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2017 Research Showcase
NB Abstracts
Abstract Title : The Role of Astrocytes in Regulating Blood-brain Barrier Properties in Response to Neural Activity
Abstract : The blood-brain barrier (BBB) is a set of properties belonging to endothelial cells that make up blood vessels in the central nervous system. The BBB’s function is to reduce the transport of nonspecific ions and molecules between the blood and brain to maintain brain homeostasis. The cerebral vasculature is also known to be tightly coupled to neural activity and dynamically changes blood flow to satisfy the energy demand of active brain regions. Although the cerebral vascular blood flow is tightly coupled to neural activity, barrier properties have largely been studied in a static context. Through manipulating glutamatergic neurons, we have shown that neural activity can dynamically regulate the expression of BBB genes in the cerebral vasculature. The goal of my project is to determine how neural activity is mediating these changes in BBB expression. Previous research has shown the importance of astrocytes, a glial cell, in mediating communication between neurons and blood vessels in the context of blood flow, as well as in the maintenance of BBB properties. However, most work has been done in vitro and in a static context. My project aims to study astrocytic regulation of BBB properties in a dynamic context in an in vivo system. I am using transgenic mice that express Gq-DREADDs to selectively activate astrocytes via intracellular calcium influx, then purifying endothelial cells to detect BBB changes via transcriptomics. Preliminary results show that Gq activation of astrocytes upregulates tight junction proteins and downregulates leukocyte adhesion molecules, both of which indicate strengthening of the BBB.
Abstract Title : Structural Differences Between MRGC Dendrites in On Sublamina vs. Off Sublamina
Abstract : In addition to rods and cones, there is a third photosensitive cell type in the mammalian retina that contributes to both circadian entrainment and pupil dilation; the cell is called a Melanopsin Expressing Retinal Ganglion Cell, or MRGC for short. Structural differences between dendrites from these cells in the On Sublamina and the Off Sublamina have been found and quantified. Both the distance between beads, or swellings containing an increased number of mitochondria, and the distance between branch points are greater in dendrites in the Off Sublamina than the On Sublamina. Statistical outliers for both the bead analysis and the branch point analysis were found to be largely the same dendrites; dendrites with exceptionally large branch point distances almost always show large inter-bead distances. The density demonstrates a strong correlation to branching frequency. The difference in both branch frequency and bead density may suggest that dendrites in the On Sublamina form more synapses than those in the Off Sublamina, as more branches means more surface area per unit volume, and mitochondria are required for synapses.
Abstract Title : Lower Nucleus Accumbens Volume is Associated with Reduced Reward-Based Decision Making in Veterans with History of Mild Traumatic Brain Injury
Abstract : Mild traumatic brain injury (mTBI) has been associated with diffuse neuronal loss, and recent research suggests that neurodegeneration in subcortical regions may play a fundamental role in commonly observed negative behavioral outcomes post-injury. The nucleus accumbens—a structure critical for motivation, reward, and higher-order decision-making— may be especially vulnerable to TBI-induced damage. Therefore, in a well-characterized sample of Veterans, we sought to (1) clarify whether volumetric differences can be observed following mTBI and (2) explore associated brain-behavior relationships. Our sample included 73 Veterans with (n = 38) and without history of mild TBI (n = 35) who underwent structural magnetic resonance imaging (MRI) and were administered a computerized version of the Iowa Gambling Task (IGT). FMRIB’s Software was used to obtain whole brain and nucleus accumbens volume estimates. Analyses of covariance controlling for intracranial volume (ICV) and post-traumatic stress disorder (PTSD) symptoms were used to determine whether there were group differences in lateralized nucleus accumbens volumes. Partial correlations were used to explore brain-behavior associations across the groups. Results revealed that independent of ICV, the mTBI group displayed significantly lower volumes of the left nucleus accumbens volumes relative to Veterans without history of mTBI (p = .04). When PTSD was taken into account, this relationship became a trend (p = .08). Additionally, across both groups, partial correlations demonstrated that independent of ICV and PTSD, lower left nucleus accumbens volume was significantly associated with poorer performance on a test of reward-based decision making (IGT Total Raw Score, r = 0.25, p = 0.04). Our results show that the nucleus accumbens may be vulnerable to neurodegenerative changes following mild neurotrauma. Importantly, gray matter volumetric changes in this region may contribute to impaired decision making in the aftermath of TBI. Findings suggest a neuroanatomical basis or mechanism for reduced executive function which is frequently observed in individuals who have sustained even mild levels of brain trauma. Future studies integrating other biomarkers of neurodegeneration and inflammation may assist in clarifying the mechanisms underlying volumetric changes post-injury.
