The underlying goal of neuroscience research is to understand how the nervous system functions to bring about behavior. A detailed map of neural circuits is required for scientists to tackle this question. To this purpose, we developed a synthetic and genetically-encoded system, TRanscellular ACtivation of Transcription (TRACT) to monitor cell-cell contact. Upon ligand-receptor interaction at sites of cell-cell contact, the transmembrane domain of an engineered Notch receptor is cleaved by intramembrane proteolysis and releases a fragment that regulates transcription in the receptor-expressing cell. We demonstrate that in cultured cells, the synthetic receptor can be activated to drive reporter gene expression by co-incubation with ligand-expressing cell or by growth on ligand-coated surfaces. We further show that TRACT can detect interactions between neurons and glia in the Drosophila brain; expressing the ligand in spatially-restricted subsets of neurons leads to transcription of a reporter in the glial cells that interact with those neurons. To optimize TRACT for neural tracing, we attempted to target the synthetic receptor to post-synaptic sites by fusion with the intracellular domain of Drosophila neuroligin2. However, this modification only facilitate the receptor to be localized homogeneously throughout the neurites. The induction data of the modified receptor shows that the new receptor has better sensitivity compared to the original receptor, but the ligand-receptor interaction still happened at non-synaptic sites of membrane contact. To further target the ligand to pre-synaptic sites, we fused the ligand to different pre-synaptic markers. We found the one fused with synaptobrevin is likely located at axon terminals, but only able to trigger moderate induction. Therefore, more examinations are required to further characterize the capability of this ligand. In summary, TRACT is useful for monitoring cell-cell interactions in animals and could also be used to genetically manipulate cells based on contact. Moreover, we believe that proper targeting of the ligand to synaptic sites will improve the specificity of TRACT for synaptic connections in the future.

RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.

Astrocytes densely infiltrate the brain and intimately associate with synaptic structures. In the past 20 years, they have emerged as critical regulators of both synapse assembly and synapse function. During development, astrocytes modulate the formation of new synapses, and later, control refinement of synaptic connections in response to activity dependent cues. In a mature nervous system, astrocytes modulate synapse function through a variety of mechanisms. These include ion buffering, neurotransmitter uptake and the release of molecules that activate synaptic receptors. Through such roles, astrocytes shape the structure and function of neuronal circuits. However, how astrocytes and synapses reciprocally communicate during circuit assembly remains an unanswered question in the field. The vast majority of our understanding of astrocyte biology has come from studies conducted in mammals, where it is challenging to dissect molecular mechanisms with cell type specificity. Drosophila melanogaster is a less established model system for studying astrocyteneuron interactions, but its vast array of genetic tools and rapid life cycle promises great potential for precisely targeted manipulations. My thesis work has utilized Drosophila melanogaster to investigate the reciprocal nature of astrocyte-synapse communication. First, I characterized Drosophila late metamorphosis as a developmental stage in which astrocyte-synapse associations can be studied. My work demonstrates that during this time, when the adult Drosophila nervous system is being assembled, synapse formation relies on the coordinated infiltration of astrocyte membranes into the neuropil. Next, I show that in a reciprocal manner, neural activity can shape astrocyte biology during this time as well and impart long lasting effects on neuronal circuit function. In particular expression of the astrocyte GABA transporter (GAT) is modulated in an activity-dependent manner via astrocytic GABABR1/2 receptor signaling. Inhibiting astrocytic GABABR1/2 signaling strongly suppresses hyperexcitability in a Drosophila seizure model, vii arguing this pathway is important for modulating excitatory/inhibitory balance in vivo. Finally, utilizing the ease of the Drosophila system, I performed a reverse genetic screen to identify additional astrocyte factors involved in modulating excitatory-inhibitory neuronal balance.

