Browsing by Subject "Nerve Tissue Proteins"
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Item ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis(December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, NealImmune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.Item ApoE Receptors in Alzheimer's and CNS Function(2017-03-24) Lane-Donovan, Courtney; Powell, Craig M.; Herz, Joachim; Eisch, Amelia J.; Huber, Kimberly M.Alzheimer's disease (AD) is a currently incurable neurodegenerative disorder and the most common form of dementia over age 65. The predominant genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4). Other genes related to lipid metabolism and lipoprotein receptor signaling have also been identified as risk modifiers for AD. Despite nearly two decades of research, the mechanisms by which lipid transport proteins cause central nervous system (CNS) disease are not completely understood. Here, the ApoE receptor family and its ligands and their roles in CNS function and neurological disease were explored. It has been previously shown that ApoE4 renders neurons resistant to the neuromodulator and ApoE receptor ligand Reelin, which enhances synaptic plasticity and protects against amyloid β (Aβ) oligomer-induced synaptic toxicity in vitro. Here, mice with reduced Reelin expression were more sensitive to amyloid-induced synaptic suppression, and had memory and learning disabilities at very low amyloid levels. However, this effect of Reelin loss did not extend to other forms a neurological insult, since the Reelin conditional knockout mice were not more susceptible to transient middle cerebral artery occlusions. Together, these findings highlight the specific physiological importance of Reelin for protecting the brain against Aβ-induced synaptic dysfunction and memory impairment. One of the continuing debates in the AD field is whether ApoE is required for synaptic function. ApoE knockout mice have synaptic loss; however, they also have severely increased plasma lipids, which could independently contribute to CNS dysfunction. A novel mouse with normal plasma ApoE, but severely depleted brain ApoE, shares a similar synaptic phenotype as ApoE knockout mice, suggesting central ApoE is required for brain function. To determine if diet can modulate ApoE levels, wildtype, ApoE3, and ApoE4 targeted replacement mice were fed a chow, high-fat, or ketogenic (high-fat, very-low-carb) diet. Surprisingly, high-fat diet reduced hippocampal ApoE levels in ApoE3 TR mice, indicating an intersection of genetic (ApoE isoform) and lifestyle (diet) risk factors on AD pathogenesis. Taken together, these findings highlight the importance of ApoE receptors and their ligands in AD biology, and future studies will have to determine how to target these mechanisms to treat AD and improve patient outcomes.Item Ceramide-Induced Alternative Translocation of TM4SF20(2015-10-29) Lee, Ching En; Pfeiffer, Julie K.; McKnight, Steven L.; Kahn, Jeffrey; Nijhawan, Deepak; Ye, JinThe polytopic membrane protein TM4SF20 (transmembrane 4 L6 family 20) is a protein containing four transmembrane helices that inhibits the Regulated Intramembrane Proteolysis (RIP) of the transcriptional factor CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor. CREB3L1 RIP is induced by several stimuli: ER stress, viral infections, the chemotherapeutic drug, doxorubicin, and the sphingolipid, ceramide. Additionally, TGF-β (transforming growth factor-β), a cytokine known to stimulate collagen production, induces the proteolytic activation of CREB3L1 in human A549 cells through inhibition of TM4SF20 expression, which normally inhibits RIP of CREB3L1. We also find that the TM4SF20 regulation of CREB3L1 RIP is regulated by ceramide. In this study we find that ceramide can regulate the ability of first transmembrane domain of TM4SF20 to determine its orientation in the membrane. Under normal conditions, TM4SF20 is synthesized as a protein that inhibits the cleavage of CREB3L1 when TRAM2 (translocation associated membrane protein 2) is associated with the ER translocon. Excess ceramide dissociates TRAM2 from the ER translocon such that the N-terminus of TM4SF20 can no longer be forced by the first transmembrane domain to function as a signal peptide. Under excess ceramide conditions, TM4SF20 adopts a completely opposite topology and allows the cleavage of CREB3L1 to proceed. We have designated this novel mechanism for transmembrane protein regulation as "alternative translocation."Item Dissecting Roles for the Macromolecular Machinery Involved in Neurotransmitter Release(2019-01-15) Prinslow, Eric Andrew; Rosenbaum, Daniel M.; Rizo-Rey, José; Rice, Luke M.; Yu, HongtaoNeurotransmitter release is a tightly regulated process that involves synaptic vesicle docking at presynaptic active zones, priming of the vesicles to a release-ready state, and calcium evoked fusion of the vesicle and plasma membranes. The probability of release is modulated by plastic changes that depend on synaptic activity; these changes shape the properties of neural