Abstract Title : 5-HT2AR Agonists May Cause an Impairment in Probabilistic Reversal Learning in Mice
Abstract : Disorders such as schizophrenia and autism spectrum disorder carry an array of cognitive detriments which include cognitive inflexibility. Identifying cortical receptors involved in impairment or overactivation along with their region specificity may uncover clinically applicable treatments. Probabilistic reversal learning tasks have been used as a model for identifying pharmacological interactions with 5-HTRs and their influence on learning and memory processes. We are investigating the function of 5-HT2A receptors and the effects of compounds that bind to these receptors. Based on our experiments so far, it seems that 5-HT2AR agonists impair probabilistic reversal learning performance. We found that 25-CN, a selective 5-HT2A agonist, impaired probabilistic reversal learning performance. 2,5-Dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A and 5-HT2C agonist, had no effect on probabilistic reversal learning performance on its own. It seemed that further investigation of DOI's effects on probabilistic reversal learning should be looked at, as its possible that the 5-HT2C interaction of DOI may have masked the effects of its 5-HT2A interaction. With DOI being an agonist of both 5-HT2A and 5-HT2C receptors, the administration of SER-082, a 5-HT2C antagonist, was administered and hypothesized to cause a blockade on the agonistic 5-HT2C effects of DOI. This blockade should unmask any 5-HT2A receptor interaction effects, which should be consistent with the results of 25-CN's interaction. This study is still in progress, with a trend in favor of the hypothesis being observed so far.
Abstract Title : Do the Acute Antipsychotic-Like Effects of Oxytocin Endure After Chronic Administration ?
Abstract : There is a pressing need for more efficacious treatments for schizophrenia (SCZ), and increasing evidence suggests that the neuropeptide oxytocin (OT) may be an effective treatment for SCZ. OT is a nine amino acid neuropeptide that, in addition to its known peripheral effects, regulates centrally-mediated behavioral and cognitive processes that are highly relevant to SCZ. Studies of patients with SCZ suggest that endogenous OT may have anti-SCZ properties and that dysregulation of the OT system may contribute to the symptoms of SCZ. These findings raise the exciting possibility that OT, or an OT analog, may be developed as a potential novel treatment for SCZ. Facilitation of prepulse inhibition of startle (PPI) is a behavioral assay that exhibits robust predictive validity for the therapeutic effects of (APDs). Consistent with the effects of APDs in rats, OT reverses natural PPI deficits exhibited by Brown Norway (BN) rats and PPI deficits produced by the psychotomimetics, amphetamine and dizocilpine. Recently, the APD efficacy of exogenous OT has been supported by several small randomized double blind clinical trials in patients with SCZ. The preliminary findings of adjunctive OT therapeutic benefit in SCZ augur very well for the possibility of developing OT, or an OT analog, as a much needed, novel treatment for SCZ. ADDITIONAL PRESENTERS: Benjamin Roberts, Ambar Avalos, Daquian Jin, Amrit Narwan.
Abstract Title : Development of a Noninvasive Blood-Based Molecular Test for Monitoring Disease Progression in Huntington’s Disease (HD)
Abstract : Huntington's disease (HD) is an inherited, neurodegenerative disorder characterized by cognitive, psychiatric, and motor dysfunction; however, the timing and rate of disease progression can be difficult to assess. Recently, cell-free (circulating) RNA (cf-RNA) has shown promise as a tissue-specific biomarker of cellular damage and degree of neurodegeneration. Working with San Diego-based company, we propose to track changes in the cell-free transcriptome as they correlate with disease progression. A small cohort of HD patients, of varying severity, and age-matched NC was assessed by sequencing to determine initial feasibility of monitoring the cell-free transcriptome in HD patients while simultaneously evaluating neuronal specific targets for further development. Initial findings demonstrated the ability to identify neuronal transcripts in these patients, and we are currently characterizing a larger cohort of samples across all three stages of Huntington’s disease, using our established protocol to identify gene targets that track with disease progression.
