Recent Submissions

  • Hypothalamic AgRP and POMC neurons modulate stress-induced depression-related behaviors

    Fang, Xing; Department of Neuroscience and Regenerative Medicine
    Depression is a common and debilitating mental disease. Currently available antidepressants are not effective for many individuals with depression and our understanding of the underlying mechanisms remain limited. Evidence suggests that hypothalamic arcuate nucleus (ARC) is highly responsive to acute stress. The ARC contains two distinct subpopulations of neurons—expressing orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC). AgRP and POMC neurons regulate food intake and the food reward system. It is unknown whether AgRP and POMC neurons are recruited by chronic stress and if their dysfunction may contribute to the development of chronic stress-induced depression-related behaviors. To address this, we have developed a mouse model of chronic unpredictable stress (CUS), which can induce anhedonia and despair behavior that mimic symptoms in human depression. Using this animal model, I investigated the roles of AgRP and POMC neurons in stress responses and stress-induced depression-related behaviors. I demonstrated that CUS decreases activity of AgRP neurons but increases activity of POMC neurons. A chemogenetic approach was used to selectively manipulate the activity of POMC and AgRP neurons, leading to opposite effects of stress-induced depression-related behaviors. These results suggest that AgRP and POMC neurons are differentially involved in stress maladaptation and related behaviors. It provides insight into the mechanisms underlying the development of depression and novel strategies for the treatment of this mental illness.
  • Neuron-derived estrogen and neural function

    Lu, Yujiao; Department of Neuroscience and Regenerative Medicine (Augusta University, 2020-05)
    17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. We created a forebrain neuron-specific aromatase knockout (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice. Under normal conditions, FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent cognitive functions. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, electrophysiological study suggested normal long-term potentiation (LTP) induction, but significantly decreased amplitude in FBN-ARO-KO mice which could be fully rescued by acute E2 treatment in vitro. Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. After global cerebral ischemia (GCI), ovariectomized female FBN-ARO-KO mice had significantly attenuated aromatase and hippocampal E2 levels. Intriguingly, FBN-ARO-KO mice exhibited a robust reduction in astrocyte activation, as well as exacerbated neuronal damage and worse cognitive dysfunction after GCI. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. In vivo E2 replacement rescued the compromised reactive astrogliosis and cognitive deficits. Moreover, neuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was dramatically increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling in astrocytes by central injection of an FGFR3 antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Collectively, our data provides novel genetic evidence for the roles of neuron-derived E2 in regulating synaptic plasticity, cognitive function in the non-injured brain, and astrocyte activation and neuroprotection in the injured brain.
  • Genetic Modeling and Pathophysiological Analysis of FAM109A, a Putative Human Disease Gene

    Ates, Kristin Marie; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
    A critical barrier in the treatment of endocytic diseases is the lack of information and understanding of the in vivo mechanisms of endocytosis. Part of this is due to the diverse array of endocytic adaptor proteins that have not yet been studied. We address this by investigating a key endocytic adaptor protein, FAM109A, which interacts with OCRL1, a causative gene for Lowe syndrome. Previous in vitro studies have identified FAM109A as a regulator for endosomal trafficking, particularly in the recycling of receptors in endosomes and sorting of cargo to lysosomes, based on knock-down studies. Here we conduct the first study into the developmental and physiological functions of FAM109A in vivo, utilizing the zebrafish model. We find that depletion of both zebrafish orthologs, zFAM109A and zFAM109B, in our maternal-zygotic homozygous mutant models (AB mutant) disrupts fluid-phase endocytosis and ciliogenesis in the pronephros. Partial knockdown of OCRL1 in the AB mutants exacerbates the endocytosis deficit, confirming that OCRL1 and FAM109 proteins are linked in a common endocytic pathway. In addition, we discover that zFAM109A/B mutant animals exhibit reduced jaw size and delay in chondrocyte maturation, indicating a novel role for zFAM109A and zFAM109B in craniofacial development. This is consistent with the phenotype in a patient within the NIH’s Undiagnosed Diseases Program (UDP). The UDP patient carries a de novo arginine (R) to cysteine (C) mutation (R6C) in FAM109A and presents with craniofacial abnormalities, developmental delay, auditory and vision impairments, and renal dysfunction. Expressing zFAM109A with the R6C mutation in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results show that FAM109A is involved in fluid-phase endocytosis and ciliogenesis in vivo. Moreover, we provide further insight into the potential pathogenesis of a UDP patient’s disease in association with a de novo mutation in FAM109A.

