treating TBI as modeled by a controlled cortical impact. We evaluated initial pharmacodynamics within the injured cortex via histone acetylation levels following QNP treatment. We observed that QNP administration acutely following injury increased histone acetylation specifically within the injury penumbra, as detected by Western blot analysis. Given this effect, we evaluated QNP therapeutic efficacy. We observed that QNP treatment dampened motor deficits as measured by increased rotarod latency to fall relative to blank nanoparticle- and saline-treated controls. Additionally, open field results show that QNP treatment altered locomotion following injury. These results suggest that HDACi therapies are a beneficial therapeutic strategy following neural injury and demonstrate the utility for nanoparticle formulations as a mode for HDACi delivery following TBI.

The highly conserved Notch signaling pathway regulates cell-cell communication pathways, cell fate, cell determination, cell death, embryonic development, and adult tissue pathways in metazoans. The Notch receptors and ligands that bind to Notch are single pass, transmembrane proteins that communicate cell to cell via juxtacrine signaling. There are reports of the divergent function and localization of the Deltalike 3 (Dll3) ligand. In Mus musculus (an eutherin mammal) the DLL3 protein inhibits the Notch signaling pathway and is localized in the Golgi apparatus. In contrast, the DLL3 protein from zebrafish, Danio rerio (a teleost) activates Notch and is located on the cell surface. This study will focus on examining the evolutionary pathway/evolutionary similarities, localization, and function of the A. carolinensis dll3 gene in comparison to other vertebrate species. This is important because there is not much known about the evolutionary divergence of the DLL3 A. carolinensis protein, its function in Notch signaling, and its subcellular localization.
Evolutionary analysis of vertebrate DLL3 protein sequences using phylogenetic trees showed that D. rerio and A. carolinensis are more evolutionarily similar in comparison to M. musculus suggesting that they may have similar intracellular localization. However, immunofluorescence staining experiments showed that the A. carolinensis DLL3 protein co-localized significantly with an endoplasmic reticulum (ER) specific primary antibody. Since this protein is localized in the secretory system, similar to that of M. musculus DLL3, it suggests that its function is to inhibit the Notch signaling pathway. Protein sequence alignments were created that suggested that there is a region in the protein sequences where the lizard and mouse sequence are conserved, while the zebrafish sequence simultaneously varies. This region of the amino acid sequence could be responsible for the difference in localization and function of the protein in these two species.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease which occurs in approximately 1 in 3,500 male births. This disease is characterized by progressive muscle wasting and causes premature death. One of the earliest symptoms of this disease is mitochondrial dysfunction. Dystrophin is a protein found under the sarcolemma. The N terminus binds to actin and the C terminus binds to dystrophin glycoprotein complex (DGC). DMD is caused by mutations in the dystrophin gene. C. elegans possess an ortholog of dystrophin, DYS-1. Though there is evidence that C. elegans can be used as a model organism to model DMD, nematode DGC has not been well characterized. Additionally, while we know that mitochondrial dysfunction has been found in humans and other model organisms, this has not been well defined in C. elegans. In order to address these issues, we crossed the SJ4103 worm strain (myo-3p::GFP(mit)) with dys-1(cx18) in order to visualize and quantify changes in mitochondria in a dys-1 background. SJ4103;cx18 nematodes were found to have less mitochondrial than SJ4103 which suggests mitochondrial dysfunction does occur in dys-1 worms. Furthermore, mitochondrial dysfunction was studied by knocking down members of the DGC, dys-1, dyb-1, sgn-1, sgca-1, and sgcb-1 in SJ4103 strain. Knock down of each gene resulted in decrease in abundance of mitochondria which suggests that each member of the DGC contributes to the overall health of nematode muscle. The ORF of dyb-1 was successfully cloned and tagged with GFP in order to visualize this DGC member C. elegans. Imaging of the transgenic dyb-1::GFP worm shows green fluoresce expressed in which suggests that dyb-1 is a functional component of the muscle fibers. This project will enable us to better understand the effects of dystrophin deficiency on mitochondrial function as well as visualize the expression of certain members of the DGC in order to establish C. elegans as a good model organism to study this disease.