Abstract Title : Circadian Alterations Impact the Regulation of Insulin Degrading Enzyme in the Brain of Alzheimer's Patients
Abstract : Alzheimer disease (AD) is the most prevalent neurodegenerative disorder of the elderly, manifested by progressive memory loss and decline in cognitive functions, with underlying accumulation of amyloid-β (Aβ) and phosphorylated Tau in the neocortex and the hippocampus. Disturbance of circadian rhythmicity is a common ailment for more than 80% of AD patients, evidenced by altered sleep/wake cycles and exacerbated cognitive impairment during the evening (sundowning). Circadian rhythms coordinate physiology with the environment. About 15% of expressed genes are controlled by the circadian clock, including those involved in insulin metabolism. Disruption of core clock genes Per2 and Bmal1 leads to insulin resistance and diabetes in mice. Normal functioning of the insulin- signaling pathway is important for the maintenance of cognitive performance during aging and hyperinsulinemia, insulin resistance and diabetes increase the risk for AD. Still, the role of circadian deregulation in insulin balance and its contribution to AD pathology is underexplored. We investigated the role of the circadian protein BMAL1 in the regulation of the insulin-degrading enzyme (IDE), which participates in the clearance of both, insulin and Aβ. We hypothesized that alterations in BMAL1 transcription associated with AD may impair brain insulin metabolism via deregulation of IDE, aggravating neurodegeneration. We pro led frontal cortex samples from mild cognitively impaired (MCI) and severe AD patients in comparison to age and gender-matched healthy control subjects (N=66), evaluating IDE transcript and protein abundance as a function of the time of death. We detected signi cant alterations in BMAL1 expression in MCI and AD cases, with reduced amplitude of oscillation and advanced phase of expression. In addition, we observed circadian-like oscillation of IDE transcripts in control brains, but the rhythmic patterns of expression were signi cantly altered in MCI and AD cases for both transcription and protein levels, with changes in amplitude and peak expression time. Moreover, IDE alterations associated with aberrant insulin signaling, including increased IRS-1 phosphorylation in AD brains, and also correlated with cognitive decline. Lastly, in silico analysis of the IDE proximal promoter identi ed multiple canonical E- boxes, for the binding of BMAL1 to the DNA, supporting the role of this clock factor on the regulation of IDE. We conclude that alteration of brain circadian rhythms impact insulin metabolism and potentially Aβ deposition by deregulation of IDE expression, a pathway mediated by BMAL1.
Abstract Title : Characterization of forelimb or hindlimb corticospinal tract regeneration into neural progenitor cell graft after upper cervical spinal cord injury
Abstract : Recently, our lab demonstrated robust regeneration of corticospinal tract (CST) axons into neural progenitor cell (NPC) grafts placed in sites of mid-cervical spinal cord injury (SCI) (Kadoya et al., Nat Med 2016). We hypothesize that forelimb and hindlimb CST axons regenerate into NPC grafts placed in the mid-cervical SCI sites, since both systems are axtomized. To test this hypothesis, we grafted rat embryonic day 14 spinal cord-derived NPCs expressing green florescent protein (GFP) into a C3 dorsal column lesion site, and then injected AAV-expressing red florescent protein (RFP) into only the forelimb or hindlimb regions of the motor cortex. To our surprise, we found that few forelimb CST axons regenerate into NPC grafts one month later, whereas hindlimb CST axons robustly regenerate into these grafts at C3. In an effort to verify this unexpected result, we utilized highly specific, Cre-mediated tracing of CST axons projecting to either C6 or L3. We first injected AAV9 viral vectors expressing Cre-dependent Td-tomato into forelimb and hindlimb motor cortex, and then injected AAV9-Cre (which undergoes retrograde transport) into either C6 (N=4 animals) or L3 (N=4 different animals). This approach specifically enabled Td-tomato expression exclusively in forelimb- or hindlimb-projecting CST axons. Preliminary studies confirm rare regeneration of forelimb CST axons into NPC grafts, whereas regeneration of hindlimb CST axons is robust. An examination of mechanisms underlying this selective regeneration is underway, and will contribute to efforts to optimize and corticospinal regeneration after SCI.