    Tucker, Lorelei; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
    Neonatal hypoxic ischemic encephalopathy (HIE), initiated by hypoxic-ischemic (HI) injury to the brain in the perinatal period, is a leading cause of infant mortality and disability. HI damage to the developing brain triggers a complex pathology, initiating with mitochondrial insult, which culminates in neuronal cell death. Photobiomodulation (PBM), the application of near-infrared light, is an experimental neuroprotective strategy targeting the activity of mitochondrial cytochrome c oxidase (CCO), but its effect on HIE is unknown. This work was designed to shed light on the effect of PBM on a neonatal rat HI injury model. Postnatal day 10 mixed-sex pups underwent HI insult followed by 7 daily PBM treatment sessions via a continuous wave diode laser (808 nm). HI pups suffered significant ipsilateral hemispheric brain shrinkage and substantial cell death in the cortex and hippocampal CA1 and CA3 subregions. PBM treatment reduced neuronal cell death in the cortex and hippocampal subregions and reduced hemispheric brain shrinkage. HI pups displayed impaired motor function and spatial learning and memory which was ameliorated by PBM. Blood-brain barrier integrity was compromised in HI animals, as evidenced by reduced extravasation of Evans blue, but was reversed by PBM. PBM also mitigated microglial activation and upregulation of pro-inflammatory cytokines in HI pups. PBM treatment induced robust reduction in oxidative damage markers and protein carbonyl production in the cortex and hippocampus. Investigation of mitochondrial function revealed that PBM markedly attenuated mitochondrial dysfunction and preserved ATP production in neonatal HI rats. Furthermore, PBM treatment profoundly suppressed HI-induced mitochondrial fragmentation. PBM administration reduced activation of pro-apoptotic caspase 3/9 and TUNEL-positive neurons in HI pups. Finally, we demonstrated that the neuroprotective action of PBM could be reversed in a primary hippocampal neuronal OGD model by application of low-dose KCN, a CCO inhibitor. Taken together, our findings demonstrated that PBM treatment contributed to a robust neuroprotection via attenuation of mitochondrial dysfunction, oxidative stress, and neuronal apoptosis in the neonatal HI brain. Additionally, we demonstrated that these effects are, in part, mediated by modulation of CCO activity. This suggests that PBM may offer a promising role as a potential treatment strategy for HIE.
  • Anterograde and Retrograde Regulation of Neuromuscular Junction Formation and Aging

    Zhao, Kai; Department of Neuroscience and Regenerative Medicine (2018-11-29)
    The neuromuscular junction (NMJ) is a chemical synapse that facilitates the neuronal control of muscle contraction. Proper NMJ formation and maintenance require both anterograde and retrograde signaling. In this study, on one hand, we characterized the role of Yes-associated protein (Yap) in the formation of neuromuscular junction (NMJ). In HSA-Yap-/- mice where Yap was mutated specifically in muscle cells, AChR clusters were smaller and distributed in a broader region in the middle of muscle fibers. In addition, HSA-Yap-/- mice also exhibited remarkable presynaptic deficits including less nerve coverage of the endplates, reduced mEPP frequency and increased paired-pulse facilitation, indicating structural and functional defects. Moreover, muscle Yap mutation prevented reinnervation of denervated muscle fibers and the phenotypes were related to compromised β-catenin signaling. Both NMJ formation and regeneration deficits of HSA-Yap-/- mice were ameliorated by inhibiting β-catenin degradation, further corroborating a role of β-catenin as a downstream molecule of Yap to regulate NMJ formation and regeneration. On the other hand, we showed that Lrp4, a receptor for agrin and critical for NMJ formation and maintenance, was reduced at the protein level in aged mice, which was associated with decreased MuSK tyrosine phosphorylation, suggesting compromised agrin-Lrp4-MuSK signaling in aged muscles. Transgenic expression of Lrp4 in muscles alleviated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. Lrp4 ubiquitination was augmented in aged muscles, suggesting increased Lrp4 degradation as a mechanism for the reduced protein level. We also found that sarcoglycan alpha (SGα) interacted with Lrp4 and delayed Lrp4 degradation in co-transfected HEK293 cells. AAV9-mediated expression of SGα in muscles mitigated Lrp4 degradation and NMJ decline in aged mice. These observations support a model where compromised agrin-Lrp4-MuSK signaling serves as a pathological mechanism of age-related NMJ decline and identify a novel function of SGα in stabilizing Lrp4 for NMJ maintenance in aged mice.