Dopamine (DA) is central to movement, reward, learning, sleep, and anxiety. The dopamine transporter (DAT) spatially and temporally controls extracellular dopamine levels by taking DA back up into the presynaptic neuron. Multiple lines of evidence from studies using pharmacological DAT blockade or genetic DAT deletion demonstrate that DAT availability at the plasma membrane is required for maintenance of homeostatic DA levels and DA tone. Therefore, intrinsic mechanisms that regulate the transporter’s availability at the plasma membrane may directly impact downstream DA signaling cascades and DA-dependent behavior. Acute, regulated DAT internalization in response to protein kinase C (PKC) activation has been well documented, however the physiological importance of this mechanism remains untested. Due to DAT’s critical role in regulating DA levels, It is essential to understand mechanisms that acutely regulate DAT function and surface expression, and further, how these mechanisms contribute to DA related behaviors. DAT has intracellular amino and carboxy termini, which contain domains for transporter phosphorylation, recruitment to and from the plasma membrane, and sites for protein-protein interactions. To test whether these domains work synergistically for DAT function and regulated endocytosis I made DAT/SERT chimeras, in which I switched DAT’s amino, carboxy, or both termini with that of SERT, a homologous transporter with highly divergent intracellular domains. I demonstrated that DAT’s amino and carboxy termini synergistically contribute to substrate and select competitive inhibitor affinities. Additionally, I demonstrated that the amino terminus is required for PKC-stimulated DAT endocytosis, and that both N- and C-termini are required for downstream Ack1-dependent regulation of DAT endocytosis. To test the physiological importance of PKC-stimulated DAT endocytosis in vivo, I knocked down Rin, a GTPase required for PKC-stimulated DAT trafficking, in mouse DA neurons. This study was the first to achieve AAV-mediated, conditional, and inducible gene silencing in neurons. Using this AAV approach, I demonstrated a critical role for Rin GTPase signaling and DAT trafficking in both anxiety and locomotor response to cocaine. Taken together, this thesis 1) adds to the understanding of DAT functional and endocytic mechanisms and 2) is the first to report the physiological impact of Rin signaling and DAT endocytosis in DA behavior.

The nervous system is composed of neurons and glia. Glial cells have been neglected and thought to have only a supportive role in the nervous system, even though ~60% of the mammalian brain is composed of glia. Yet, in recent years, it has been shown that glial cells have several important functions during the development, maintenance and function of the nervous system. Glial cells regulate both pre and post mitotic neuronal survival during normal development and maintenance of the nervous system as well as after injury, are necessary for axon guidance, proper axon fasciculation, and myelination during development, promote synapse formation, regulate ion balance in the extracellular space, are required for normal synaptic function, and have immune functions in the brain. Although glia have crucial roles in nervous system development and function, there are still much unknown about the underlying molecular mechanisms in glial development, function and glial-neuronal communication. Drosophila offers great opportunity to study glial biology, with its simple yet sophisticated and stereotypic nervous system. Glial cells in flies show great complexity similar to the mammalian nervous system, and many cellular and molecular functions are conserved between flies and mammals. In this study, I use Drosophila as a model organism to study the function of one subtype of glia: astrocytes. The role of astrocytes in synapse formation, function and maintenance has been a focus of study. However, their role in engulfment and clearance of neuronal debris during development remains unexplored. I generated a driver line that enables the study of astrocytes in Drosophila.In chapter two of this thesis, I characterize astrocytes during metamorphosis, when extensive neuronal remodeling takes place. I found that astrocytes turn into phagocytes in a cell-autonomous, steroid-dependent manner, by upregulating the phagocytic receptor Draper and forming acidic phagolysosomal structures. I show that astrocytes clear neuronal debris during nervous system remodeling and that this is a novel function for astrocytes during the development of nervous system. I analyzed two different neuronal populations: MB γ neurons that prune their neurites and vCrz+ neurons that undergo apoptosis. I discovered that MB γ axons are engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 pathways in a partially redundant way. Interestingly, Draper is required for clearance of vCrz+ cell bodies, while Crk/Mbc/dCed-12, but not Draper, are required for clearance of vCrz+ neurites. Surprisingly, I also found that loss of Draper delayed vCrz+ neurite degeneration, suggesting that glia facilitate neurite destruction through engulfment signaling. Taken together, my work identifies a novel function for astrocytes in the clearance of synaptic and neuronal debris during developmental remodeling of the nervous system. Additionally, I show that Crk/Mbc/dCed-12 act as a new glial signaling pathway required for pruning, and surprisingly, that glia use different engulfment pathways to clear neuronal debris generated by cell death versus local pruning.

