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Dysregulated ERK/MAPK Signaling in RASopathy Animal Model Systems Leads to a Decrease in mTOR Expression and Activation of Translational Machinery

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The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development

The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development of the nervous system. Activation of ERK is indispensable for the differentiation of Embryonic Stem Cells (ESC) into neuronal precursors (Li z et al, 2006). ERK signaling has also shown to mediate Schwann cell myelination of the peripheral nervous system (PNS) as well as oligodendrocyte proliferation (Newbern et al, 2011). The class of developmental disorders that result in the dysregulation of RAS signaling are known as RASopathies. The molecular and cell-specific consequences of these various pathway mutations remain to be elucidated. While there is evidence for altered DNA transcription in RASopathies, there is little work examining the effects of the RASopathy-linked mutations on protein translation and post-translational modifications in vivo. RASopathies have phenotypic and molecular similarities to other disorders such as Fragile X Syndrome (FXS) and Tuberous Sclerosis (TSC) that show evidence of aberrant protein synthesis and affect related pathways. There are also well-defined downstream RAS pathway elements involved in translation. Additionally, aberrant corticospinal axon outgrowth has been observed in disease models of RASopathies (Xing et al, 2016). For these reasons, this present study examines a subset of proteins involved in translation and translational regulation in the context of RASopathy disease states. Results indicate that in both of the tested RASopathy model systems, there is altered mTOR expression. Additionally the loss of function model showed a decrease in rps6 activation. This data supports a role for the selective dysregulation of translational control elements in RASopathy models. This data also indicates that the primary candidate mechanism for control of altered translation in these modes is through the altered expression of mTOR.

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2017-05

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MicroRNA regulation of addiction-related gene expression and motivation for cocaine in rats

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MicroRNAs are small, non-coding transcripts that post-transcriptionally regulate expression of multiple genes. Recently microRNAs have been linked to the etiology of neuropsychiatric disorders, including drug addiction. Following genome-wide sequence analyses, microRNA-495 (miR-495) was found to target several genes within the

MicroRNAs are small, non-coding transcripts that post-transcriptionally regulate expression of multiple genes. Recently microRNAs have been linked to the etiology of neuropsychiatric disorders, including drug addiction. Following genome-wide sequence analyses, microRNA-495 (miR-495) was found to target several genes within the Knowledgebase of Addiction-Related Genes (KARG) database and to be highly expressed in the nucleus accumbens (NAc), a pivotal brain region involved in reward and motivation. The central hypothesis of this dissertation is that NAc miR-495 regulates drug abuse-related behavior by targeting several addiction-related genes (ARGs). I tested this hypothesis in two ways: 1) by examining the effects of viral-mediated miR-495 overexpression or inhibition in the NAc of rats on cocaine abuse-related behaviors and gene expression, and 2) by examining changes in NAc miR-495 and ARG expression as a result of brief (i.e., 1 day) or prolonged (i.e., 22 days) cocaine self-administration. I found that behavioral measures known to be sensitive to motivation for cocaine were attenuated by NAc miR-495 overexpression, including resistance to extinction of cocaine conditioned place preference (CPP), cocaine self-administration on a high effort progressive ratio schedule of reinforcement, and cocaine-seeking behavior during both extinction and cocaine-primed reinstatement. These effects appeared specific to cocaine, as there was no effect of NAc miR-495 overexpression on a progressive ratio schedule of food reinforcement. In contrast, behavioral measures known to be sensitive to cocaine reward were not altered, including expression of cocaine CPP and cocaine self-administration under a low effort FR5 schedule of reinforcement. Importantly, the effects were accompanied by decreases in NAc ARG expression, consistent with my hypothesis. In further support, I found that NAc miR-495 levels were reduced and ARG levels were increased in rats following prolonged, but not brief, cocaine self-administration experience. Surprisingly, inhibition of NAc miR-495 expression also decreased both cocaine-seeking behavior during extinction and NAc ARG expression, which may reflect compensatory changes or unexplained complexities in miR-495 regulatory effects. Collectively, the findings suggest that NAc miR-495 regulates ARG expression involved in motivation for cocaine. Therefore, using microRNAs as tools to target several ARGs simultaneously may be useful for future development of addiction therapeutics.