    Fox, Grace Ellen; Department of Neuroscience and Regenerative Medicine (8/7/2018)
    Fear is an adaptive response that permits organisms to reduce or avoid danger. In many animal species, behavioral correlates of fear can be seen, which highlights its essential role in survival; conversely, inappropriate and exaggerated fears are a hallmark of psychiatric disorders, such as anxiety-related disorders. Recently, the retrosplenial cortex (RSC) has been implicated for long-term storage of fear-related memories. However, the basic physiological properties of RSC cells as well as their response to fearful events remained largely unexplored. Our experiments demonstrate that excitatory principal cells within RSC layers 2&3 and 5&6 contain multiple physiologically distinct sub-populations that are differentially affected by ketamine. We also demonstrate the novel finding that the RSC utilizes the specific-to-general cell-assembly logic for processing neural information about fearful experiences. Taken together, these results illustrate that the RSC generates a cell assembly-level representation of fear memory engrams and supports the pursuit of the RSC as a therapeutic target for anxiety-related disorders.
  • Identifying the Function of Nasal Embryonic LHRH factor (NELF) in Immortalized GnRH Neurons

    Ko, Eun Kyung; Department of Neuroscience and Regenerative Medicine (7/30/2018)
    Hypothalamic gonadotropin releasing hormone (GnRH) is crucial for the proper function of the hypothalamic-pituitary-gonadal (HPG) axis, puberty, and reproduction. When GnRH neuron migration or GnRH regulation is impaired, hypogonadotropic hypogonadism results. Mutations in the gene for nasal embryonic LHRH factor (NELF) have been identified in GnRH-deficient humans. NELF is a predominantly nuclear protein that may participate in gene transcription, but it is unlikely to be a transcription factor. Thus, our hypothesis is that NELF may indirectly regulate transcription via protein-protein interaction within a transcription complex. To address this question, RNA was extracted from NLT GnRH neuronal cells following either stable Nelf knockdown or scrambled control and subjected to cDNA arrays. Expression of transcription factors and cell migration gene expression was most commonly altered. Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, including Stat1, Stat2, Stat5a, Jak2, Irf7 and Irf9, were significantly down-regulated as assessed by RT-qPCR. Protein levels of STAT1, phospho-STAT1, and JAK2 were reduced, but the levels of phospho-JAK2 were not. These findings suggest a role for NELF in the regulation of the JAK/STAT signaling pathway, which has important functions in GnRH neurons. Jacob, the rat orthologue of NELF, is phosphorylated at the serine 180 residue by phosphorylated Erk1/2 which is activated by synaptic NMDA receptor activation and then translocates to the nucleus. Phosphorylated Jacob in the nucleus interacts with CREB and regulates the expression of BDNF, which is associated with synaptic plasticity in the brain. Proteins, such as caldendrin, importin-α and α-interxin, have been identified as binding proteins with Jacob. However, binding proteins, phosphorylation sites and/or kinases for NELF have not yet been reported. To demonstrate novel and putative functions of NELF in the nucleus, identification of binding proteins and phosphorylation sites is required. To address this question, co-immunoprecipitation (Co-IP), mass spectrometry and in vitro kinase assays were performed. We found six putative binding proteins that could interact with NELF, including 14-3-3ε. We also identified phosphorylation sites on NELF, including serine 288, which could be phosphorylated by AKT1 kinase. These new findings will be helpful to understand the function of NELF in the nucleus
  • Using peptide-based vaccines to enhance adoptive cell therapy with genetically engineered T cells

    Fan, Aaron; Department of Neuroscience and Regenerative Medicine (6/27/2018)
    Adoptive cell therapy (ACT) of retrovirally transduced (RV) CD8 T cells is a powerful technique that has shown promise in tumor eradication in cancer patients. However, some major barriers to current methods are that ACT is expensive, time consuming, and requires harmful and toxic adjunct procedures. The Celis laboratory has demonstrated the use of TriVax, a potent peptide vaccination strategy that dramatically expands ACT cell populations and bypasses the necessity for adjunct procedures. The purpose of my thesis project was to enhance current methods of ACT+TriVax by testing an antigen-specific antitumor response of RV CD8 T cells and if it could be improved with constitutively active STAT5 (CA-STAT5) expression, a protein activated downstream several cytokine pathways that have been shown to play a role in increasing CD8 T cell persistence and resistance to apoptosis. Here, I aimed to test the hypothesis that CA-STAT5 in CD8 T cells enhances an antitumor effect by increasing T cell persistence and efficacy. My results show that TriVax administration selectively expanded frequencies of the ACT cell population expressing gp100-TCR in both blood and spleen. When co-transduced with CA-STAT5, an even higher fold expansion of antigen-specific cells was observed. +CA-STAT5 T cells were able to expand more robustly than -CA-STAT5 T cells upon repeated antigen stimulation (vaccine boost), demonstrating nearly 4000-fold increases in antigen-specific CD8 T cells. +CA-STAT5 T cells also seemed to persist longer in vivo over time, and they expressed lower levels of surface PD-1. Using B16F10 melanoma, ACT+TriVax of these cell populations into tumor-bearing mice demonstrated a powerful antitumor effect, leading to tumor regression in treated groups. CA-STAT5 seemed to recapitulate similar antitumor effects our laboratory observed previously with combinatorial anti-PD-L1 treatment or IL2/anti-IL2 mAb complexes (IL2Cx), suggesting a potential role for STAT5 in resisting the PD-1/PD-L1 inhibitory pathway. Altogether, these results demonstrate that RV CD8 T cells expressing gp100-TCR and CA-STAT5 are capable of antigen-dependent expansion in response to TriVax. CA-STAT5 plays a role in increasing T cell proliferation and persistence, as well as increasing efficacy through resistance to PD-1/PD-L1 inhibition.
  • NLRP3 Inflammasome-Mediated Uncoupling of Hippocampal Vasoneuronal Communications in Diabetes: Relevance to Cognitive Impairment and Stroke