networks and underlie multiple forms of information processing in the brain. Elucidating the mechanisms of neurotransmitter release and its regulation is thus critical for understanding brain function and establishing fundamental principles of neuronal communication. I have investigated the mechanism by which neurotransmitters send messages between neurons as a specific model system to study the general mechanism of intracellular membrane fusion. In one project, I investigated whether trans-SNARE complexes can be disassembled by NSF-αSNAP. I showed that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, and is facilitated by synaptotagmin-1. I proposed a model whereby Munc18-1 and Munc13-1 are critical for mediating vesicle priming as well as precluding de-priming by preventing trans-SNARE complex disassembly. Complexin-1 also impaired de-priming, while synaptotagmin-1 may have assisted in priming and hindered de-priming. Additionally, I used various biophysical approaches including ITC and NMR to shed light into how Complexin has dual roles in fusion. One of my projects investigated the inhibitory role of Complexin and solved a controversy over conflicting ITC data. Another project focused on the Complexin N-terminal and C-terminal domains to try and develop a complete model of how Complexin functions that incorporates all of its known interactions and activating/inhibiting properties. I observed cooperative interactions between Complexin, the SNARE complex, and lipids by forming SNARE complexes anchored on nanodiscs and liposomes. Such cooperative binding of Complexin to membranes and SNAREs may be critical for releasing the inhibition caused by the accessory helix, although the molecular mechanism of action has yet to be determined. Overall, these experiments highlight the importance of interactions between numerous accessory proteins and the trans-SNARE for proper regulation of SNARE complex formation, and therefore fusion.Item Dissecting the Function of FARP1 and Rho GTPases in Semaphorin-Plexin Signaling: Structural Perspective(2017-11-30) Kuo, Yi-Chun; Albanesi, Joseph P.; Huang, Jun-Shen; Rizo-Rey, José; Sternweis, Paul C.The Semaphorin-Plexin signaling is important for regulating axon guidance. Binding of Semaphorin to the Plexin receptor induces the dimerization of Plexin and stimulates its cytoplasmic GAP activity towards Rap, resulting in cytoskeleton re-organization. The growth cone of an axon then turns around, which ultimately leads to the repulsive guidance of the axon. Structural studies by our laboratory and others have revealed how the RapGAP activity of Plexin cytoplasmic region is stimulated by dimerization, how the Semaphorin ligand interacts with Plexin ectodomain, and how extracellular domains prevent Plexin from premature activation. Several downstream effectors interacting with Plexin cytoplasmic domain were found; however, the molecular mechanisms by which these effectors regulate Plexin signaling remain largely unknown. Our laboratory was interested in one of the effectors named FARP. Previous studies in our laboratory determined the crystal structures of the functional units of FARP1 and FARP2 but failed to detect any GEF activity in vitro. A recent screening with yeast two-hybrid identified a RhoGTPase, Rif, as a novel interacting protein for FARP1. To investigate how Rif/FARP1 interaction is involved in Plexin signaling, I determined the crystal structure of Rif-bound FARP1. This complex structure explains the functional roles that FARP1 and Rif might exert in dendritic spine formation and neurite outgrowth. Also, the N-terminal FERM domain of FARPs is known to interact with the cytoplasmic domain of Plexin. To interrogate how FERM interacts with Plexin and how this interaction might regulate Plexin signaling, I characterized the interaction of FERM with Plexin both biophysically and biochemically. The last question that I attempted to address in this dissertation is how a RhoGTPase, RhoD, inhibits activation of Plexin. RhoD was known for its remarkable inhibitory effect on Plexin signaling presumably through binding to the defined RhoGTPase binding domain of Plexin. The structure of Rac1 or Rnd1 in complex with Plexin did not explain how this binding regulates Plexin activity. I solved the crystal structure of the RhoD/Plexin complex. Modeling of this structure with that of Plexin active dimer in the context of the plasma membrane reveals a mechanism by which RhoD inhibits Plexin activation. Structure studies in this dissertation added another layer of comprehension on how the Semaphorin-Plexin signaling is regulated.Item Does Reelin Affect Recovery from a Stroke?