No description available.

All multicellular life begins as a single cell—the fertilised egg, from which the adult organism develops. As a general priniciple, as embryos progess through development, changes in cellular status seem to be effected by cell specific transcription factors which regulate specific gene subsets. The SOX (Sry box) family of transcription factors are one such developmentally important class of transcription factors, consisting of twenty mammalian proteins that each contain a single High Mobility Group (HMG) DNA binding domain that is >50% homologous to that of Sry, the mammalian testes determining factor. SOX proteins are multi-functional developmental regulators that sequence specifically bind DNA, and can function both as classical transcription factors and as architectural components of chromatin (Kiefer et al. 2007, Lefebvre et al. 2007). We have been modeling SOX gene function using the Drosophila SOX gene Dichaete (D). D has similar biochemical properties to mammalian SOX proteins, and is essential for embryonic segmentation and cell fate specification (Ma et al. 1998, Russell et al. 1996). In this thesis I detail novel functions of D in oogenesis and adult olfactory system development. Chapter two details D expression and function during oogenesis in Drosophila. We show that D is transiently expressed in the oocyte cytoplasm from region 2 of the germarium through stage 8. We demonstrate that D protein can bind gürken mRNA, which was mislocalised in D mutant egg chambers. These studies contribute to our understanding of the establishment of dorsal/ventral polarity and significantly detail a cytoplasmic role for SOX proteins in binding mRNA (Mukherjee et al., 2006). Chapter three details the expression and function of D in the adult Drosophila nervous system. I show that D is prominently expressed in a mixture of excitatory and inhibitory local neurons (LNs) and central complex ring neurons. Hypomorphic D alleles were generated, and the mutant brains exhibited misplacement and mistargeting of specific olfactory projection neurons. These data greatly enhance our understanding of the development of neuronal connectivity in a discrete neural map represented by the fly antennal lobe, and represent a detailed report of SOX gene expression in the adult brain.

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (α-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized into two groups based on their incidence of occurrence. First is the familial form that occurs due to known genetic aberrations like mutation, gene duplication/triplication in important PD associated gene like SNCA which in turn leads to early-onset PD (EOPD). Second is the late-onset idiopathic or sporadic form, whose origin of occurrence is often unknown. Interestingly, more than 90%-95% of reported PD cases belong to the latter category. Although, the familial and the idiopathic form of PD are different in their respective cause of occurrence, aggregation of α-SYN into Lewy body is a common pathologic hallmark seen in both. Aggregation of α-SYN in turn is strongly implicated by the transcriptional upregulation of the gene as seen in both familial forms as well as idiopathic forms. In this thesis, we first describe the designing and functioning of a novel tool to monitor real-time SNCA transcription in Human Embryonic Kidney (HEK) 293T cells. In the next part, we shed light into a novel transcriptional deregulation phenomenon called transcriptional mutagenesis, which leads to accelerated aggregation of α-SYN as seen in sporadic PD. In brief, the focus of this work is to highlight the importance of transcriptional regulation of SNCA gene, through development of a tool and a mechanism affecting the fidelity of transcription under pathologic condition. In the first study, we developed a stable cell line in HEK293T cells in which α-SYN was tagged with Nanoluc luciferase reporter using CRISPR/Cas9-mediated genome editing. Nanoluc is a small stable reporter of 19KDa size, which is 150 fold brighter compared to firefly and Renilla luciferase, thus making it a very good candidate for endogenous monitoring of gene regulations. We successfully integrated the Nanoluc at the 3'end of the SNCA before the stop codon. Successful integration of the Nanoluc was demonstrated by the fusion α-SYN protein containing the Nanoluc. This allowed efficient monitoring of α-SYN transcription keeping its native epigenetic landscape unperturbed which was otherwise difficult using exogenous luciferase reporter assays. The Nanoluc activity monitored by a simple two-step assay faithfully reflected the endogenous deregulation of SNCA following treatment with different drugs including epigenetic modulators and dopamine which were already known to up-regulate SNCA transcription. Interestingly, use of exogenous promoter-reporter assays (firefly luciferase assays) failed to reproduce the similar outcomes. In fact, exogenous system showed contradictory results in terms of the α-SYN regulation which aroused from spurious effects of the drug on the reporter system. To our knowledge, this is the first report showing endogenous monitoring of α-SYN transcription, thus making it an efficient drug screening tool that can be very effectively used for therapeutic intervention in PD. In the next study, we investigated the effect of oxidative DNA damage in the form of 8-hydroxy-2-deoxyguanosine (8-oxodG, oxidized guanine) on aggregation of α-SYN through a novel phenomenon called transcriptional mutagenesis. It is already known that 8-oxodG is repaired by a specific component of the base excision repair machinery of the cell called 8-oxodG-DNA glycosylase 1 (OGG1). If left unrepaired, 8-oxodG can lead to misincorporation of adenine instead of cytosine (C→A transversion) in the synthesized mRNA during transcription for post-mitotic cells like neurons. This phenomenon is called transcriptional mutagenesis (TM) and can generate novel mutant variants of any functional protein. α-SYN, which is implicated very strongly in the pathogenesis of PD, has been shown to become aggregation prone by specific point mutation. Previous studies have shown that certain point mutations can make α-SYN more prone to aggregation and can affect the aggregation of the parental protein as a template directed misfolding mechanism. We used SNCA as a model gene and predicted the generation of forty-three different positions that can be mutated by the TM event. We investigated the generation of three out of the forty-three possible TM mutants from the SN of post-mortem PD and age-matched control brain cohorts based on their potential to aggregate as predicted by aggregation prediction software TANGO. The three mutants were Serine42Tyrosine (S42Y), Alanine53Glutamate (A53E) and Serine129Tyrosine (S129Y). We confirmed the presence of all the three mutant α-SYN (S42Y, A53E and S129Y) in SNCA mRNA from the SN of human post-mortem PD brain using a PCR-based detection technique. As expected, analysis of the overall distribution of the three mutants showed a higher rate of occurance in the PD cohort compared to the age-matched controls. Sequencing genomic DNA of the same PD sample from the same region of ?-SYN revealed no mutations at the genomic DNA level, thus implying its generation during transcription. Although we could detect the presence of S42Y, A53E and S129Y α-SYN in the cohort of PD patients, we focused to analyse the contribution of S42Y towards the aggregation of wild-type (WT) α-SYN parental protein based on its higher potential to aggregate. By using cell-based biochemical and recombinant protein assays, we saw that S42Y-α-SYN can accelerate the aggregation process involving the WT protein even when present in significantly lower proportion (100 times less compared to the WT). Importantly, we developed antibody to specifically detect the S42Y α-SYN in human PD cohort. Immunohistochemical analysis of serial post-mortem PD brain sections with Hematoxylin and Eosin staining (H&E), anti-ubiquitin staining and anti-S42Y α-SYN staining, showed Lewy bodies that stained positively with S42Y α-SYN. To our knowledge, this is the first report about TM related mutations of α-SYN in Parkinson's disease and their role in the pathogenesis.