    Ward, Rebecca; Department of Neuroscience and Regenerative Medicine (5/22/2018)
    Diabetes is a prevalent chronic disease that affects over 29 million individuals in the United States and 422 million people worldwide as of 2017. Given the high mortality and morbidity associated with diabetes due to its complications including retinopathy, chronic kidney disease, peripheral neuropathy, heart disease and stroke, this increase in incidence of diabetes creates many clinical, social and economic problems. A silent, but unrecognized complication of diabetes is cognitive impairment, which ranges from mild cognitive impairment to dementia. The increased risk and incidence of stroke amplifies these cognitive deficits. The objectives of this dissertation were to 1) determine the role and mechanism(s) by which diabetes worsens cognitive decline and 2) determine the extent and mechanism by which NLRP3 activation contributes to poor cognitive function after stroke in diabetes. To investigate these objectives, feeding rats a high fat diet and administering a low dose of streptozotocin was used as a clinically relevant diet-enhanced model of diabetes. Stroke was induced through either transient MCAO (60 or 90 min) or embolic MCAO. Embolic stroke caused more severe hippocampal neurovascular injury, microglial activation and cognitive decline in diabetes as compared to stroke induced by a shorter 60 min suture occlusion of the MCA. Diabetic females were more sensitive to ischemic injury than males. Furthermore, hippocampal vascularization patterns at baseline and after ischemic injury differed in males and females and despite these sex differences in the extent and patterns of hippocampal neurovascular injury, diabetes worsened cognitive outcomes in both sexes. Collectively, these first studies provide a preclinical foundation for future studies addressing cognitive impairment in diabetes in both sexes. NLRP3 inflammasome, which cleaves IL-1β and IL-18 into their active forms, was upregulated in diabetes and amplified following ischemia. Inhibition of the NLRP3 inflammasome with MCC950, a specific small molecule inhibitor of NLRP3 activation, improved post-stroke cognition, reduced hippocampal cell death, was associated with less leaky vasculature, and blunted chronic inflammation in the hippocampus after 90-min MCAO. MCC950 did not seem to provide neuroprotection to the neuron through the mBDNF, but did reduce cell death after hypoxia/reoxygenation in vitro. These results are the first to provide essential data showing that MCC950 has the potential to become a therapeutic agent to prevent neurovascular remodeling and worsened cognitive decline in diabetic patients following stroke. Collectively, this work may provide a piece of the puzzle explaining how diabetes leads to cognitive impairment and worsens outcome following acute ischemic injury, and it provides a potential therapeutic target to treat cognitive impairment after stroke, especially in diabetic patients.
  • Early Life Environmental Exposure and Hormonal Exposure and Race-Related Influence on the Human Stem Cell Populations in Fibroid and Myometrial Tissues Lead to Compromised Genomic Integrity and Increased Tumorigenesis

    Prusinski Fernung, Lauren; Department of Neuroscience and Regenerative Medicine (5/22/2018)
    Though benign, uterine fibroids (UF) are the most significant benign neoplastic threat to women’s health and most common indication for hysterectomy. The elusive etiology of UF inhibits significant improvement in quality of care for affected women. Somatic mutations in the MED12 gene are currently thought to arise in myometrial stem cells (MSCs) converting them into UF tumor-initiating cells. Defective DNA repair increases the risk of tumorigenic somatic mutations, suggesting that additional mutations arising in fibroid stem cells (FSCs) ultimately contribute further to tumor growth and development. In addition, a significant ethnic disparity exists in UF prevalence, occurring in African American (AA) four times more as compared to Caucasian (CA) women, a phenomenon that has been observed for more than 120 years, but the molecular attributes behind UF’s ethnic disparity are still not fully realized. Our goal is to determine the mechanism by which the physiology of these human uterine MSCs is altered by changes in utero during early development of the epigenetic regulators of DNA-damage repair genes and how these stem cells lead to the origination of MED12 mutations and, ultimately, UF development later in adult life. Using a rat model of early-life environmental exposure, in which rats undergoing early uterine development were exposed to an endocrine disruptor, we compared the DNA repair capacity of exposed, "at-risk" myometrial stem cells to those from unexposed animals. In addition, we utilized human myometrial and fibroid tissue samples to characterize the myometrial stem cell populations from normal versus fibroid-containing uteri and compared the DNA repair capacity of human fibroid stem cells to the stem cells of adjacent myometrium. We determined that DNA repair in both exposed rat MSCs and human FSCs was decreased/altered compared to unexposed murine MSCs and human adjacent MSCs, respectively. In exposed rat MSCs, DNA double-strand break (DSB) repair was significantly impaired both in untreated cells and in cells in which DNA DSB damage was induced. Similar phenomena were observed in human FSCs as compared to adjacent MSCs. These data suggest impaired DNA repair in exposed MSCs and in human FSCs may contribute to initiation and perpetuation of UF tumorigenesis.
  • Role of NADPH Oxidase following Traumatic Brain Injury