(2016-01-19) DeSai, Charisma; Herz, Joachium; Stowe Ann M.; Lane-Donovan, CourtneyReelin is an extraceullular glycoprotein that modulates synaptic plasticity and increases long-term potentiation. Since Reelin has neuroprotective effects, we were interested to see if Reelin plays a role in recovery after significant neurological damage. Earlier studies with reeler mice showed that mice lacking Reelin have increased susceptibility to stroke and suffer more damage post-stroke. Since Reelin is important in neuronal migration during development, it is possible that the effects seen were due to improper brain development, instead of Reelin deficiency itself. Instead of using Reeler mice, we used the Reelin conditional knockout(cKO) mouse model. Thus, we were able to see the effects of Reelin loss while permitting normal brain development. Stroke size and post-stroke behavior were investigated after inducing transient middle cerebral artery occlusion in four-month-old Reelin cKO mice. No significant difference was seen between wild type and Reelin cKO mice in infarct size or behavior, suggesting that while Reelin does play in important function in the brain, it does not play a significant role in post-stroke recovery.Item Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein(2009-06-17) Jivan, Arif; Cobb, Melanie H.Initially identified in Chlamydomonas, radial spoke protein 3 (RSP3) is one of at least twenty identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be identified and fully characterized. Mammalian RSP3 was found to interact with ERK2 through a yeast two-hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2. Confirming this finding, the human homolog long form, RSP3H, co-immunoprecipitates with ERK1/2 in HEK293 cells. Human RSP3, and its larger alternative start site gene product, radial spoke protein 3 homolog (RSP3H), are phosphorylated by ERK1/2 on threonine 286 in vitro and in cells. RSP3/RSP3H are also phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Additionally, we showed that human RSP3H functions as an A-kinase anchoring protein (AKAP), and its ability to bind to the regulatory subunits of PKA, RII and RII, is regulated by ERK1/2 activity and phosphorylation. Interestingly, expression analysis of mRNA suggests RSP3/RSP3H are also present in cells that are thought to contain a single primary cilium but not motile cilia. Immunofluorescence staining of primary cilia-containing cells indicates that RSP3/RSP3H localize to nuclear punctae, specifically promyelocytic leukemia (PML) bodies, suggesting a non-cilia related role for RSP3/RSP3H in these cells. Functionally, RSP3/RSP3H may localize ERK1/2 to a distinct site of action within the cell and serve as a point of convergence of cAMP-dependent and PKA-mediated influence upon ERK1/2 downstream signaling or vice versa. These data are the first to establish a connection between ERK1/2 and what was once ostensibly thought to only be a ciliary component as well as to identify a novel ERK1/2-interacting AKAP.Item Insights into the Functions of Munc18-1 in Neurotransmitter Release(2013-03-07) Su, Lijing; Rizo-Rey, José; Luo, Xuelian; Kavalali, Ege T.; Liu, YiNeurotransmitter release is an exquisitely regulated process that transmits signals between neurons. The release process includes: docking of synaptic vesicles at the active zone of the pre-synaptic plasma membrane, priming to a release ready state, and then membrane fusion and release of neurotransmitters triggered by Ca2+. Several conserved proteins are involved in regulating the entire process. The central membrane fusion machinery in neurons includes Munc18-1 and the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin. The SNAREs form tight SNARE complexes that bring the vesicle membrane and plasma membrane into close proximity and provide forces to induce membrane fusion. Munc18-1 is essential because deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion. However, its molecular mechanism of action is still unclear. This work is aimed to unravel the critical roles of Munc18-1 in regulating neurotransmitter release. The functions of Munc18-1 discovered so far are related to the SNAREs. Recently we found that Munc18-1 interacts with synaptobrevin and the SNARE four-helix bundle with week affinity, which have been shown to stimulate in vitro SNARE-dependent liposome fusion. Biophysical characterization of these two interactions could provide important information to uncover the roles of Munc18-1 in membrane fusion. Cross-linking and NMR spectroscopy experiments showed that Munc18-1 interacts with the C-terminus of the synaptobrevin SNARE motif through some positively charged residues located in its domain 3a. NMR spectroscopy and ITC experiments revealed that the Munc18-1 N-terminal domain interacts with the C-terminal part of synaptobrevin and syntaxin-1 on the SNARE four-helix bundle, and that the affinity is higher than full length Munc18-1. In our in vitro reconstitution experiments that try to establish the vital functions of Munc18-1 and Munc13 in neurotransmitter release, I found that Munc18-1 displaces SNAP-25 from syntaxin-1/SNAP-25 complex to form Munc18-1/syntaxin-1 complex on liposomes in the presence of NSF/α-SNAP/ATP. When NSF/α-SNAP were incorporated in the lipid mixing assays between synaptobrevin-liposmes and syntaxin-1/SNAP-25-liposomes, Munc18-1 together with Munc13 activate lipid mixing that is inhibited by NSF/α-SNAP. These results suggest that Munc18-1 functions with