Circadian clocks are biological timekeepers that help maintain an organism’s behavior and physiological state optimally timed to the Earth’s day/night cycle. To do this, these internal pacemakers must accurately keep track of time. Equally importantly, they must be able to adjust their oscillations in response to external time cues to remain properly synchronized with the environment, and correctly anticipate environmental changes. When the internal clock is offset from its surrounding day/night cycle, clinically relevant disruptions develop, ranging from inconveniences such as jet-lag to more severe problems such as sleep disorders or mood disorders. In this work, I have used the fruit fly, Drosophila melanogaster, as a model organism to investigate how light and temperature can synchronize circadian systems. My initial studies centered on an intracellular photoreceptor, CRYPTOCHROME (CRY). CRY is a blue light photoreceptor previously identified as a major component of the primary light-input pathway into the Drosophila circadian clock. We used molecular techniques to show that after light-activation, CRY binds to the key circadian molecule TIMELESS (TIM). This interaction irreversibly targets TIM, but not CRY, for degradation. Further studies characterizing a newly isolated cry mutant, crym, showed that the carboxyl-terminus of CRY is not necessary for CRY’s ability to impart photic information to the molecular clock. Instead, the C-terminus appears to be necessary for normal CRY stability and protein-protein interactions. Thus, we conclude that in contrast to previous reports on CRYs of other species, where the C-terminal domain was required for transduction of photic information, the C-terminus of DrosophilaCRY has a purely modulatory function. During the second part of my dissertation work, I focused my studies on circadian thermoreception. While the effects of light in synchronization of the Drosophilaclock to environmental cycles have been extensively characterized, significantly less is known about temperature input pathways into the circadian pacemaker. I have used two approaches to look at how temperature affects the circadian system. First, I conducted a series of behavioral analyses looking at how locomotor rhythms can be phase-shifted in response to temperature cycles. By examining the behavior of genetically ablated flies, we determined that the well-characterized neurons controlling morning and evening surges of activity during light/dark cycles are also implicated in morning and evening behaviors under temperature cycles. However, we also find evidence of cells that contribute to modulating afternoon and evening behavior specifically under temperature cycles. These data contribute to a growing number of studies in the field suggesting that pacemaker cells may play different roles under various environmental conditions. Additionally, we provide data showing that intercellular communication plays an important role in regulating circadian response to temperature cycles. When the morning oscillator is absent or attenuated, the evening cells respond abnormally quickly to temperature cycles. My work thus provides information on the roles of different cell groups during temperature cycles, and suggests that beyond simply synchronizing individual oscillating cells, intercellular network activity may also have a role in modulating proper response to environmental time cues. Finally, I present some preliminary work looking at effects of temperature on known circadian molecules. Using a combination of in vivo and cell culture techniques, I have found that TIM protein levels decrease at higher temperatures. My cell culture data suggest that this is a proteasome-independent degradation event. As TIM is also a key molecule in the light-input pathway, the stability of TIM proteins may be a key point of integration for light and temperature input pathways. While additional research needs to be conducted to confirm these effects in vivoin wild-type flies, these preliminary results identify a possible avenue for further study. Taken together, my work has contributed new data on both molecular and neuronal substrates involved in processing light and temperature inputs into the Drosophila circadian clock.

Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations). The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are also involved in generating mammalian circadian rhythms. In addition, there are only six neuronal groups in the adult fly brain (comprising about 75 pairs of cells) that express high levels of clock genes. The simplicity of this system is ideal for the study of the neural circuitry underlying behavior. The first half of this dissertation focuses on a genetic screen designed to identify novel genes involved in the circadian light input pathway. The screen was based on previous observations that a mutation in the circadian photoreceptor CRYPTOCHROME (CRY) allows flies to remain rhythmic in constant light (LL), while wild type flies are usually arrhythmic under this condition. 2000 genes were overexpressed and those that showed a rhythmic behavior in LL (like crymutants) were isolated. The candidate genes isolated in the screen present a wide variety of biological functions. These include genes involved in protein degradation, signaling pathways, regulation of transcription, and even a pacemaker gene. In this dissertation, I describe work done in order to validate and characterize such candidates. The second part of this dissertation focuses on identifying the pacemaker neurons that drive circadian rhythms in constant light (LL) when the pacemaker gene period is overexpressed. We found that a subset of pacemaker neurons, the DN1s, is responsible for driving rhythms in constant light. This attractive finding reveals a novel role for the DN1s in driving behavioral rhythms under constant conditions and suggests a mechanism for seasonal adaptation in Drosophila.