    Ma, Merry Wenlan; Department of Neuroscience and Regenerative Medicine (5/22/2018)
    Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Despite intense investigation, no neuroprotective agents for TBI have yet translated to the clinic. Recent efforts have focused on identifying potential therapeutic targets that underlie the secondary TBI pathology that evolves minutes to years following the initial injury. Oxidative stress is a key player in this complex cascade of secondary injury mechanisms and prominently contributes to neurodegeneration and neuroinflammation. In addition, the NLRP3 inflammasome, which produces pro-inflammatory signals, can become activated in response to oxidative stress and may exacerbate secondary pathology. NADPH oxidase (NOX) is a unique family of enzymes whose primary function is to produce reactive oxygen species (ROS). Human post-mortem and animal studies have identified elevated NOX2 and NOX4 levels in the injured brain, suggesting that NOX is involved in the pathogenesis of TBI. Our experiments demonstrate that targeting NOX, specifically NOX2 and NOX4, can reduce oxidative stress, attenuate neuroinflammation, reduce lesion size, and promote neuronal survival following TBI. In particular, deletion of NOX2 or inhibition of NOX can attenuate the increased expression and activation of the NLRP3 inflammasome via TXNIP- mediated pathway and decrease the production of pro-inflammatory factors, such as caspase-1 and IL-1β. We also demonstrate the novel findings that deletion of NOX4 can reduce neuronal oxidative damage evidenced by decreased DNA oxidation, lipid peroxidation, and protein nitration in the injured cerebral cortex. Mice lacking NOX4 also showed reduced cell death and neurodegeneration following TBI. Collectively, our results support the notion that targeting NOX enzymes can suppress neuroinflammatory secondary TBI pathology in addition to alleviating oxidative damage following injury. In addition, our inhibitor studies extend the critical window of efficacious TBI treatment, which further supports the pursuit of NOX as therapeutic targets.

    Thakkar, Roshni Dinesh; Department of Neuroscience and Regenerative Medicine (2017)
    17β-estradiol (E2) is a well-known neuroprotective hormone, but its role in regulation of neuroinflammation is less understood. In the current study, we examined whether E2, acting via PELP1, can exert anti-inflammatory effects in the ovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation, cytokine production and microglial M1/M2 phenotype after global cerebral ischemia (GCI). The results showed that activation of the NLRP3 inflammasome pathway and expression of its downstream products, cleaved caspase-1, and IL-1β, are temporally increased in the hippocampus after GCI, with peak levels observed at 6-7 days. E2 robustly inhibited NLRP3 inflammasome pathway activation, caspase-1 and pro-inflammatory cytokine production, as well as gliosis after GCI at gene as well as protein levels. Moreover, E2 also profoundly suppressed the pro-inflammatory M1 microglial phenotype, while increasing the anti-inflammatory M2 microglial phenotype after GCI. Intriguingly, the ability of E2 to exert all of these anti-inflammatory effects was lost in PELP1 forebrain-specific knockout mice. These robust effects of E2 may be mediated directly upon microglia, as we found that E2 suppressed the M1 while enhancing the M2 microglia phenotype in LPS-activated BV2 microglia cells. Furthermore, E2 treatment also prevented the neurotoxic effects of BV2 microglia cells upon hippocampal HT-22 neurons, suggesting a novel E2-mediated neuroprotective effect via regulation of microglia activation and phenotype. Mechanistically, E2 strongly suppressed expression and activation of the transcription factor NF-κB in BV2 microglia cells, which is known to be a critical regulator of both microglia pro-inflammatory effects and M1/M2 microglia phenotype. Additional studies revealed that NF-κB inhibition also prevents the cytotoxic effects of BV2 microglia cells upon hippocampal HT-22 neurons. Collectively, our study suggests a novel E2-mediated neuroprotective effect via regulation of inflammasome and microglia activation and promotion of the M2 “anti-inflammatory” phenotype in the brain. KEY WORDS: Estrogen, global cerebral ischemia, NLRP3 inflammasome, microglia phenotype, cytokines, neuroprotection.
  • Role of GluN2C-containing N-methyl-D-aspartate (NMDA) receptor in Oligodendrocyte Differentiation and Myelination