Munc13 to promote SNARE complex formation in an NSF/α-SNAP resistant manner and to guide the synaptic vesicle exocytosis through a tightly regulated pathway.Item Mechanistic Insights into the Role of Munc13 in Synaptic Vesicle Docking, Priming, and Fusion(2019-03-06) Quade, Bradley Jackson; Luo, Xuelian; Rizo-Rey, José; Rice, Luke M.; Zhang, XuewuNeurotransmitter release is a fundamental aspect of neuronal communication that relies on the fusion of synaptic vesicles with the presynaptic membrane. These fusion events are tightly regulated by the influx of Ca2+, which is sensed by the complex protein machinery at the axon terminal. In order for these Ca2+-mediated fusion events to occur in the correct time and place, protein machines interact with neurotransmitter filled vesicles to dock and prime them for release. Munc13 is one of the essential components of the docking, priming, and fusion machinery. To understand the role of Munc13 in docking and priming I attempted to structurally characterize the MUN domain and SNARE protein interactions using nuclear magnetic resonance spectroscopy. Paramagnetic relaxation enhancement and pseudocontact shift experiments were performed to identify the binding site of SNAREs or the SNARE complex on the MUN domain and in both cases the data suggested that there may be binding in multiple locations or that the interactions are promiscuous. I also attempted to crystallize the C2C domain of Munc13 alone and in the context of larger fragments. I was able to grow crystals of various fragments of Munc13 containing C2C and adjacent domains, but these crystals were fragile and diffracted poorly. In lieu of a crystal structure, I modeled the C2C domain based on homologous C2 domains and performed sequence conservation analysis to identify functionally important regions of C2C that may bind membranes. Using structural information coupled with reconstitutions, dynamic light scattering, and cryo-electron tomography I explored the functional relevance of membrane binding within the C1, C2B, and C2C domains of Munc13. The C2C domain was identified as a critical component of Munc13 that enables bridging between liposomes with synaptic vesicle and plasma membrane composition in vitro and this bridging ability is integral for synaptic vesicle docking in vivo. The C1C2B area has a large membrane binding interface that changes depending on the ligands present in the system and this enables Munc13 to modulate the distance between membranes. The ability of Munc13 to regulate the distance between membranes in response to ligands may underlie its role in synaptic plasticity.Item Neuronal PAS Domain 1 Protein and Its Hypothetical Relationship to Autism(2013-02-08) Walker, Jamie M.; Hsieh, Jenny; McKnight, Steven L.; Brown, Michael S.; Yanagisawa, MasashiNeuronal PAS domain 1 (NPAS1) was discovered in 1997 as a brain-specific transcription factor first detected at embryonic day 15 of the developing mouse embryo. Its expression was observed to peak in the early post-natal days, and in situ hybridization assays in adult mice revealed expression localized to the cortex, hippocampus, thalamus, hypothalamus and superior colliculus. Subsequent immunohistochemical staining assays employing antibodies to NPAS1 revealed an expression pattern largely restricted to inhibitory neurons in the aforementioned brain regions, as well as localized expression in the subgranular region of the dentate gyrus. NPAS1 and NPAS3 transcription factors are paralogues that have evolved from the Drosophila gene Trachealess. Despite the high homology in their bHLH, PAS-A and PAS-B domains, the NPAS1 and NPAS3 proteins appear to be endowed with diametrically opposing functional properties. Interestingly, a translocation that disrupts the NPAS3 gene has been reported in a family suffering from schizophrenia. NPAS3-deficient mice have been reported to have behavioral abnormalities reminiscent of schizophrenia, as well as a distinct deficit in hippocampal neurogenesis. In contrast, several variations in NPAS1 have been found in autistic children. It was found that NPAS1-deficient mice are born with larger than normal brains that contain an over-abundance of neurons in the cortex, as well as a significant increase in hippocampal neurogenesis. The NPAS1-deficient mice also exhibited enhanced sensitivity to acoustic and tactile stimuli. The behavioral and neuroanatomical phenotypes observed in the NPAS1-deficient mice appear to be reminiscent of phenotypes seen in autistic children.Item Novel Roles for the Activity-Regulated Genes Arc and Npas4 in Stress- and Cocaine-Induced Plasticity(2016-04-18) Kumar, Jaswinder Singh; Huber, Kimberly M.; Cowan, Christopher W.; Olson, Eric N.; Zinn, Andrew R.Mood, anxiety, and substance abuse disorders are chronic medical illnesses that contribute significantly to morbidity and mortality worldwide. Currently, these conditions are treated symptomatically using pharmacological and psychotherapeutic approaches; however, the efficacy of these modalities is limited by the dearth of understanding of