BK channels are well studied targets of acute ethanol action. They play a prominent role in neuronal excitability and have been shown to play a significant role in behavioral ethanol tolerance in invertebrates. The focus of my work centers on the effects of alcohol on the BK channel and comprises studies that examine how subcellular location affects acute ethanol sensitivity and how duration of acute alcohol exposure impacts the development of rapid tolerance. My results also provide potential mechanisms which underlie acute sensitivity and rapid tolerance. I first explore BK channel sensitivity to ethanol in the three compartments (dendrite, cell body, and nerve terminal) of magnocellular neurons in the rat hypothalamic-neurohypophysial (HNS) system. The HNS system provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins because the cell bodies are physically separated from the nerve terminals. Using electrophysiological and immunohistochemical techniques I characterize the BK channel in each of the three primary compartments and find that dendritic BK channels, similar to somatic channels, but in contrast to nerve terminal channels, are insensitive to alcohol. Furthermore, the gating kinetics, calcium sensitivity, and iberiotoxin sensitivity of channels in the dendrite are similar to somatic channels but sharply contrast terminal channels. The biophysical and pharmacological properties of somatodendritic vs. nerve terminal channels are consistent with the characteristics of exogenously expressed αβ1 vs. αβ4 channels, respectively. Therefore, one possible explanation for my findings is a selective distribution of β1 subunits to the somatodendritic compartment and β4 subunits to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate β1 or β4 channel clusters in the membrane of somatodendritic or nerve terminal compartments, respectively. In conclusion, I found that alcohol sensitivity of BK channels within the HNS system is dependent on subcellular location and postulate that β-subunits modulate ethanol sensitivity of HNS BK channels. In the second and primary focus of my thesis I explore tolerance development in the striatum, a brain region heavily implicated in addiction. Numerous studies have demonstrated that duration of drug exposure influences tolerance development and drug dependence. To further elucidate the mechanisms underlying behavioral tolerance I examined if BK channel tolerance was dependent on duration of alcohol exposure using patch clamp techniques in cultured striatal neurons from P8 rats. I found that persistence of rapid tolerance is indeed a function of exposure time and find it lasts surprisingly long. For example, after a 6 hr exposure to 20 mM ethanol, acute sensitivity was still suppressed at 24 hrs withdrawal. However, after a 1 or 3 hr exposure period, sensitivity had returned after only 4 hrs. I also found that during withdrawal from a 6 hr but not a 3 hr exposure the biophysical properties of BK channels change and that this change is correlated with an increase in mRNA levels of the alcohol insensitive STREX splice variant. Furthermore, BK channel properties during withdrawal from a 6 hr exposure to alcohol closely parallel the properties of STREX channels exogenously expressed in HEK293 cells. In conclusion I have established that BK channels develop rapid tolerance in striatal neurons, that rapid tolerance is dependent upon exposure protocol, and is surprisingly persistent. These findings present another mechanism underlying BK channel tolerance and possibly behavioral tolerance. Since these phenomena are dependent on duration of drug exposure my results may find relevance in explaining how drinking patterns impact the development of alcohol dependence in humans.

Metazoan genomes contain thousands of protein-coding and non-coding RNA genes, most of which are differentially expressed, i.e., at different locations, at different times during development, or in response to environmental signals. Differential gene expression is achieved through complex regulatory networks that are controlled in part by two types of trans-regulators: transcription factors (TFs) and microRNAs (miRNAs). TFs bind to cis-regulatory DNA elements that are often located in or near their target genes, while microRNAs hybridize to cis-regulatory RNA elements mostly located in the 3’ untranslated region (3’UTR) of their target mRNAs. My work in the Walhout lab has centered on understanding how these trans-regulators interact with each other in the context of gene regulatory networks to coordinate gene expression at the genome-scale level. Our model organism is the free-living nematode Caenorahbditis elegans, which possess approximately 950 predicted TFs and more than 100 miRNAs Whereas much attention has focused on finding the protein-coding target genes of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. To this end, we have embarked in the task of mapping the first genome-scale miRNA regulatory network. This network contains experimentally mapped transcriptional TF=>miRNA interactions, as well as computationally predicted post-transcriptional miRNA=>TF interactions. The work presented here, along with data reported by other groups, have revealed the existence of reciprocal regulation between these two types of regulators, as well as extensive coordination in the regulation of shared target genes. Our studies have also identified common mechanisms by which miRNAs and TFs function to control gene expression and have suggested an inherent difference in the network properties of both types of regulators. Reverse genetic approaches have been extensively used to delineate the biological function of protein-coding genes. For instance, genome-wide RNAi screens have revealed critical roles for TFs in C. elegans development and physiology. However, reverse genetic approaches have not been very insightful in the case of non-coding genes: A single null mutation does not result in an easily detectable phenotype for most C. elegans miRNA genes. To help delineate the biological function of miRNAs we sought to determine when and where they are expressed. Specifically, we generated a collection of transgenic C. elegans strains, each containing a miRNA promoter::gfp (Pmir::gfp) fusion construct. The particular pattern of expression of each miRNA gene should help to identify potential genetic interactors that exhibit similar expression patterns, and to design experiments to test the phenotypes of miRNA mutants.