    Luo, Tong; Department of Neuroscience & Regenerative Medicine (1/25/2018)
    Myelination by oligodendrocytes (OLs) is critical for rapid nerve signal conduction. Abnormalities of OLs mediate a variety of central nervous system (CNS) diseases, including multiple sclerosis. N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors expressed in neurons and are key regulators for neuron survival and normal brain functions. Recently, NMDA receptors were identified in OLs and contribute to OL migration, differentiation and myelination. However, the exact function of NMDA receptors on OLs remains unclear. Previous studies have shown that GluN2C is one of the predominant NMDA receptor subunits expressed in OLs. Here we report that NMDA treatment promotes OL differentiation in vitro, but fails to increase mature OL percentage in the absence of GluN2C. In addition, we observed an early developmental myelination delay and long-term recovery in the optic nerve of GluN2C-knockout (KO) mice in vivo, which was closely related to the impairment of OL differentiation. Overall, these results indicate a functional involvement of GluN2C-containing NMDA receptors in OL differentiation and myelination.
  • Genetic labeling reveals novel cellular targets of schizophrenia susceptibility gene ERBB4 and neuregulin-1 – ERBB4 signaling in monoamine neurons

    Bean, Jonathan C; Department of Neuroscience and Regenerative Medicine (2015)
    Neuregulin 1 (NRG1) and its receptor ErbB4 are schizophrenia risk genes. NRG1-ErbB4 signaling plays a critical role in neural development and regulates neurotransmission and synaptic plasticity. Nevertheless, its cellular targets remain controversial. ErbB4 was thought to be expressed in excitatory neurons although recent studies have disputed this view. Utilizing mice that express a fluorescent protein under the promoter of the ErbB4 gene, I determined in what cells ErbB4 is expressed and their identity. ErbB4 was widely expressed in the mouse brain, being highest in amygdala and cortex. Almost all ErbB4-positive cells were GABAergic in cortex, hippocampus, basal ganglia, and most of amygdala in neonatal and adult mice, suggesting GABAergic transmission as a major target of NRG1-ErbB4 signaling in these regions. Non-GABAergic, ErbB4-positive cells were present in thalamus, hypothalamus, midbrain and hindbrain. In particular, ErbB4 was expressed in both dopamine neurons in the substantia nigra and ventral tegmental area and in serotoninergic neurons of raphe nuclei, but not in norepinephrinergic neurons of the locus coeruleus. In hypothalamus, ErbB4 was present in neurons that express oxytocin. ErbB4 was expressed in a group of cells in the subcortical areas that are positive for S100β. These results identify novel cellular targets of NRG1-ErbB4 signaling. Finally, perfusion of NRG1 into the medial prefrontal cortex enhanced both dopamine and serotonin release but with differing time courses.

    Lin, Thiri W.; Department of Neuroscience and Regenerative Medicine (11/2/2017)
    GABA activity has been implicated in neural development; however, in vivo genetic evidence is missing because mutant mice lacking GABA activity die prematurely. Here, we studied postnatal synapse development in ErbB4-Vgat-/- mice where Vgat was deleted in ErbB4+ interneurons. We show that the number of inhibitory axo-somatic synapses onto pyramidal neurons is layer-specific; however, inhibitory synapses on axon initial segments (AISs) were similar from layer to layer. On the other hand, PV+ErbB4+ interneurons and PV-only interneurons receive higher number of inhibitory synapses from PV+ErbB4+ interneurons, compared with ErbB4-only interneurons. Erbb4-Vgat-/- mice exhibited fewer inhibitory synapses from PV+ErbB4+ interneurons onto excitatory neurons (either axo-somatic or axo-axonic), compared with control mice. The Vgat mutation seemed to have little effect on inhibitory synapses onto PV+ and/or ErbB4+ interneurons. These morphological alterations were associated with concomitant changes in neurotransmission. Finally, perineuronal nets were increased in the cortex of ErbB4-Vgat-/- mice. These results demonstrate that GABA activity from ErbB4+ interneurons specifically regulates the development of inhibitory synapses onto excitatory neurons and provides in vivo evidence for a critical role of GABA activity in circuit assembly.
  • Developmental and Behavioral Analyses of clarin-2: A Novel Somatosensory Neuron Subtype-Enriched Gene