No description available.

The cerebellum is the most commonly reported brain region to demonstrate pathology in autism. Multiple reports have indicated a lower density of Purkinje cells (PCs) in the posterolateral cerebellar hemispheres. We performed a stereological technique to precisely quantify PCs in postmortem autism cases compared to age- and sex-matched controls. We found that although PC density was lower in all cerebellar regions studied, the most significant difference was in lobule VIIa of the posterior lobe. The PCs in this region project to deep cerebellar nuclei that reciprocally connect to all aspects of the established broader sensorimotor gating network. Sensorimotor gating abnormalities are commonly observed in individuals diagnosed with autism, and may contribute to problems with sensory processing and behavioral inhibition in these individuals. We studied a rat model of sensorimotor gating impairment, in which the histamine H1 receptor antagonist, pyrilamine, improved sensorimotor gating. Using autoradiography, we found that pyrilamine treatment altered H1 receptor and α7 nicotinic receptor binding in the anterior cingulate and insular cortex, respectively, an effect which correlated with improved sensorimotor gating. Histamine functions as both a neurotransmitter as well as a regulator of glial activity throughout the brain. Using western blots, we quantified H1 receptor levels in lobule VIIa from postmortem autism cases but found no difference compared to controls. We further quantified additional proteins to investigate theories of neuroimmune and neuroendocrine dysregulation in the cerebellum in autism. These included IBA-1, GFAP, IL-6, androgen receptor, estrogen receptor β, and aromatase. We found no evidence to support these theories: all protein levels tested were found to be similar in the autism and control groups. We suggest further studies to better understand cerebellar pathogenesis and regulation of sensorimotor gating in autism. The implications of sensorimotor gating impairment in autism are discussed in relation to the established symptomatology of this neurodevelopmental behavioral disorder.