Abstract Title : Target-specificity of auditory cortex projections
Abstract : Sensory information processed in the auditory cortex (AC) is directed to a variety of spatially and functionally distinct targets including contralateral AC, basolateral amygdala, inferior colliculus, striatum, and thalamus. These areas play distinct roles in sound-guided behavior and cognition. However, it is unclear whether the AC transmits the same auditory information to all target regions, or if specific sensory information is conveyed to different parts of the brain. To address this question, we used a retrograde tracer, cholera toxin B (CTB), conjugated to several different fluorescent markers to simultaneously visualize multiple subpopulations of AC projection neurons in the adult mouse brain. We find that AC pyramidal cells projecting to the inferior colliculus, a subcortical relay area, very rarely project to other targets. Similarly, only a small subset of pyramidal cells targeting the striatum also project to the contralateral AC. Taken together, our results suggest that individual AC neurons transmit information via distinct channels corresponding to the function of the downstream target area. We are currently using in vivo calcium imaging in awake mice to determine the sound-evoked response properties of pyramidal cells that project to different targets. Additionally, we are performing anterograde tracing to further characterize the spatial specificity of AC projections within known downstream regions and to potentially identify previously unreported targets. ADDITIONAL PRESENTER: Marissa Justen
Abstract Title : Apolipoprotein E-ε4 Genotype and Pulse Pressure Interact to Affect Cortical Thickness in Alzheimer’s Disease Vulnerable Regions in Veterans with Mild Traumatic Brain Injury
Abstract : Objective: Vascular, genetic, and environmental (e.g., mild traumatic brain injury [mTBI]) factors have all been shown to increase risk for development of Alzheimer’s disease (AD) in late life. Although there has been extensive research linking the APOE-ε4 allele and risk for AD risk in mTBI samples, it remains unclear how vascular risk might interact with the presence of the APOE-e4 allele in the expression of brain changes in participants with history of head-injury. Thus, the present study explored the independent and interactive effects of elevated vascular risk and APOE-ε4 positivity on cortical thinning in brain regions vulnerable to AD pathology in Veterans with history of mTBI. Participants and Methods: Participants included 48 Veterans (mean age: 32; range: 23-46) with history of mTBI who underwent magnetic resonance imaging (MRI), APOE genotyping, and blood pressure (BP) assessment. Pulse pressure (PP), an index of arterial stiffening, was derived from blood pressure values. FreeSurfer was used to generate cortical thickness values for brain regions of interest (ROIs). Results: Multiple linear regression, adjusting for age and PTSD, showed that APOE-ε4 positivity modified the relationship between PP and cortical thickness in medial orbitofrontal (p = .023), precentral (p = .091), supramarginal (p =.082), and precuneus (.045) regions. For each of these ROIs, lower PP was associated with greater cortical thinning in ε4-positive participants. Conversely, higher PP was associated with cortical thinning in these same regions in non-ε4 carriers. Conclusions: Results revealed that, even within a relatively young sample of participants with mild head trauma histories, vascular and genetic risk interact to affect cortical thickness in AD vulnerable regions. Specifically, mTBI participants with lower PP and who possessed at least one copy of the ε4 allele showed cortical thinning across several regions known to be predilection sites for AD pathology. However, contrary to our hypothesis, higher PP appeared to be protective against cortical thinning in APOE-ε4 carriers, but not non-carriers. While additional research is needed to clarify these findings, they add to a growing literature that highlights the complexity of vascular and genetic contributions to AD pathology following neurotrauma.