neurobiological mechanisms underlying mental illness. The high rate of mortality associated with mood, anxiety, and substance abuse disorders is compounded by their shared comorbidity, warranting an investigation into potential shared pathophysiological mechanisms. Studies from human patients and rodent models suggest that these mechanisms may be attributed to disrupted structural and functional plasticity in brain regions involved in mood, reward, and motivation, including the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC). However, the molecules and signaling pathways within these structures that regulate these behaviors, and how they are dysregulated in pathological psychiatric conditions, have yet to be fully identified and characterized. Here, we focus on two key proteins that participate in activity-dependent synaptic plasticity, the neuronal Per Arnt Sim Domain protein 4 (NPAS4) and the activity-regulated cytoskeleton-associated protein (Arc). We utilize a series of ethologically relevant behavioral paradigms to identify Arc and NPAS4 as two important mediators of stress, anxiety, and addiction-related behaviors. Npas4 and Arc, two activity-regulated genes, are robustly induced by stressful, anxiogenic stimuli. Loss of either gene confers an antidepressant and anxiolytic response in mice, and these behavioral phenotypes are mediated by local function of these two proteins in limbic forebrain regions. In a related study, we ask whether loss of Arc influences behavioral responses to cocaine administration. We find that Arc knockout (KO) animals exhibit increased sensitivity to the locomotor activating and rewarding effects of cocaine, and these two phenotypes are associated with a selective increase in synaptic strength in the NAc. Taken together, our results highlight a heretofore-unidentified role for Arc and NPAS4 in stress- and anxiety-like behaviors, as well as Arc in cocaine-related behavioral adaptations. We propose that these two molecules play a vital role in regulating synaptic and behavioral plasticity evoked by exposure to stress and drugs of abuse, likely via experience-dependent synaptic remodeling.Item Regulation and Lineage Analysis of Neurog1 in the Developing Spinal Cord(2007-05-23) Quiñones-Figueroa, Herson Isaac; Johnson, Jane E.The bHLH transcription factor Neurog1 is involved in neuronal differentiation and cell-type specification in distinct regions of the developing nervous system. I developed mouse models that efficiently drive expression of GFP or Cre recombinase in all Neurog1 (Ngn1, NeuroD3) domains. Deleting highly conserved sequences from a BAC containing 113kb 5' and 71kb 3' genomic sequence surrounding the Neurog1 coding region allowed the identification of enhancer elements required to drive Neurog1 expression. I show that a 3.8 kb fragment located 4.2 kb 5' of Neurog1 is required for efficient reporter expression in all Neurog1 domains. This sequence contains previously identified enhancer elements for midbrain, hindbrain and dorsal neural tube, and has two sequences conserved from human to fish. A 16kb fragment containing 8.9 kb 5' and 5.2 kb 3' of the Neurog1 coding sequence was not sufficient to drive expression in all domains. Reporter expression was observed in the dorsal neural tube, the midbrain, hindbrain and trigeminal ganglia, but was missing in the olfactory epithelium, dorsal root ganglia, dorsal telencephalon, and ventral neural tube. A 2.3 kb enhancer element located 8 kb 5' of the Neurog1 coding region was identified that is necessary to direct expression in the ventral neural tube. In addition, these mouse models allowed both short-term and long-term lineage analyses. I show that derivatives of Neurog1-expressing progenitor cells in the neural tube largely comprise the interneuron populations dI2, dI6, V0, V1, and V2, and to a lesser extent motorneurons. This is seen in the co-expression of GFP driven by Neurog1 regulatory sequences with the neuronal identity markers Brn3a, Islet1/2, Lhx1/5, Lhx3, Pax2, and Chx10. Genetic fate mapping in vivo using Cre recombinase reveals that although Neurog1-expressing cells primarily give rise to neurons, minor populations of oligodendrocytes and astrocytes are also identified in the lineage by adult stages in the spinal cord. Adding temporal control to the fate mapping strategy demonstrates that the neurons are generated from Neurog1-expressing cells prior to E13, and glial cells after E13, placing Neurog1 in lineage restricted precursor cells during embryogenesis.Item Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling(2017-04-14) Rich, Shannon Kay Good; Johnson, Jane E.; Terman, Jonathan R.; Krämer, Helmut; Alto, NealDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology.Item A Reverse Translation Mouse Model for Schizophrenic Psychosis: Contribution of Hippocampal Subfield Pathology(2016-11-30) Southcott, Sarah Ann; Hsieh, Jenny; Huber, Kimberly M.; Rothenfluh, Adrian; Tamminga, CarolSchizophrenia is a serious and lifelong psychotic illness that affects all aspects of cognitive and affective function