    Roberts, Rachel; Department of Neuroscience and Regenerative Medicine (2017-06)
    The trigeminal ganglion (TG) is a somatosensory organ that relays stimuli in the head to the hindbrain and spinal cord, and it comprises multiple subtypes of sensory neurons that respond to different somatosensory stimuli and establish distinct neuronal circuits. The Trpa1b subtype of TG sensory neurons (TGSNs) are responsible for sensing noxious chemicals, but the molecular cues that specify the development of this neuronal subtype remain poorly understood. Zebrafish were previously established as a robust model for studying the development of TGSNs due to its small size, translucency, and robust somatosensory behaviors. A previous microarray study in zebrafish found a novel four transmembrane-domain protein, clarin-2, to be enriched in Trpa1b-expressing cells. Nothing is known about the function of clarin-2, but a close homolog, clarin-1, is one of the causative genes for Usher Syndrome Type 3, a disorder characterized by progressive hearing and vision loss. We hypothesize that clarin-2 may play a role in the development and sensory function of TGSNs. To test this hypothesis, we examined the expression of clarin-2 within the TG during development and used clarin-2 knockout (KO) fish to study the genesis and neurite outgrowth of Trpa1b TGSNs. We found that clarin-2 is indeed enriched in a subset of TGSNs but is not required for the morphogenesis of the TG or the specification of nociceptive sensory neurons. Furthermore, axon projections from Trpa1b neurons were normal in clarin-2 KO fish, compared to control siblings. To test whether clarin-2 is required for the function of TGSNs, we tested somatosensory behaviors in larval zebrafish, including chemo-, thermo-, and mechanosensation. Behavioral analyses showed that clarin-2 is not required for the ability of Trpa1b neurons to detect the chemical irritant mustard oil. Additionally, the detection of heat or vibration was not affected in clarin-2 KO fish. Together, these results suggest that although clarin-2 is enriched in a subset of TGSNs, it is not required for the general morphogenesis of TGSNs or for somatosensation.
  • The Contributions of Microglial Vps35 to Adult Hippocampal Neurogenesis and Neurodegenerative Disorders

    Erion, Joanna Ruth; Department of Neuroscience and Regenerative Medicine (5/10/2017)
    The retromer complex is a multimeric protein complex which facilitates intracellular trafficking of a variety of transmembrane proteins. Vacuolar protein sorting-associated protein 35 (VPS35) is a critical component of retromer’s cargo recognition subunit and has been implicated in neurodegenerative disease pathology, including Alzheimer’s disease (AD). Without functional VPS35, trafficking of retromer cargo is often impaired, compromising cargo function and/or downstream signaling events. VPS35 expression is ubiquitous and can be found throughout the central nervous system (CNS). Microglia express VPS35, and microglia from AD patients exhibit reduced VPS35 expression. We sought to determine how microglial VPS35 loss-of-function might contribute to neurodegenerative disease pathology. We examined the CNS of a mouse model of heterozygous VPS35 deletion and found evidence suggesting upregulated microglial activity. To specifically examine the effects of microglial VPS35 loss-of-function, we developed VPS35CX3CR1 mice, a model which induces microglial-specific VPS35 depletion. Microglial VPS35 loss-of-function upregulated microglial density in a region-specific manner, which we determined to be an effect of increased microglial differentiation/survival. Upon further inspection, we found evidence of upregulated microglial inflammatory activity in the hippocampus, including increased levels of proinflammatory cytokine interleukin-6. Microglial density was increased within the subgranular zone (SGZ) of VPS35CX3CR1 mice, so we sought to determine if microglial VPS35 loss-of-function has any effect on hippocampal neurogenesis. While we found decreased doublecortin+ (DCX) immature neurons, an increase was observed in the differentiation/survival of neural progenitor cells (NPCs). Further analysis suggested the cell cycle exit of VPS35CX3CR1 hippocampal NPCs may be compromised. To examine morphology of newborn neurons, hippocampi were labeled with a retroviral vector, revealing impaired dendritic development in newborn hippocampal VPS35CX3CR1 neurons. Aberrant regulation of hippocampal neurogenesis in VPS35CX3CR1 mice was associated with a depressive behavioral phenotype and long-term memory impairment. These findings implicate a novel microglial-specific role of VPS35 that might contribute to neurodegenerative disease pathogenesis. The full extent of this role remains to be determined, as well as the mechanisms underlying our observations. It also remains to be determined what role microglial VPS35 might play in other brain regions, and how microglial VPS35 depletion might contribute to other aspects of neurodegenerative disease pathology.
  • Estrogen Receptor Beta and Autism Spectrum Disorder