Abstract Title : Neural stem cell graft differentiation and interaction with host central nervous system after spinal cord injury
Abstract : Neural stem cells (NSCs) have the clinical potential to restore lost connections after spinal cord injury. In this study, human NSCs were transduced with green fluorescent protein (GFP) and embedded in fibrin matrices containing a growth factor cocktail. These human NSC grafts were then implanted into C5 lateral hemisection sites of spinal cord injury in immunodeficient rats. The subjects were euthanized at 1, 3, 6, 12, and 18 months post-implantation to study differentiation, migration, and maturation of graft-derived neurons and glia. We focused on the development of migrated glial cells from the NSC graft at 6, 12, and 18 months post-grafting by immunohistochemistry. The migrated human cells can be clearly observed at 6 months post- grafting and identified as glial progenitor cells at this early time point. Human cells migrated continuously at a rate of 2-3 mm per month and gradually matured as GFAP positive astrocytes. We also used a combination of immunohistochemistry and electron microscopy to observe how these migrating NSC-derived glial cells interacted with host neuron soma, axons, myelin, and blood vessels, over the same time period. Understanding how NSCs differentiate and interact with the host spinal cord will allow researchers and clinicians to optimize the use of NSC as a potential treatment for spinal cord injury, where clinical trials are currently ongoing.
Abstract Title : Evaluating Learned Behavior of Flies using aversive operant conditioning
Abstract : Learning and memory can be measured through the changes in behavior. Such changes can be used to quantify the learning outcome of an animal after training. In our studies, we used Drosophila melanogaster, or fruit fly, as a model to evaluate learning outcomes using behavioral changes in an aversive conditioning setting. Aversive operant conditioning uses punishment, such as heat, as a means of reinforcing particular behavior responses to the punishment. Heat is a natural punishment to the flies. The goal of the experiment is to measure the behavioral changes of the flies during conditioning. A laser increases the fly’s temperature when the fly is moving and the laser is turned off when the fly stops walking. The fly’s behavior is recorded before and after each training session.
Abstract Title : Lipocalin-2, Energy Homeostasis, and HIV-induced Neuronal Damage
Abstract : The development of combination antiretroviral therapy (cART) transformed human immunodeficiency virus-1 (HIV-1) from a fatal diagnosis to a chronic but manageable disease. However, the prolonged life expectancy brought by cART comes with another challenge for those diagnosed with HIV-1: brain injury and cognitive impairment. To help characterize the pathological mechanism of HIV-induced neuronal degeneration and brain injury, transgenic (tg) mice expressing HIV envelope protein gp120 in their central nervous system (CNS) have been developed. Through study of the gp120tg mice model, the acute phase protein lipocalin-2 (LCN2) was identified as one of the most up-regulated factors in gp120tg brains that may play a significant role in the progression of HIV-induced brain injury. Since the function of LCN2 in the context of viral infection is incompletely understood, gp120tg mice were cross-bred with a genetic knock out of lipocalin-2 (LCN2KO). When the LCN2KO and LCN2KOgp120tg mice were compared to wild-type and gp120tg mice of the same age, the LCN2KO and LCN2KOgp120tg mice appeared to be of heavier weight and increased body fat levels, and were observed to have significantly decreased brain mass. With this observation in mind and other studies identifying LCN2 as an adipokine with a role in energy metabolism in adipose tissue, a set of qPCRs was designed to determine if the absence of LCN2 influenced energy homeostasis in the brain. The results show a significant up-regulation of all tested energy homeostasis genes in mice with LCN2 knocked-out compared to wild-type, regardless of if the gp120 viral protein was present. This suggests that LCN2 plays a crucial role in maintaining energy homeostasis in the brain and in innate inflammation and injury response beyond HIV. A better understanding of the role of lipocalin-2 in pathological mechanisms underlying HIV-induced brain injury is expected to help the identification of new therapeutic targets and approaches. Supported by NIH R01 MH087332, MH104131 and MH105330
Abstract Title : Evaluation of Neural Stem Cell Impacts on Pain Modulation Following Spinal Cord Injury
Abstract : Neural progenitor cell (NPC) grafts have demonstrated successful spinal integration in spinal cord injury (SCI) models, projecting axons over long distances and forming functional relays in the central nervous system to improve motor outcomes (Lu et al., 2012; Kadoya et al., 2016). However, because neuropathic pain remains a common detriment to the quality of life of SCI patients, careful screening of cell therapies is necessary to ensure that NPC implantation does not exacerbate nor produce pain states. Such assessment is particularly relevant as previous reports have reported the development of neuropathic pain in SCI models following stem cell treatment (Hofstetter et al., 2005). To evaluate the effects of NPC grafts on neuropathic pain outcomes, behavioral responses to sensory stimuli were assessed in animals that received either a severe spinal cord lesion associated with the development of neuropathic pain in the absence of a graft or a mild cervical lesion that did not result in neuropathic pain. Immunohistological characterization of pain biomarkers in the cervical spinal cord was then conducted to correlate sensory data with glial proliferation and CGRP fiber sprouting. No significant differences in sensory pain outcomes nor biochemical analysis was detected between grafted and ungrafted injury models across tactile, heat, cold, and spontaneous pain modalities. These findings indicate that NPC therapies neither generate nor worsen post-SCI neuropathic pain states, supporting the feasibility of such grafting approaches for clinical translation.