and whose etiology and brain mechanisms remain elusive. Schizophrenia affects not only those who express the condition, but also their family members, friends, and society as a whole. There is a worldwide prevalence of 1%, and the illness in 2012 alone, cost the USA an estimated $62.7 billion in medical care cost and lost wages. Schizophrenia is an extremely complex disease with a heterogeneous mixture of symptoms, including cognitive dysfunction, mood dysfunction, negative symptoms, and the defining symptom set, positive psychotic symptoms. The antipsychotic effects of dopamine receptor antagonists led people to hypothesize that schizophrenia is a disorder of dopamine hyperfunction, but considerable research has generated no strong evidence to support such a simple mechanistic hypothesis. Most recently, the glutamate system has become an etiologic focus in schizophrenia research and its research is proving more promising. We have studied the molecular basis of psychosis in human post mortem hippocampus in schizophrenia, and its related proteins important for learning and memory, especially the n-methyl-d-aspartate (NMDA) glutamate receptor system. Based on our findings we have developed a testable hypothesis of psychosis, formulated as a learning and memory disorder. In order to fully test this hypothesis we first needed to create a dynamic animal model based on our tissue findings that could be manipulated and probed. We found that knocking out the obligate subunit (GluN1) of the NMDA receptor selectively in the dentate gyrus paradoxically led to an increase in neuronal activity in the CA3 and several behavioral changes parallel to those we observe in schizophrenia. Furthermore we combined a pharmacological risk factor (phencyclidine) and a genetic risk factor (DISC1) with the knockout mouse that we believed would have the highest probability of interacting in a manner reminiscent of schizophrenia. These particular combinations did not exacerbate the symptoms of the dentate gyrus-specific GluN1 knockout mouse. Now we plan to use this dynamic mouse preparation to study the mechanisms whereby the reduction in GluN1 protein in dentate gyrus sensitizes and stimulates neuronal activity downstream within the hippocampus to better understand psychosis processes.Item The Role of NeuroD1 in Physiological and Pathological Neurogenesis(2016-12-05) Brulet, Rebecca R.; Schneider, Jay W.; Hsieh, Jenny; Eisch, Amelia J.; Ge, Woo-PingNeurogenesis in the adult brain is a complex and highly regulated process. Under normal physiological conditions neurogenesis in the hippocampal subgranular zone (SGZ) is important for learning, memory, and mood regulation. What is not well understood, however, is whether in certain disease contexts, like epilepsy, aberrant neurogenesis can contribute to the progression of spontaneous reoccurring seizures (SRS) and associated memory decline. In this work, I present evidence that aberrant hippocampal neurogenesis is causative in the perpetuation of SRS. In an effort to target a select stage of adult neurogenesis I identified the bHLH transcription factor NeuroD1, known to be important in adult neurogenesis, as being strongly upregulated after status epilepticus (SE). Additionally, I show expression of NeuroD1 in aberrant ectopically localized granule cells suggesting a potential role for this transcription factor in the progression of epilepsy. NeuroD1 conditional knockout (cKO) in progenitor cells of the hippocampus may be sufficient to reduce the number of immature and mature neurons amongst the labeled population, however the total number of immature and mature neurons was not significantly changed aside from the immature neurons ectopically localized to the hilus. Consistent with this, the total SRS was unchanged in the NeuroD1 cKO. Transdifferentiation, or the direct inter-lineage conversion of adult somatic cells is a powerful tool with the potential to be used in neuronal replacement strategies in certain neurological disorders or CNS injuries. Transdifferentiation of reactive astrocytes into glutamatergic neurons via retroviral targeting in the cortex can be accomplished by overexpression of the transcription factor NeuroD1. However, what is not well understood is whether the state of reactive gliosis is necessary to "prime" these cells for the transdifferentiation process. In this work I present evidence to suggest that overexpression of NeuroD1 in the absence of reactive gliosis is capable of astrocyte to neuron transdifferentiation, however the total number of converted cells is vastly lower than what was previously published, suggesting that reactive gliosis does indeed enhance and facilitate the conversion process.Item Role of Neuroligin in Synapse Formation and Autism(2006-08-11) Chubykin, Alexander Anatoly; Südhof, Thomas C.Neuroligins mediate synaptogenesis through formation of a trans-synaptic complex with presynaptic neurexins. Interaction of neuroligin 1 with neurexins is regulated by alternative splicing of both neuroligin 1 (at splice site B) and of neurexins (at splice site #4). Full-length neuroligin 1 that binds only beta -neurexin more potently promotes synapse formation in hippocampal neurons, whereas neuroligin 1 lacking splice site B, which binds both alpha - and beta -neurexins, is more efficient at synapse expansion. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta and blocked synapse formation. Neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synaptogenic activity. In hippocampal neurons, overexpressed neuroligin 1 enhances excitatory but not inhibitory synaptic responses, and increases the ratio of NMDA to AMPA receptor-mediated synaptic currents. In contrast, genetic deletion of neuroligin 1 in mice decreases NMDA receptor-mediated synaptic currents and the NMDA/AMPA receptor ratio. Contrary to neuroligin 1, neuroligin 2 potentiates inhibitory but not excitatory synaptic responses. The synaptic actions of neuroligin 1 are suppressed by chronic blockade of NMDA receptors or of CaM-kinase II. Neuroligin 1 with an autistic-spectrum syndrome mutation decreases excitatory synaptic responses, consistent with a role for endogenous neuroligin 1 in synapse development. Taken together, our data suggest that neuroligin-neurexin interaction regulated by their alternative splicing promotes formation of specific synapses; synaptogenic function of neuroligin is regulated by NMDA receptor and Cam-kinase II activation, suggesting a critical role for neuroligins in synaptic plasticity and modulation of neural circuits.Item The Role of SHANK3 at the Synapse and Its Implications in Autism-Associated Behaviors and Synaptic Transmission(2015-04-10) Kouser, Mehreen; Rothenfluh, Adrian; Huber, Kimberly M.; Bibb, James A.; Powell, Craig M.Autism is a neurodevelopmental disorder characterized by an increase in repetitive behaviors and impairments in social interaction and communication. Since its discovery, a multitude of studies have linked SHANK3 to autism. Moreover, deletion of SHANK3 has been shown to cause Phelan McDermid Syndrome (22q13 Deletion Syndrome) by several human studies. Shank3 is a multi domain post-synaptic scaffolding proteins that is found in excitatory synapses and plays a critical role in forming the post-synaptic density by connecting the necessary machinery together. In this study, I have characterized a homozygous Shank3 mutation in mice that deletes exon 21(Shank3ΔC) including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major, naturally occurring Shank3 protein bands. Shank3ΔC/ΔC mice exhibit an increased localization of mGluR5 to the synapses in the hippocampus, a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. I also report on a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G) which causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse like the Shank3ΔC/ΔC mice. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, and altered response to novelty. Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity. Finally, Shank3G/G mice were designed to be genetically rescued to wild-type at various times during development. In this study, I also report on the biochemical and behavioral results of the genetic rescue in Shank3G/G mice after the completion of neurodevelopment. I was able to achieve a biochemical rescue in the Shank3G/G mice. Interestingly, not all the behavioral impairments observed in Shank3G/G mice were replicated in the Reversible-Shank3G/G mutation mice making the interpretation of the data more challenging which is discussed in detail in this thesis.Item Sensory Neurogenesis and the Role of PRDM12 in Nociceptor Development and Function(August 2021) Landy, Mark Andrew; Johnson, Jane E.; Henkemeyer, Mark; Price, Theodore; Lai, HelenNociceptors are a set of peripheral neurons responsible for the detection of noxious stimuli. They function to alert us to the presence of potentially damaging internal and environmental threats, thereby playing an important role in allowing us to react and avoid danger. Unfortunately, for over 30% of the population, the sense of pain derived from nociception becomes maladaptive, leading to chronic painful conditions which carry an estimated economic burden of over USD 600 billion. While opioids are the current mainstay analgesic medication, they have a vast array of side effects, including risk of overdose and death, which makes their use in treatment of chronic conditions far from ideal. Recently, studies of genetic mutations leading to congenital insensitivity to pain (CIP) have provided a springboard for the development of novel analgesics targeting nociceptor function in the periphery. The identification of additional such mutations in the gene Prdm12 raised questions about processes inherent to nociceptor development and sensory neurogenesis as a whole, and what function this gene plays in mature primary afferent neurons. Over the course of my dissertation work I sought to address some of these questions by studying the development of nociceptive neurons in mice, and the effect of Prdm12-knockout on pain sensation. To establish a framework for sensory neuronal development, I