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2016

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Traumatic brain injury induces rapid enhancement of cortical excitability in juvenile rats

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Following a traumatic brain injury (TBI) 5-50% of patients will develop post traumatic epilepsy (PTE). Pediatric patients are most susceptible with the highest incidence of PTE. Currently, we cannot prevent the development of PTE and knowledge of basic

Following a traumatic brain injury (TBI) 5-50% of patients will develop post traumatic epilepsy (PTE). Pediatric patients are most susceptible with the highest incidence of PTE. Currently, we cannot prevent the development of PTE and knowledge of basic mechanisms are unknown. This has led to several shortcomings to the treatment of PTE, one of which is the use of anticonvulsant medication to the population of TBI patients that are not likely to develop PTE. The complication of identifying the two populations has been hindered by the ability to find a marker to the pathogenesis of PTE. The central hypothesis of this dissertation is that following TBI, the cortex undergoes distinct cellular and synaptic reorganization that facilitates cortical excitability and promotes seizure development. Chapter 2 of this dissertation details excitatory and inhibitory changes in the rat cortex after severe TBI. This dissertation aims to identify cortical changes to a single cell level after severe TBI using whole cell patch clamp and electroencephalogram electrophysiology. The work of this dissertation concluded that excitatory and inhibitory synaptic activity in cortical controlled impact (CCI) animals showed the development of distinct burst discharges that were not present in control animals. The results suggest that CCI induces early "silent" seizures that are detectable on EEG and correlate with changes to the synaptic excitability in the cortex. The synaptic changes and development of burst discharges may play an important role in synchronizing the network and promoting the development of PTE.

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2014

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The Functions of LKB1 in the Development of Inhibitory Interneurons in the Cerebral Cortex

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LKB1/STK11 is a serine/threonine kinase first identified in C.elegans as a gene important for cell polarity and proliferation. Mutations in LKB1 are the primary cause of Peutz-Jegher’s cancer syndrome, an autosomal dominantly inherited disease, in which patients are predisposed to

LKB1/STK11 is a serine/threonine kinase first identified in C.elegans as a gene important for cell polarity and proliferation. Mutations in LKB1 are the primary cause of Peutz-Jegher’s cancer syndrome, an autosomal dominantly inherited disease, in which patients are predisposed to benign and malignant tumors. Past studies have focused on defining LKB1 functions in various tissue types, for example LKB1 regulates axonal polarization and dendritic arborization by activating downstream substrates in excitatory neurons of the developing neocortex. However, the implications of LKB1, specifically in the developing cortical inhibitory GABAergic interneurons is unknown. LKB1 deletion was achieved by using Cre-lox technology to induce LKB1 loss in cells localized in the medial ganglionic eminence (MGE) that express Nkx2.1 and generate cortical GABAergic neurons. In this research study it is suggested that LKB1 plays a role in GABAergic interneuron specification by specifically regulating the expression of parvalbumin during the development of fast-spiking interneurons. Preliminary evidence suggest LKB1 also modulates the number of Nkx2.1-derived oligodendrocytes in the cortex. By utilizing a GABAergic neuron specific LKB1 deletion mutant, we confirmed that the loss of parvalbumin expression was due to a GABAergic neuron autonomous function for LKB1. These data provide new insight into the cell specific functions of LKB1 in the developing brain.

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2019

Severe traumatic brain injury induces cortical remodeling in the pediatric inhibitory network

Description

Pediatric traumatic brain injury (TBI) is a leading cause of death and disability in children. When TBI occurs in children it often results in severe cognitive and behavioral deficits. Post-injury, the pediatric brain may be sensitive to the

Pediatric traumatic brain injury (TBI) is a leading cause of death and disability in children. When TBI occurs in children it often results in severe cognitive and behavioral deficits. Post-injury, the pediatric brain may be sensitive to the effects of TBI while undergoing a number of age-dependent physiological and neurobiological changes. Due to the nature of the developing cortex, it is important to understand how a pediatric brain recovers from a severe TBI (sTBI) compared to an adult. Investigating major cortical and cellular changes after sTBI in a pediatric model can elucidate why pediatrics go on to suffer more neurological damage than an adult after head trauma. To model pediatric sTBI, I use controlled cortical impact (CCI) in juvenile mice (P22). First, I show that by 14 days after injury, animals begin to show recurrent, non-injury induced, electrographic seizures. Also, using whole-cell patch clamp, layer V pyramidal neurons in the peri-injury area show no changes except single-cell excitatory and inhibitory synaptic bursts. These results demonstrate that CCI induces epileptiform activity and distinct synaptic bursting within 14 days of injury without altering the intrinsic properties of layer V pyramidal neurons. Second, I characterized changes to the cortical inhibitory network and how fast-spiking (FS) interneurons in the peri-injury region function after CCI. I found that there is no loss of interneurons in the injury zone, but a 70% loss of parvalbumin immunoreactivity (PV-IR). FS neurons received less inhibitory input and greater excitatory input. Finally, I show that the cortical interneuron network is also affected in the contralateral motor cortex. The contralateral motor cortex shows a loss of interneurons and loss of PV-IR. Contralateral FS neurons in the motor cortex synaptically showed greater excitatory input and less inhibitory input 14 days after injury. In summary, this work demonstrates that by 14 days after injury, the pediatric cortex develops epileptiform activity likely due to cortical inhibitory network dysfunction. These findings provide novel insight into how pediatric cortical networks function in the injured brain and suggest potential circuit level mechanisms that may contribute to neurological disorders as a result of TBI.