How do organisms attain the capacity to regenerate a structure, entire body, or not to regenerate? These are fundamental questions in biology for understanding how replicative systems are evolved to renew, age, and/or die. One outstanding question in regenerative biology that attracts attention is how and why the human central nervous system fails to regenerate after injury. Nervous system injuries are characterized by axonal damage and loss of synaptic function that contribute to debilitating neuronal dysfunctions. Although the molecular underpinnings of axon regeneration are well characterized, very little is known about how and what molecular pathways modulate reformation of synapses within regenerating axons to restore function. Thus, understanding the fundamental molecular and genetic mechanisms of functional axon regeneration (FAR), restoration of both axon and synapse, for the functional recovery of the nervous system remains elusive. In Chapter I, I outline the biology of regeneration and provide evolutionary perspectives of this phenomenon. Then, I provide clinical perspectives of central nervous system regeneration and therapeutic innovations. I next introduce the regulators of axon regeneration and how C. elegans as a genetic system allows detailed characterization of axon regeneration. In Chapter II, using C. elegans as a platform, I show how axon regeneration and synaptic reformation are controlled by distinct genetic pathways. I show how Poly-ADP ribose polymerase (PARP) pathway modulates functional restoration by regulating divergent genetic pathways leading to axon regeneration and synapse restoration. Finally, in Chapter III, I summarize the model of axon regeneration, evolutionary perspectives, and epistemic limitations of C. elegans axon regeneration.

Nucleocytoplasmic transport (NCT) declines during aging and in the context of age-dependent neurodegenerative diseases. However, the mechanisms underlying NCT decline in the disease are poorly understood. FUS is an RNA binding protein that shuttles between the nucleus and cytoplasm and is actively involved in NCT. Mutations in FUS cause amyotrophic lateral sclerosis (ALS), a fatal and incurable motor neuron disorder. We sought to understand the disease mechanism underlying FUS-induced NCT decline in ALS. Here, I uncovered NCT-related defects in motor neurons derived from human induced pluripotent stem cells (iPSCs) harboring an ALS-linked FUS mutation. Importantly, these NCT defects were rescued by genetically correcting the FUS mutation in iPSCs. To gain insight into how expression of mutant FUS causes nuclear pore defects, I demonstrated an altered localization where FUS and nucleoporins (Nups) interact in situ within patient-derived human neurons. Moreover, FUS became aggregation-prone when interacting with Nup62 in vitro, and RNA further alleviated their aggregation propensity. Importantly, NCT-related defects and neuronal toxicity induced by ALS-FUS were ameliorated by modulating Nup expression in vivo. Collectively, these findings implicate aberrant Nup interactions in the pathogenic mechanism of ALS-FUS, and direct targeting the gain-of-function protein interactions could be therapeutic for multiple causes of neurodegeneration.

Rotating shift work, irregular sleep patterns and jetlag disrupt circadian rhythms, induce or aggravate disease, and produce deficits in cognitive function. Internal misalignment, a state in which abnormal phase relationships prevail between and within organs, is widely proposed to account for these adverse effects of circadian disruption. This hypothesis has been difficult to test because phase shifts of the entraining environmental cycle lead to transient desynchrony. Thus, it remains possible that phase shifts, regardless of internal desynchrony, account for adverse effects of circadian disruption. I have used the duper mutant hamster, whose locomotor activity rhythms re-entrain 5-fold faster than wild types after a phase shift of 8 hours, to test whether internal desynchrony can account for adverse effects of jet lag on adult neurogenesis. I subjected wild type and duper female hamsters to alternating 8h phase advances and delays of the LD cycle at 16-day intervals. I injected 5-Bromo-2’-deoxyuridine (BrdU, a thymidine analogue) after the 4th shift and collected brains after the 8th shift. As expected, mutants re-entrained activity rhythms more rapidly than did wild types. On the other hand, estrous cycles, as assessed by vaginal smears, were rarely disrupted by repeated phase shifts in either genotype. I next compared cell proliferation and neurogenesis in the subgranular zone of the hippocampus between Duper mutants and wild type siblings using the S-phase marker BrdU and the neuronal marker NeuN. I assessed the total number of BrdU cells in the subgranular zone of the hippocampus, as the proportion that expressed NeuN. Duper mutants had more BrdU-ir cells, and more BrdU+/NeuN+ cells than did wild types, whether or not they experienced phase shifts, revealing an unexpected increase in neurogenesis. Surprisingly, repeated phase shifts increased neurogenesis in WT but not duper hamsters. Despite the increase in neurogenesis, phase shifts reduced the number of adult-born non-neuronal (BrdU+/NeuN-) cells in WT hamsters but had no such effect on duper mutants. In addition, the duper mutation increases hippocampal neurogenesis regardless of circadian. Our results suggest that adult-born non-neuronal cells are most vulnerable to circadian disruption, and that internal desynchrony promotes their demise. disruption.