Thirty percent of individuals with autism spectrum disorder (ASD) have recurrent spontaneous seizures (SS). The relationship between the etiology of ASD and epilepsy is an active area of study and one that our laboratory is interested in pursuing as evidence points to a shared disturbance in brain inhibitory systems using the neurotransmitter g-aminobutyric acid (GABA). Most patients suffer from temporal lobe epilepsy and although many molecular changes have been ascribed to its etiology, why certain individuals are at high risk and others are spared is unknown. Temporal lobe epilepsy can be modeled in animals through use of chemically induced prolonged seizures, called status epilepticus. After status epilepticus there is a seizure-free latent period, where ongoing molecular changes occur, followed weeks later by SS, a hallmark of epilepsy. Our laboratory has shown that major changes occur in the composition of GABA-A receptors (GABARs) during the latent period that impairs the function of synaptic receptors in the dentate gyrus, a gatekeeper of excitation in the hippocampus. In my thesis research I demonstrate for the first time that specific changes in GABAR expression are also present during the chronic period of SS, suggesting that altered GABAR composition is preserved throughout the disease process. A major molecular feature of ASD, and one that is seen in multiple ASD models, is marked disruption in development of GABAergic interneurons emerging from their birthplace in ganglionic eminence (GE), and the altered expression of GABARs. In my thesis research using RNA-sequencing I identified that GE expresses almost all of the genes coding for GABAR subunits laying the foundation for future studies on the role of GABARs during development. In addition, using sequencing of chromatin immunoprecipitated fragments containing binding sites for Engrailed 2, a major ASD candidate gene, I show that En2 may be a master regulator of multiple genes in the SFARI database; and, using mouse in utero electroporation, we provide the first evidence that En2 may control the fate of neurons emanating from GE at E12.5. Taken together, my thesis research has uncovered two major areas for future investigation into the overlapping fields of epilepsy and ASD.

Approximately 795,000 Americans experience a new or recurrent stroke each year (American Heart Association 2016; Mozaffarian et al. 2016). However, the only experimental therapeutic to have gained FDA approval for treatment of stroke in humans is the thrombolytic agent tPA that can dissolve clots and restore blood flow, if given within a narrow therapeutic window of a few hours following stroke onset (AHA 2016, Li et al. 2016). Nevertheless, in many cases with or without tPA there is significant residual impairment, and there are currently no FDA approved therapeutic agents that facilitate functional recovery following stroke (Zhang L et al. 2012). Recent studies have suggested that neural plasticity and neurogenesis following stroke may play a role in recovery of function, and promising findings have been demonstrated with cell therapies for enhancing recovery after stroke (Kokaia and Darasalia, 2015; Kozorovitskiy et al, 2013; Zhao et al, 2012). Our recent study (Moore et al. 2013) showed significant recovery of function following a reproducible ischemic lesion limited to the hand representation of the motor cortex in non-human primates (NHPs) treated with the investigational cell drug product CNTO 0007, that contains human umbilical tissue-derived cells (hUTC). While the treatment group in this study demonstrated significantly better recovery of motor function, the mechanism of recovery remains unclear. Previous studies conducted with brain tissue from these monkeys have suggested that functional recovery may be related to cortical reorganization induced by the hUTC treatment. To explore the possibility that neurogenesis may have also played a role in the enhanced recovery, these same monkeys received an injection of the thymidine analog Bromodeoxyuridine (BrdU), which was visualized in the brain tissue to investigate cell proliferation in the subventricular zone and hippocampus. Results show that there is no significant difference in the number of BrdU positive cells in the hUTC treated vs. untreated monkeys, however there is a trend towards significant increase in BrdU labeling in the granule cell layer of the hippocampus of the hUTC treated animals. Clusters of proliferating cells were also found in the GCL of treated monkeys, but not in the untreated monkeys. These findings support the hypothesis that enhanced recovery of function may be related to a combination of reorganization of undamaged cortical motor regions and generation of new cells in the brain.

Breathing is a vital rhythmic behavior essential for life. The preBötzinger complex (preBötC) of the lower brainstem houses a cluster of interneurons that generate the respiratory rhythm in humans and all terrestrial mammals. Neurons of the preBötC produce rhythmic bursts of neural activity that drive inspiration, the only inexorable active phase of the breathing cycle (expiration is normally passive). preBötC interneurons that comprise the core inspiratory rhythm generator are derived from precursors that express the transcription factor developing brain homeobox 1 (Dbx1). Neurons in the preBötC derived from Dbx1-expressing progenitors (hereafter referred to as Dbx1 preBötC neurons) produce rhythmic bursts of spike activity as well as the fundamental output that then drives inspiratory-related pump and airway resistance muscles. A longstanding question in respiratory neuroscience is whether rhythm (inspiratory frequency) and pattern (motor output to inspiratory-related muscles) are controlled by distinct sets of neurons and their neural mechanisms. Here, I test the burstlet theory of inspiratory rhythm- and pattern-generation that demonstrates that inspiratory rhythm in preBötC microcircuits occurs independently without obligatory neural bursts that generate motor output responsible for airway control, which affirms the discrete separation of preBötC neurons into “rhythm-related” and “pattern-related” camps. The question articulated above has been refined over the past decade to consider whether rhythm and pattern might be separately mediated by discrete sets of Dbx1 neurons. Next, I delve deeper in order to distinguish the inspiratory rhythm- and pattern- generating populations of Dbx1 preBötC neurons, starting with their classification electrophysiologically on the basis of key intrinsic properties associated with either rhythmogenesis or motor pattern (i.e., output). I then perform patch-seq, which involves the whole-cell patch-clamp recording of Dbx1 preBötC neurons and then by extraction of their cytoplasmic contents, followed by RNA sequencing and transcriptome analyses. I show that 123 genes significantly differentiate the putative rhythm- and pattern-generating populations of Dbx1 preBötC neurons. Surprisingly, the differentially expressed genes do not pertain to the ion channels that give rise to their distinct electrophysiological disparities. Nevertheless, the expression of synaptic receptors and neuromodulators appears to delineate how these discrete sets of neurons are programmed to play different roles in breathing rhythm vs. pattern. The deliverable of the project provides high-quality transcriptomes at the cellular origin of breathing, a key physiological behavior to the scientific commons, and provide distinct genetic targets to manipulate and perturb breathing’s cellular and molecular underpinnings. I conclude that discrete sets of Dbx1 preBötC neurons generate rhythm and mediate fundamental aspects of inspiratory output pattern. These neuron types are distinct in terms of physiology and transcriptome, which answers a longstanding question in respiratory neurobiology about the neural origins of inspiratory breathing movements.