Aberrant signaling through the canonical RAS/RAF/MEK/ERK (ERK/MAPK) pathway leads to the pathology of a group of neurodevelopmental disorders called RASopathies. RASopathies are caused by germline mutations in the ERK/MAPK pathway and have an incidence of approximately 1:2000 births. The majority of RASopathies stem from mutations that cause gain-of-function in the ERK/MAPK pathway. In this study, we have begun to unravel the roles that GABAergic interneurons play in the pathology of RASopathies. Our data demonstrate that gain-of-function ERK/MAPK signaling expressed in a GABAergic interneuron-specific fashion leads to forebrain hyperexcitability in mutant mice. Further, some GABAergic interneurons experience activated-caspase 3 mediated apoptosis in the embryonic subpallium, leading to a loss of PV-expressing interneurons in the somatosensory cortex. We found that pharmaceutical intervention during embryogenesis using a MEK1 inhibitor may be effective in preventing apoptosis of these neurons. Future work is still needed to understand the mechanism of the death of GABAergic interneurons and to further pursue therapeutic approaches. Taken together, this study suggests potential roles of cortical GABAergic interneurons in ERK/MAPK-linked pathologies and indicates possible approaches to provide therapy for these conditions.

Pantothenate kinase-associated neurodegeneration, PKAN, is a neurological disease that is caused by biallelic mutations in the PANK2 gene, which codes for a pantothenate kinase. Some PANK2 mutations that cause PKAN retain enzymatic activity. A possible explanation for the mutations that have residual activity but still cause the disease is that they do not have the correct cellular localization. The localization of PANK2 was studied through cellular fractionation. We found the precursor form of PANK2, pPANK2, appears to be anchored to the inner membrane of the mitochondria, and the mature form, mPANK2, is located in the inter-membrane space, IMS. However, the IMS of the PKAN causing mutants is completely devoid of mPANK2 which suggests some disease-causing mutations may be mislocalized. In addition, PANK2 catalyzes the first and rate limiting step in Coenzyme A biosynthesis, and in other studies, it has been shown that the CoA biosynthesis enzymes form a complex in yeast. Therefore, we also considered the possibility that PKAN-causing mutations that retain activity have altered interactions with the other CoA biosynthesis enzymes. Coimmunoprecipitation of the proteins in the pathway was done to determine if there were any interactions with PANK2. The results indicate that PANK2 does not directly interact with either PPCS or CoASY, the second and final enzymatic activities in the CoA biosynthesis pathway.

Serotonin 1B receptors (5-HT1BRs) are a novel target for developing pharmacological therapies to reduce psychostimulant craving. 5-HT1BRs are expressed in the mesolimbic pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), which is involved in reward and motivation. 5-HT1BR agonists modulate both cocaine- and methamphetamine-seeking behaviors in rat models of psychostimulant craving. In this dissertation, I tested the central hypothesis that 5-HT1BRs regulate cocaine and methamphetamine stimulant and rewarding effects in mice. I injected mice daily with cocaine for 20 days and then tested them 20 days after their last injection. The results showed that the 5-HT1BR agonist CP94253 attenuated sensitization of cocaine-induced locomotion and cocaine-seeking behavior, measured as a decrease in the ability of a cocaine priming injection to reinstate extinguished cocaine-conditioned place preference (CPP). Subsequent experiments showed that CP94253 given prior to conditioning sessions had no effect on acquisition of methamphetamine-CPP, a measure of drug reward; however, CP94253 given prior to testing attenuated expression of methamphetamine-CPP, a measure of drug seeking. To examine brain regions and cell types involved in CP94253 attenuation of methamphetamine-seeking, I examined changes in the immediate early gene product, Fos, which is a marker of brain activity involving gene transcription changes. Mice expressing methamphetamine-CPP showed elevated Fos expression in the VTA and basolateral amygdala (BlA), and reduced Fos in the central nucleus of the amygdala (CeA). In mice showing CP94253-induced attenuation of methamphetamine-CPP expression, Fos was increased in the VTA, NAc shell and core, and the dorsal medial caudate-putamen. CP94253 also reversed the methamphetamine-conditioned decrease in Fos expression in the CeA and the increase in the BlA. In drug-naïve, non-conditioned control mice, CP94253 only increased Fos in the CeA, suggesting that the increases observed in methamphetamine-conditioned mice were due to conditioning rather than an unconditioned effect of CP94253 on Fos expression. In conclusion, 5-HT1BR stimulation attenuates both cocaine and methamphetamine seeking in mice, and that the latter effect may involve normalizing activity in the amygdala and increasing activity in the mesolimbic pathway. These findings further support the potential efficacy of 5-HT1BR agonists as pharmacological interventions for psychostimulant craving in humans.