Abstract Title : Investigation of kisspeptin neurons as mediators of stress induced disruption of reproductive function
Abstract : In female mice, the treatment of the stress steroid, corticosterone, inhibits both estrous cyclicity and the LH surge required for ovulation. These functions are regulated by the hypothalamic-pituitary-gonadal (HPG) axis which begins with GnRH signaling in the hypothalamus. We hypothesize that corticosterone inhibits this axis by acting directly on hypothalamic kisspeptin neurons. Kisspeptin neurons are key regulators of GnRH neurons and play an important role in regulating both LH pulsatility and the LH surge. Through immunofluorescence we found expression of glucocorticoid-receptors (GR) within kisspeptin neurons which confirmed that kisspeptin neurons can respond to corticosterone signaling. We are currently validating the knockdown of GR in kisspeptin neurons in the GR flox kisspeptin cre mouse model which, if validated, can be used to more thoroughly test this hypothesis. These findings can help us better understand the actions of corticosterone on the HPG axis and the mechanisms behind stress-induced disruption of reproductive function.
Abstract Title : Identification and classification of PACSIN-1 as a novel ligand for LRP1 in Schwann cells
Abstract : In the United States there are an estimated 20 million cases of peripheral neuropathy with symptoms ranging from numbness to burning pain. The majority of cases are caused by physical trauma. There are currently no catch-all treatments, and the mechanisms underlying natural repair mechanisms remain to be elucidated. In peripheral nervous system injury, Schwann cells act as "first-responders" by de-differentiating, migrating, proliferating and secreting growth factors. These processes are orchestrated by low-density lipoprotein receptor-related protein 1 (LRP1), an endocytic cell-signaling receptor for diverse ligands. LRP1 is up-regulated in Schwann cells (SC) after peripheral nervous system injury and orchestrates many elements of the SC repair program, including functional regeneration. To identify novel LRP1 ligands that may be responsible for activating LRP1 signaling in PNS injury, the Campana Lab applied a proteomics-based discovery approach. Injured peripheral nerves were immunoprecipitated with Fc-fusion proteins containing the extracellular ligand-binding domains of LRP1. The same samples were incubated with free Fc protein as a control. Consecutive analysis by tandem mass spectrometry (MS/MS) revealed that Protein kinase C and casein kinase substrate in neurons 1 (PACSIN-1) may be involved in the LRP1-triggered SC repair program. To determine whether PACSIN-1 activates LRP1-dependent cell signaling, primary rat SC cultures were treated with recombinant human PACSIN-1 at various concentrations. Cells were then lysed and subjected to immunoblotting for ERK1/2 activation. Preliminary results indicate that depending on concentration, PACSIN-1 potentially acts as an agonist for LRP1-dependent cell signaling. Future studies include: 1) identifying the time-course of PACSIN1-induced activation of ERK1/2; 2) confirming that PACSIN1 mediated activity in SCs is specifically mediated by LRP1 in vitro and in vivo. These studies will include use of the LRP1 antagonist receptor associated protein (RAP) and genetic silencing of LRP1 in SCs.