During my thesis work I studied the expression and potential function of brain expressed microRNAs (miRNAs) in human prefrontal cortex (PFC). Initially, I used combinatorial computational analysis and microarray data to identify miRNAs that are predicted with high probability to target the human Brain Derived Neurotrophic Factor (BDNF) 3’ Untranslated Region (3’UTR) and are expressed in moderate to high levels in adult human prefrontal cortex. A subset of 10 miRNAs segregating into 5 different miRNA families (miR-30a-d, miR-103/107, miR-16/195, miR-191 and miR-495) met the above criteria. I then designed a protocol to detect these miRNAs with Locked Nucleic Acid (LNA) in situ hybridization in human prefrontal cortex and determine their layer and cellular expression patterns. LNA in situ revealed differential lamina and cellular enrichment of BDNF-related miRNAs. As an example, miR-30a-5p was found to be enriched in large pyramidal neurons of layer 3, which was verified using laser capture microdissection of layer 3 pyramidal neurons and quantitative Real Time Polymerase Chain Reaction (qRT-PCR) following dissection of upper and deeper layers of human PFC. Parallel to this, I used miRNA qRT-PCR to determine the developmental expression of miRNAs using postmortem PFC tissues ranging from embryonic age to old adulthood and compared miRNA to BDNF protein levels. My results revealed a robust inverse correlation between BDNF-related miRNAs and BDNF protein during late maturation and aging of human prefrontal cortex. In vitro luciferase assays and/or lentivirus mediated neuronal miRNA overexpression experiments validated that at least two miRNAs, miR-30a-5p and miR-195, target human BDNF 3’UTR and mediate its translational repression. In the second part of my thesis work I measured levels of miR-30a and miR-195 in the prefrontal cortex of patients with schizophrenia and compared them with levels of BDNF protein and BDNF-related GABAergic mRNAs. According to my results differences in miR-195 levels in a subset of subjects diagnosed with schizophrenia were found to be associated with disease related changes in BDNF protein levels and deficits in BDNF dependent GABAergic gene expression. In the last part of my work I focused on miR-30b, another member of the miR-30 family, which I found to be reduced in the prefrontal cortex of female but not male subjects with schizophrenia. More importantly, disease related changes in miR-30b levels were strongly associated with the age of onset of the disease. Additional experiments in mouse cortex and hippocampus revealed a gender dimorphic expression pattern of this miRNA with higher expression in female brain. Collectively, my results suggest that miRNAs could participate in novel molecular pathways that play an important role during cortical development and maturation and are potentially linked to the pathophysiology of neuropsychiatric disease.

Generation of the primitive neuroectoderm into specialized brain subdivisions, such as the hindbrain primordium, involves the regulated coordination of complex morphogenetic and molecular mechanisms. These processes are evident in the segregation of the zebrafish hindbrain into seven distinct lineage-restricted compartments, termed rhombomeres (r), which are established by the interplay of several spatially-restricted expressed genes. These include transcription factors, members of specific signaling pathways and specialized molecules that mediate cell adhesion and identity. Despite their extensive characterization, it is evident that other genes are involved to mediate the proper specification and segregation of individual rhombomeres. One candidate that likely fits this role is related to the no ocelli/l(2)35Ba gene in Drosophila, termed nlz (nocA-like zinc-finger). Nlz-related proteins behave as transcriptional repressors and are related to the vertebrate Sp1-like family of transcription factors. nlz is dynamically expressed in the zebrafish hindbrain, residing in the caudal hindbrain at gastrula stages and rostrally expanding from presumptive r3/r4 boundary to encompass r3 and r2 at segmentation stages. Nlz localizes to the nucleus and associates with the co-repressors Groucho and histone deacetylases, suggesting that Nlz acts as a repressor. Consistent with this, misexpression of nlz into zebrafish embryos results in a loss of gene expression in the rostral hindbrain (rl-r3). Taken together, the findings in this thesis suggest that Nlz functions as a transcriptional repressor to control segmental gene expression in the rostral hindbrain.