The ability to detect and appropriately respond to chemical stimuli is important for many organisms, ranging from bacteria to multicellular animals. Responses to these stimuli can be plastic over multiple time scales. In the short-term, the synaptic strengths of neurons embedded in neural circuits can be modified and result in various forms of learning. In the long-term, the overall developmental trajectory of the olfactory network can be altered and synaptic strengths can be modified on a broad scale as a direct result of long-term (chronic) stimulus experience. Over evolutionary time the olfactory system can impose selection pressures that affect the odorants used in communication networks. On short time scales, I measured the effects of repeated alarm pheromone exposure on the colony-level defense behaviors in a social bee. I found that the responses to the alarm pheromone were plastic. This suggests that there may be mechanisms that affect individual plasticity to pheromones and regulate how these individuals act in groups to coordinate nest defense. On longer time scales, I measured the behavioral and neural affects of bees given a single chronic odor experience versus bees that had a natural, more diverse olfactory experience. The central brains of bees with a deprived odor experience responded more similarly to odorants in imaging studies, and did not develop a fully mature olfactory network. Additionally, these immature networks showed behavioral deficits when recalling odor mixture components. Over evolutionary time, signals need to engage the attention of and be easily recognized by bees. I measured responses of bees to a floral mixture and its constituent monomolecular components. I found that natural floral mixtures engage the orientation of bees’ antennae more strongly than single-component odorants and also provide more consistent central brain responses between stimulations. Together, these studies highlight the importance of olfactory experience on different scales and how the nervous system might impose pressures to select the stimuli used as signals in communication networks.

Neuroinflammation is an important secondary injury response occurring after traumatic brain injury (TBI). Anxiety-like disorders are commonly exacerbated after TBI and are mediated through the amygdala; however, the amygdala remains understudied despite its important contribution in processing emotional and stressful stimuli. Therefore, we wanted to study neuroinflammation after experimental TBI using midline fluid percussion in rodent models. We assessed microglia morphology over time post-injury in two circuit related nuclei of the amygdala, the basolateral amygdala (BLA) and central amygdala of the nucleus (CeA), using skeletal analysis. We also looked at silver staining and glial fibrillary acidic protein (GFAP) to evaluate the role of neuropathology and astrocytosis to evaluate for neuroinflammation in the amygdala. We hypothesized that experimental diffuse TBI leads to microglial activation in the BLA-CeA circuitry over time post-injury due to changes in microglial morphology and increased astrocytosis in the absence of neuropathology. Microglial cell count was found to decrease in the BLA at 1 DPI before returning to sham levels by 28 DPI. No change was found in the CeA. Microglial ramification (process length/cell and endpoints/cell) was found to decrease at 1DPI compared to sham in the CeA, but not in the BLA. Silver staining and GFAP immunoreactivity did not find any evidence of neurodegeneration or activated astrocytes in the respectively. Together, these data indicate that diffuse TBI does not necessarily lead to the same microglial response in the amygdala nuclei, although an alternative mechanism for a neuroinflammatory response in the CeA likely contributes to the widespread neuronal and circuit dysfunction that occurs after TBI.