    Crider, Amanda; Department of Neuroscience and Regenerative Medicine (2017-05-08)
    Autism Spectrum Disorders are more prevalent in boys than in girls, with ratios of 4.5:1, suggesting the possible role of sex hormones in the pathophysiology of this. In addition to the extreme male brain theory on the high levels of testosterone during early development as a risk factor for ASD, a number of recent studies have shown the role of estrogens in the development of ASD. Many studies have suggested an important role of endoplasmic reticulum (ER) stress in the pathophysiology of ASD, but the underlying mechanism(s) is not known. This thesis aims to determine A) the role of estrogen receptor beta in the development of Autism Spectrum Disorder and B) the role of ER stress in the regulation of ERβ and in the development of ASD pathophysiology.
  • Ceramide Compartments and Protein Interaction: Structure Meets Function

    Kong, JiNa; Department of Neuroscience and Regenerative Medicine (12/27/2016)
    Ceramide is a key sphingolipid, regulating a variety of critical cellular processes. Although exosomes and cilia are derivatives of the membrane, little is known about the role of lipids in their formation. Here we examined the novel role of ceramide in two ceramide-enriched, subcellular compartments: 1) secreted, extracellular vesicles (EVs) termed exosomes, and 2) cell membrane protrusions termed cilia. Firstly, we attempted to address the role of ceramide in exosome secretion and breast cancer. Breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP). We show that incubation of human breast cancer MDA-MB-231 cells with the farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, which is known to induce exosome secretion. Ceramide elevation by combined treatment with gug and bex induced BCRP secretion in exosomes and reduced cellular BCRP in cancer and cancer stem-like cells. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex treatment increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death. Our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Secondly, to investigate the role of ceramide in ciliogenesis, in particular motile cilia, we used Chlamydomonas reinhardtii (Chlamydomonas) and murine ependymal cells as models. Motile cilia are specialized organelles formed by cell membrane protrusions to function in movement of body fluids. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway. Ceramide depletion, by the SPT inhibitor myriocin and a neutral sphingomyelinase deficiency (fro/fro mouse), led to glycogen synthase kinase-3 (GSK3) dephosphorylation and defective flagella and cilia, respectively. A novel activation mechanism for GSK3 by the sphingolipids phytoceramide and ceramide is shown to be critical for ciliogenesis in Chlamydomonas and ependymal cells, respectively. We conclude that ceramide promotes exosome secretion to reduce MDR in MDA-MB-231 cells and regulates GSK3-mediated ciliogenesis in Chlamydomonas and murine ependymal cells.
  • DNA sensing via STING regulates immunity

    Mohamed, Eslam; Department of Neuroscience and Regenerative Medicine (2016)
    The stimulator of interferon genes (STING) is an adaptor protein downstream of an array of cytosolic DNA sensors such as cyclic GMP-AMP synthase (cGAS). STING activation by the second messengers, cyclic dinucleotides (CDNs) induces interferon type I (IFN-I). STING/IFN-I signaling incites autoimmunity in mice lacking the DNA catabolizing enzyme Trex-1. Paradoxically, we find the DNA sensing to activate STING/IFN-I signaling induces dendritic cells (DCs) to express indoleamine 2,3 dioxygenase (IDO), which activates regulatory T cells (Tregs). Thus treatment with DNA nanoparticles (DNPs) or CDNs to activate STING attenuated experimental autoimmune encephalomyelitis (EAE) and therapeutic responses were dependent on STING/IFN-I signaling to induce IDO. DNP and CDNs treatments were also effective in slowing type I diabetes (T1D) progression in susceptible female non-obese diabetic (NOD) mice. Recent reports revealed that DNA sensing to activate STING in DCs that engulfed dying tumor cells impeded growth of immunogenic tumors and potentiated responses to therapy. Consistent with these findings synthetic STING agonists enhanced tumor regression. Paradoxically, lewis lung carcinoma (LLC), grew slower in STING-deficient mice, revealing that STING is required for optimal LLC growth. Mechanistically, STING ablation abolished IDO upregulation in DCs located in tumor draining lymph nodes during LLC growth. Consequently, expression of the regulatory cytokine IL-10 and infiltration of myeloid derived suppressor cells (MDSCs) into the tumor microenvironment (TME) were diminished in mice lacking STING. In contrast, STING was not required for optimal growth of LLC tumors expressing neo-antigens, revealing a pivotal role for tumor antigenicity in influencing responses to DNA in the TME. Thus, DNA from dying cells are sensed in the TME to activate STING, which induces dominant regulatory responses via IDO when tumor antigenicity is low and dominant immunogenic responses when tumor antigenicity is enhanced. Collectively, these findings support the hypothesis that DNP cargo DNA and DNA from dying tumor cells is sensed to activate STING\IFN-I in regulatory DCs that suppresses T cell immunity and autoimmunity at sites of chronic inflammation associated with autoimmunity and tumor growth.

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