In order to ascertain the degree of compatibility in developmental restructuring and behavioral plasticity between two fish species frequently made subject of laboratory research (Metriaclima zebra & Danio rerio), alternative trophic niche exposure experiments utilizing novel three-prong feeding treatments were conducted to obtain morphometric data, which demonstrated both species do bear some degree of plasticity. The results are somewhat complicated by differences in locality of detectable restructuring, which may be due to disparity in the form-function relationship for each species’ lineage. Each is notable in the manner of respective species’ jaw protrusion, as it is driven by anterior kinethmoid rotation in D. rerio. as opposed to force imparted upon the rostral cartilage of the premaxilla’s articular process in M zebra. Each is markedly distinct in the pharyngeal jaw as well, as zebrafish (also toothless at the oral jaw) bear teeth only on the lower set at the posterior of the mouth, while cichlids bear teeth on all jaws and additionally possess a unique, fused lower pharyngeal jaw. However, accounting for this difference in experimental models does allow for direct comparison, both at the morphological/behavioral and potentially the genetic level, though additional research is necessary. The evidence provided here also provides encouragement that more nuanced approaches to laboratory trophic niche exposure experiments could elucidate further evidence on the nature of phenotypic plasticity.

Development of functional neural circuits involves a series of complicated steps, including neurogenesis and neuronal morphogenesis. To understand the molecular mechasnims of neurite complexity, especially neurite branching/arborization, the Drosophila brain, especially MBNs (mushroom body neurons) and PNs (projection neurons) in olfactory circuitry, was used in this dissertation work as the model system to study how two molecules, Dscam and Kr-h1 affect neurite complexity in the Drosophilabrain. For the Drosophila Dscam, through alternative splicing it could encode up to 152,064 distinct immunoglobulin/fibronectin type cell adhesion molecules. Each Dscam isoform is derived from one of the 19,008 ectodomain variants connected with one of the two alternative transmembrane segments and one of the four possible endodomain portions. Recent studies revealed that Dscam was widely required for neurite branching/arborizaiton. However, due to the technical difficulty, the functional roles of Dscam transmembrane variants and ectodomain variants remain unclear. In this thesis work, a microRNA based RNA interference was used to knock down distinct subsets of Dscam isoform. First, loss of Dscam[TM1] versus Dscam[TM2], two distinct Dscam transmembrane variants, disrupted the dendritic versus axonal morphogenesis, respectively. Furthermore, structural analysis suggested that the juxtamembrane portion of transmembrane segment was required for the Dscam protein targeting in dendrites/axons and this differential protein targeting might account for the functional distinction between Dscam[TM1] and Dscam[TM2]. Second, to further address the functional significance of having two Dscam transmembrane variants in axons versus dendrites, the possibility that there might be different usage of Dscam repertoire between axons and dendrites that lead to different levels of morphological complexity between axons and dendrites in the same neuron was examined. To this end, end-in targeting approaches were used to exchange Dscam populations between axons and dendrites. Though the genetic data suggested that Dscam populations were exchanged between axons and dendrites, the phenotypic analysis in various neuronal types revealed that depending on the neuronal types, exchange of Dscam populations between axons and dendrites might primarily affect either axonal or dendritic morphology, suggesting that different usage of Dscam population between axons and dendrites might regulate complex patterns of neurite morphology. Finally, the functions of Dscam exon 4 variants had been addressed in different model neurons in the Drosophilabrain. First, 12 Dscam exon 4 variants were divided into three groups based on their phylogenetic distance. Then, three miRNA constructs were engineered to knock down one group at a time. The genetic data suggested that different Dscam exon 4 variants are differentially required in different neurons to support their proper neuronal morphogenesis. In summary, this part of my thesis work identified and characterized previously unrecognized functions of all these distinct Dscam variants and provided novel insights into how diverse Dscam isoforms regulate the different aspects of neuronal morphogenesis. In the honey bee brain, Kr-h1 is upregulated during the behavioral shift from nursing to foraging when there is increased neurite branching in the brain. To directly examine the hypothesis that altered Kr-h1 expression might regulate morphological complexity of neurites, this research work involved the MARCM (mosaic analysis with a repressible cell marker) and TARGET (temporal and regional gene expression targeting) techniques to analyze the roles of Kr-h1 in Drosophila neuronal morphogenesis. Interestingly, increased expression of Kr-h1 blocked the axon branching and further disrupted the lobe formation in the mushroom body whereas the loss-of-Kr-h1 did not show any apparent neuronal morphogenetic defects. In addition, it was observed that Kr-h1 was expressed when MB (mushroom body) did not undergo active morphogenesis, suggesting its potential anti-morphogenetic activity. Indeed, loss of Kr-h1 (Kruppel homolog 1) enhanced the neuronal morphogenesis that was otherwise delayed due to the defective TGF-beta signaling. Furthermore, Kr-h1 expression was closely linked to ecdysone dependent signaling: Kr-h1 was first regulated by usp (ultraspiracle), which dimerized with various ecdysone receptors and then Kr-h1 expression was essential for proper ecdysone patterning in the larval CNS (central nervous system). Together, though Kr-h1could potentially regulate the neurite complexity, it seems primarily involved in the coordinating ecdysone signaling. In conclusion, the powerful genetic toolkit available in the Drosophila has allowed the investigation in the molecular mechanisms of neuronal morphogenesis and understanding of these mechanisms will enhance our understanding of how the complex nervous system is wired to perform the delicate behaviors.