Egr3 is an immediate early gene transcription factor that shows genetic association with schizophrenia, and is found in decreased levels in the brains of schizophrenia patients. Schizophrenia patients also exhibit cognitive and memory deficits, both of which Egr3 has been shown to play a crucial role in. Additionally, high levels of DNA damage are found in the brains of schizophrenia patients. A recent study has shown that DNA damage occurs as a result of normal physiological activity in neurons and is required for induction of gene expression of a subset of early response genes. Also, failure to repair this damage can lead to gene expression in a constitutive switched on state. Egr3 knockout (Egr3-/-) mice show deficits in hippocampal synaptic plasticity and memory. We were interested in characterizing downstream targets of EGR3 in the hippocampus. To determine these targets, electroconvulsive seizure (ECS) was carried out in Egr3 -/- versus wild type (WT) mice, and a microarray study was first done in our lab. ECS maximally stimulates Egr3 expression and we hypothesized that there would be gene targets that are differentially expressed between Egr3 -/- and WT mice that had been subjected to ECS. Two separate analyses of the microarray yielded 65 common genes that were determined as being differentially expressed between WT and Egr3 -/- mice after ECS. Further Ingenuity Pathway Analysis of these 65 genes indicated the Gadd45 signaling pathway to be the top canonical pathway, with the top four pathways all being associated with DNA damage or DNA repair. A literature survey was conducted for these 65 genes and their associated pathways, and 12 of the 65 genes were found to be involved in DNA damage response and/or DNA repair. Validation of differential expression was then conducted for each of the 12 genes, in both the original male cohort used for microarray studies and an additional female cohort of mice. 7 of these genes validated through quantitative real time PCR (qRT-PCR) in the original male cohort used for the microarray study, and 4 validated in both the original male cohort and an independent female cohort. Bioinformatics analysis yielded predicted EGR3 binding sites in promoters of these 12 genes, validating their role as potential transcription targets of EGR3. These data reveal EGR3 to be a novel regulator of DNA repair. Further studies will be needed to characterize the role of Egr3 in repairing DNA damage.

Traumatic brain injury is the leading cause of mortality and morbidity in children and adolescents. Adolescence is a critical time in development where the body and brain undergoes puberty, which not only includes reproductive maturation, but also adult social and cognitive development. Brain-injury-induced disruptions can cause secondary inflammation processes and as a result, pediatric TBI can lead to significant life-long and debilitating morbidities that continue long after initial injury. In this study, neuroinflammation following diffuse brain injury was explored in prepubertal and peripubertal rats using an adapted method of midline fluid percussion injury (mFPI) for juvenile rats to further understand the relationship between pediatric TBI and puberty disruption due to endocrine dysfunction. We expect the adapted mFPI model to be effective in producing diffuse, moderate brain injury in juvenile rats and hypothesize that pre-pubertal rats (PND35) will have increased neuroinflammation compared to peri-pubertal rats (PND17) and shams because of the potential neuroprotective nature of sex steroids. Male Sprague-Dawley rats (n=90) were subjected to either a diffuse midline fluid percussion injury (mFPI) or sham injury at post-natal day (PND) 17 (pre-puberty) or PND35 (peri-puberty). Animals were sacrificed at different time points defined as days post injury (DPI) including 1DPI, 7DPI and 25DPI to represent both acute and chronic time points, allowing for comparisons within groups (injury vs. sham) and across groups (PND17 vs PND35). Body weight of the rats was measured postoperatively at various time points throughout the study to follow recovery. Tissue was collected and subjected to Heamatoxylin and Eosin (H&E) stain to visualize histology and evaluate the application of diffuse mFPI to juvenile rats. In addition, tissue underwent immunohistochemical analysis using 3,3'-diaminobenzidine (DAB) to stain for ionized calcium binding proteins (Iba1) in order to assess injury-related neuroinflammation in the form of microglia activation. Diffuse brain injury using the mFPI model did not affect rat body weight or cause overt cell death, suggesting adaption of the adult mFPI model for juvenile rats is representative of moderate diffuse brain injury. In addition, diffuse TBI lead to morphological changes in microglia suggesting there is an increased inflammatory response following initial insult, which may directly contribute to improper activation of pubertal timing and progression in adolescent children affected. Since there is little literature on the full effects of puberty dysfunction following TBI in the pediatric population, there is a significant need to further assess this area in order to develop improved interventions and potential therapies for this affected population.