One of the goals of neuroscience is to classify all of the neurons in the brain. Neuronal types can be defined using a combination of morphology, electrophysiology, and gene expression profiles. Gene expression profiles allow differentiation between cells that share similar characteristics. Leveraging the advantage of Berghia stephanieae (Gastropoda; Nudibranchia), which has around 28,000 neurons, I constructed high-throughput single-neuron transcriptomes for its whole brain. I produced a single-cell dissociation protocol and a custom data analysis pipeline for data of this nature. Around 129,000 cells were collected from 18 rhinophore ganglia and 20 circumesophageal ring ganglia (brain), consisting of the cerebropleural, pedal, and buccal ganglia. Messenger RNA libraries were constructed using the 10X Genomics’ Chromium platform. After library preparation, around 1,000 cells were recovered and sequenced. The HTStream package was utilized to trim off unwanted sequences from the raw reads and remove PCR duplicates and other contamination, then the salmon alevin package was employed to construct gene-by-cell matrices containing all the transcripts for each gene in each cell. The Seurat pipeline was used to extract this expression data from the matrices, normalize it, and perform dimensionality reduction. The cells were clustered based on similarities in their gene expression profiles. The cells formed eight clusters on a UMAP graph, each having distinct marker genes. Additionally, one cluster was composed of almost exclusively cells from the rhinophore ganglia, accounting for 30% of all rhinophore ganglion cells in the sample. Cells from the rhinophore ganglia are as heteregenous as cells from the rest of the brain, with cells forming six clusters. Cell populations that express the same neurotransmitter were identified for a wide range of both small-molecule neurotransmitters and neuropeptides. In a separate project, the locomotion of Berghia was recorded over 9 days with 2 lighting regimes: LD first and DD first. The results suggest that locomotion of Berghia is governed by circadian clock and that Berghia is nocturnal. Hunger state likely plays a role in modulating this circadian rhythm.

Stroke is the world's leading cause of adult disability, caused by lack of oxygen and nutrients to the brain due to a blood clot in a major artery. This leads to ischemic damage of neuronal cells that leads to paralysis, motor, and speech deficits. While most stroke therapies aim at removing or reducing the blood clots in the brain, few treatments target cell damage. Neuroglobin (NGB) is a protein in the brain that is able to aid in neuroprotection following oxidative stress. Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor that serves as a marker for cell recovery after hypoxia or low oxygen levels. Exosomes are microscopic extracellular vesicles that can help deliver proteins across the blood-brain barrier. This thesis focuses on finding a correlation between exosomal-delivered neuroglobin to ischemic cells and the regulation of HIF-1 in order to develop an innovative treatment using exosomes. The specific aims of this thesis are as follows: Aim 1: Package NGB in exosomes of healthy cell The XPAK-NGB plasmid will be used to transfect NGB DNA into wild-type human embryonic kidney (HEK-293 cell line) cells. Exosomes will be harvested from the spent media. The exosomes will be analyzed to ensure that the protein is packaged inside the exosomes. Aim 2: Determine the limit of hypoxic conditions and effects of NGB on damaged cells A literature review will be performed to determine the ideal concentration of H2O2 for the survival of neuronal cells. This will include the composition of hypoxia as well as the length of time that cells can be exposed to and remain viable. Aim 3: Correlate NGB concentration and HIF-1 concentration Another literature review will determine the specific markers of NGB and HIF-1.