first completed a birthdating study using a thymidine analog to permanently label cells undergoing their final mitotic divisions and identify them at later timepoints. With this, I show that there is no temporal difference in the birth rates of different nociceptor subtypes, but that most are born after touch and proprioceptive neurons. Next, using multiple models to knockout Prdm12 at various timepoints ranging from conception to adulthood, I then provide evidence that this is a key transcription factor for specification of the nociceptor population, but that its function likely changes over time. Loss of Prdm12 during development causes defects in nociception, but no behavioral phenotype was readily discernible following adult knockout. Instead, I show that Prdm12 appears to regulate a population of stem cell-like neurons, with an as-yet unknown role in dorsal root ganglia. Altogether my work highlights the role of Prdm12 in nociceptor development and lays the groundwork for additional studies to investigate the clinical relevance of this gene.Item Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming(2016-07-12) Smith, Derek Kurtis; Johnson, Jane E.; Kim, Tae-Kyung; Olson, Eric N.; Zhang, Chun-LiThe activity of pro-neural signaling molecules and transcription factors is sufficient to induce the transdifferentiation of lineage-restricted fibroblasts into functional neurons; however, a mechanistic model of the immediate-early events that catalyze this conversion has not been well defined. We utilized a high-efficiency reprogramming system of NEUROG2, forskolin (F), and dorsomorphin (D) to characterize the genetic and epigenetic events that initiate an acquisition of neuronal identity in fetal human fibroblasts. NEUROG2 immediately activates a neurogenic program, but is only sufficient to impart a functional identity in the presence of FD. These small molecules promote NEUROG2 and CREB1 co-transcription, induce SOX4 expression, and promote SOX4-dependent chromatin remodeling. Genome-wide occupancy analysis revealed that SOX4 targets numerous SWI/SNF complex subunits and co-binds with NEUROG2 to enhance the expression of diverse neurogenic factors. The overexpression of SWI/SNF chromatin remodeling factors or treatment with small molecules that modify chromatin accessibility enhanced NEUROG2-mediated neuronal reprogramming of adult human skin fibroblasts. This work represents the first comprehensive mechanism for the immediate events that catalyze neuronal transdifferentiation.Item Structural and Functional Studies of C2-Domain Proteins Involved In Neurotransmitter Release(2007-05-22) Dai, Han; Rizo-Rey, JoséNeurotransmitter release is mediated by synaptic vesicle exocytosis at presynaptic nerve terminals. This process is extremely fast and strictly regulated by the intracellular Ca2+ concentration. To achieve this exquisite regulation, many proteins are involved. The central fusion machinery includes the SNARE proteins synaptobrevin, syntaxin and SNAP-25, as well as Munc18. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered release. Interestingly, most of these regulatory proteins contain C2 domains, versatile protein modules with Ca2+-dependent and Ca2+-independent activities. However, the mechanism of regulation by these C2-domain proteins remains unclear. My research has focused on understanding the structural and functional properties of two types of C2-domain proteins, synaptotagmins and RIMs. With NMR spectroscopy and biochemical assays, we demonstrated that synaptotagmin 4 is a Ca2+ sensor in Drosophila but not in rat, in contrast to the prediction based on sequence alignments. X-ray crystallography revealed that changes in the orientations of critical Ca2+-ligands render the rat synaptotagmin 4 C2B domain unable to form full Ca2+-binding sites. We also analyzed the structural and biochemical properties of the RIM2 C2A domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM2 C2A domain and Ca2+, phospholipid, synaptotagmin 1, and SNAP-25. However, the crystal structure of RIM C2A domain exhibits a strikingly dipolar distribution of the surface electrostatic potential. Several lines of evidence from the crystal structure suggested a potential target binding site around the bottom 310-helix. With fluorescence microscopy and microfluidic channel technology, we demonstrated that synaptotagmin 1 binds simultaneously to phospholipids and the SNARE complex reconstituted in membranes in the presence of Ca2+, forming a quaternary SSCAP complex, and that the membrane penetration of synaptotagmin 1 into phospholipids is independent of the reconstituted SNARE complex. We also showed that synaptotagmin 1 displaces complexin from the reconstituted SNARE complex in the presence of Ca2+, and that the synaptotagmin 1 C2B domain is primarily responsible for SNARE binding. Moreover, NMR spectroscopy and site-directed mutagenesis studies yielded structural information of the potential binding interface, allowing us to use computational modeling and docking to generate a preliminary model of the SSCAP complex.