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2015

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A Mouse Model of Serotonin 1B Receptor Modulation of Cocaine and Methamphetamine Craving

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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

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.

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2018

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Effects of Odorant-environment Complexity on Behavioral and Neural Plasticity at Different Time Scales

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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

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.

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2018

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Diffuse Brain Injury Incites Sexual Differences and Hypothalamic-Pituitary-Adrenal Axis Disruptions

Description

Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective

Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective disorders. Preliminary data indicate TBI causes neuroinflammation in the hippocampus, likely due to axonal damage, and in the paraventricular nucleus of the hypothalamus (PVN), where no axonal damage is apparent. Mechanisms regulating neuroinflammation in the PVN are unknown. Furthermore, chronic stress causes HPA dysregulation and glucocorticoid receptor (GR)-mediated neuroinflammation in the PVN. The goal of this project was to evaluate neuroinflammation in the HPA axis and determine if GR levels change at 7 days post-injury (DPI).

Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.

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2019

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Disrupted synaptic transmission and abnormal short-term synaptic plasticity in an Angelman syndrome mouse model

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Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein,

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein, E6-AP) in neurons. Currently, the mechanism on how loss-of-function of the enzyme influences the nervous system development remains unknown. We hypothesize that impaired metabolism of proteins, most likely those related to E6-AP substrates, may alter the developmental trajectory of neuronal structures including dendrites, spines and synaptic proteins, which leads to disrupted activity/experience-dependent synaptic plasticity and maturation. To test this hypothesis, we conducted a detailed investigation on neuronal morphology and electrophysiological properties in the prefrontal cortex (PFC) layer 5 (L5) corticostriatal pyramidal neurons (target neurons). We found smaller soma size in the maternal Ube3a deficient mice (m-/p+; 'AS' mice) at postnatal 17-19 (P17-19), P28-35 and older than 70 days (>P70), and decreased basal dendritic processes at P28-35. Surprisingly, both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) decreased on these neurons. These neurons also exhibited abnormalities in the local neural circuits, short-term synaptic plasticity and AMPA/NMDA ratio: the excitatory inputs from L2/3 and L5A, and inhibitory inputs from L5 significantly reduced in AS mice from P17-19; Both the release probability (Pr) and readily-releasable vesicle (RRV) pool replenishment of presynaptic neurons of the target neurons were disrupted at P17-19 and P28-35, and the change of RRV pool replenishment maintained through adulthood (>P70). The AMPA/NMDA ratio showed abnormality in the L5 corticostriatal neurons of PFC in AS mice older than P28-35, during which it decreased significantly compared to that of age-matched WT littermates. Western Blot analysis revealed that the expression level of a key regulator of the cytoskeleton system, Rho family small GTPase cell division control protein 42 homolog (cdc42), reduced significantly in the PFC of AS mice at P28-35.These impairments of synaptic transmission and short-term synaptic plasticity may account for the impaired neuronal morphology and synaptic deficits observed in the PFC target neurons, and contribute to the phenotypes in AS model mice. The present work reveals for the first time that the E6-AP deficiency influences brain function in both brain region-specific and age-dependent ways, demonstrates the functional impairment at the neural circuit level, and reveals that the presynaptic mechanisms are disrupted in AS model. These novel findings shed light on our understanding of the AS pathogenesis and inform potential novel therapeutic explorations.

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2017

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The Role of ERK/MAPK In The Postnatal Development of Lower Motor Neurons

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The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and

The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction.

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2017