The expression of lordosis in rats, the receptive component of female sexual behavior, requires sufficient levels of circulating estradiol and progesterone and the appropriate tactile stimuli usually provided by the male during mounting. Estradiol and progesterone mediate many of their effects on female sexual behavior by acting through their respective receptors in neurons, located predominantly in the preoptic area, hypothalamus and midbrain. Tactile stimuli also appear to regulate lordosis by acting in the brain. Taken together, this suggests that hormonal and tactile stimuli associated with female reproduction may converge on specific neurons in the brain. This dissertation investigated the intraneuronal convergence of information associated with estradiol and vaginal-cervical stimulation (VCS), an important aspect of tactile stimuli provided by the male during copulation. In the first experiment, an increase in the number of neurons expressing Fos, an indicator of neuronal response, was observed in the medial preoptic area, medial bed nucleus of the stria terminalis, posterodorsal medial amygdala, ventromedial hypothalamus and midbrain following VCS. In the second experiment, these same brain regions responded to VCS provided by manual probing, which elicits many of the same behavioral and endocrine changes related to female reproduction as VCS by mating. In the third experiment it was determined that steroid hormones influence the number of neurons in the ventromedial hypothalamus which respond to VCS. In the final experiment, it was found that many of the neurons that respond to VCS also contain estrogen receptors, suggesting that they can also respond to estradiol. These results suggest that sensory and hormonal information associated with female reproduction converge on specific populations of neurons and may be integrated at the molecular level within these neurons.

Food availability is one of the most important environmental variables in controlling reproduction in mammals including humans. Undernutrition causes a delay of puberty, irregular menstrual/estrous cyclicity, and an impairment of sexual behavior. In female Syrian hamsters, food deprivation or treatment with metabolic inhibitors (2-deoxy- scD-glucose (2DG) and methyl palmoxirate (MP)) during estrous cycle days 1 and 2 suppresses the proestrous luteinizing hormone (LH) surge, blocks ovulation rate and inhibits lordosis. Taken together, this suggests that 48 h of fuel restriction cause a deficit in various aspects of reproduction function in Syrian hamsters. This dissertation investigated the possible neural mechanisms and neural pathways associated with fuel restriction-induced suppression of estrous behavior in Syrian hamsters. In the first experiment, 48 h of food deprivation or treatment with 2DG and MP inhibited steroid-induced lordosis in ovariectomized hamsters. However, neither 2DG nor MP given alone affected lordosis duration. To test the hypothesis that fuel restriction results in altered estradiol binding in the brain, immunocytochemistry for estrogen-receptor immunoreactivity (ERIR) was developed for hamster brain. Dense populations of ERIR cells were observed in the preoptic area, bed nucleus of stria terminalis, mediobasal hypothalamus, and amygdala. In the third experiment, fuel restriction for 48 h decreased the number of detectable ERIR cells in the ventromedial/ventrolateral hypothalamus (VMH/VLH), increased ERIR in the medial preoptic area (mPOA), but did not change ERIR in the nucleus of the solitary tract. The final experiment was designed to explore the possible neural relay loci in transmitting metabolic fuel information into the VMH/VMH and mPOA. The results showed that the area postrema, but not the vagus nerves, is required for the VMH/VLH to detect fuel availability. However, the vagus nerves, but not the area postrema, are required for the mPOA to detect fuel information. These results suggest that fuel restriction-induced suppression of lordosis is partially due to an alteration of estradiol binding in the brain and the level of VMH/VLH ERIR is correlated with the expression of lordosis. Furthermore, caudal hindbrain and the vagus nerves play an essential role for the transmission of metabolic fuel information to the ERIR cells in the VMH/VLH and mPOA, respectively.