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease that affects motor neurons causing progressive muscle weakness and respiratory failure. In 2011, the presence of a hexanucleotide repeat expansion within chromosome 9 open reading frame 72(C9ORF72) was identified in ALS patient samples, becoming the major known genetic cause for ALS and frontotemporal dementia (FTD). Carriers of this mutation present reduced levels of C9ORF72 mRNA, RNA foci produced by the aggregating expansion and toxic dipeptides generated through repeat-associated non-ATG translation. These findings have led to multiple hypotheses on the pathogenesis of C9ORF72: 1) Haploinsufficiency, 2) RNA gain-of-function, 3) RAN Translation, and 4) Disrupted nucleocytoplasmic trafficking. Due to lack of treatments for this disease, we have pursued an AAV-RNAi dependent gene therapy approach, using an artificial microRNA (amiR) packaged in a recombinant adeno-associated virus (rAAV). After validating our in vitro results, we advanced to in vivo experiments using transgenic mice that recapitulate the major histopathological features seen in human ALS/FTD patients. Adult and neonate mice were injected through clinically relevant routes and our results indicate that AAV9-mediated amiR silencing not only reduced mRNA and protein levels of C9ORF72 but also the expansion derived toxic GP dipeptides. Although our amiR is not targeting the expansion itself but exon 3, we illustrate here that the evident dipeptide decrease is achievable due to the presence of aberrant transcripts in the cytoplasm containing miss-spliced Intron-HRE-C9ORF72 species. These encouraging results have led to the continued testing of this treatment as a therapeutic option for C9ORF72 - ALS patients.

The Tec family tyrosine kinase, Itk, plays an important role in signal transduction following T cell receptor engagement. Several prior studies have established the importance of Itk in immune system processes, such as T cell development and T cell activation. Additional biochemical studies have found that Itk specifically functions within a multi-molecular signalosome complex, which ultimately functions to provide a platform by which Itk can phosphorylate and activate PLC-γ1, a crucial step in T cell activation. To further study how Itk regulates distinct immune outcomes via T cell effector processes within the peripheral immune system, and to further understand how Itk functions in T cells in response to a physiological ligand-receptor interaction, I crossed Itk-deficient mice to mice transgenic for a TCR specific for a moth cytochrome C peptide. My studies have established a unique role for Itk in several important aspects of T cell function. Following T cell activation, I identified an imperative role for Itk in activation-induced cell death via FasL, a mechanism of immune homeostasis. Furthermore, I found Itk plays a unique role in the process of T cell differentiation, where Itk positively regulates the induction of cytokine genes, such as IL-4, while negatively regulating the induction of T-bet, a transcription factor important for Th1 differentiation. Lastly, following T cell differentiation, I found that Itk mRNA and protein are up-regulated during Th2 differentiation, while Rlk, a related Tec kinase, disappears rapidly from Th2 cells, indicating a critical role for Itk in Th2 cell function. Collectively, my thesis work has more clearly defined an important function for Itk not only in TCR signaling, but also in immune processes such as T cell differentiation and activation-induced cell death that are required for proper immune function.