Medical Neuroscience Department Theses and Dissertations
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Item Novel Strategies for the Prevention of Post-Stroke Epilepsy and Sudden Unexpected Death in Epilepsy Patients(2022-10) Adhikari, Yadav Prasad; Truitt, William; Witkin, Jeffrey M.; Gupta, Kunal; Brutkiewicz, Randy; Jin, XiaomingStroke is the second leading cause of mortality worldwide, accounting for 5.5 million deaths annually. In addition to its high mortality rate, stroke is the most common cause of acquired epilepsy. Three to thirty percent of stroke survivors develop post-stroke epilepsy. Although currently available therapies such as thrombolytics and mechanical thrombectomy prevent immediate mortality by restoring blood flow after stroke, these treatments do not target the cellular and molecular mechanisms that lead to post-stroke epileptogenesis. With the increasing number of stroke survivors, there is an urgent need for therapies that prevent epilepsy development in this population. Here, we showed that homeostatic plasticity is involved in the development of hyperexcitability after stroke and can be targeted to prevent the development of post-stroke epilepsy. Using two-photon calcium imaging, we found that homeostatic regulation leads to cortical hyperexcitability after stroke. We also found that activity enhancement by optogenetic and pharmacological approaches can target homeostatic plasticity to prevent post-stroke epilepsy. This study demonstrates the high translational potential of activity enhancement as a novel strategy to prevent post-stroke epilepsy through regulating cortical homeostatic plasticity. Sudden premature death is a leading cause of death in patients with medically refractory epilepsy. This unanticipated death of a relatively healthy person with epilepsy in which no structural or toxicological cause of death can be identified after postmortem analysis is referred to as sudden unexpected death in epilepsy patients (SUDEP). Respiratory failure during seizures is an important underlying mechanism of SUDEP. Here, we showed that LPS-induced peripheral inflammation is protective against SUDEP. This protection is mediated at least in part via enhancing serotonergic function in the brain stem. To the best of our knowledge, this is the first study demonstrating the relationship between peripheral inflammation and SUDEP prevention.Item Impact of PLCG2 Alzheimer's Disease Risk and Protective Variants on Microglial Biology and Disease Pathogenesis(2022-09) Tsai, Andy Po-Yi; Oblak, Adrian L.; Landreth, Gary E.; Lamb, Bruce T.; Liu, Yunlong; Mckinzie, David L.; Nho, KwangsikAlzheimer’s disease (AD) is typified by a robust microglial-mediated immune response. Genetic studies have demonstrated that many genes that alter AD risk are involved in the innate immune response and are primarily expressed in microglia. Among these genes is phospholipase C gamma 2 (PLCG2), a critical element for various immune receptors and a key regulatory hub for immune signaling. PLCG2 genetic variants are associated with altered AD risk. The primary objective of this thesis was to determine the role of PLCG2 in AD pathogenesis. We observed significant upregulation of PLCG2 expression in three brain regions of late-onset AD (LOAD) patients and a significant positive correlation of PLCG2 expression with amyloid plaque density. Furthermore, the differential gene expression analysis highlighted inflammatory response-related pathways. These results suggest that PLCG2 plays an important role in AD. We systematically investigated the impact of PLCG2 haploinsufficiency on the microglial response and amyloid pathology in the amyloidogenic 5xFAD mouse model. The results demonstrated that Plcg2 haploinsufficiency altered the phenotype of plaqueassociated microglia, suppressed cytokine levels, increased compact X34-positive plaque deposition, and downregulated the expression of microglial genes associated with immune cell activation and phagocytosis. Our study highlights the role of PLCG2 in immune responses; loss of function of PLCG2 exacerbates the amyloid pathology of AD. Genetic studies demonstrated that the hypermorphic P522R variant is protective and that the loss of function M28L variant confers an elevated risk for AD. Our results demonstrated that PLCG2 variants modulate disease pathologies through specific transcriptional programs. In the presence of amyloid pathology, the M28L risk variant impaired microglial response to plaques, suppressed cytokine release, downregulated disease-associated microglial genes, and increased plaque deposition. However, microglia harboring the P522R variant exhibit a transcriptional response endowing them with a protective immune response signature linked to their association with plaques and Aβ clearance, attenuating disease pathogenesis in an amyloidogenic mouse model of AD. Collectively, our study provides evidence that the M28L variant is associated with accelerated and exacerbated disease-related pathology, and conversely, the P522R variant appeared to attenuate disease severity and progression.Item Spinophilin Cell Type-Specifically Mediates Metabotrophic Glutamate Receptor 5-dependent Excessive Grooming(2022-09) Morris, Cameron W.; Truitt, William; Atwood, Brady; Baucum, Anthony J., II; Ma, Yao-Ying; McKinzie, DavidCompulsive and repetitive behaviors in obsessive-compulsive spectrum disorders (OCSDs) are associated with perturbations in the sensorimotor striatum. Repetitive behaviors are associated with cell type-specific adaptations in striatal direct- and indirect-pathway medium spiny neurons (dMSNs and iMSNs, respectively). Furthermore, preclinical models for understanding OCSDs, such as constitutive knockout of disks large associated protein 3 (SAPAP3), suggest repetitive motor dysfunction, such as excessive grooming, is associated with increased metabotropic glutamate receptor 5 (mGluR5) activity that increases dMSN function relative to iMSNs in the sensorimotor striatum. However, MSN subtype-specific signaling mechanisms that mediate mGluR5-dependent adaptations underlying excessive grooming are not fully understood. Reversible phosphorylation of mGluR5’s C-terminal domain is one mechanism to regulate mGluR5 signaling, however, unlike kinases, promiscuous phosphatases require targeting proteins to shuttle them into contact with their targets. Therefore, phosphatase targeting proteins may be intimately involved in mediating mGluR5-dependent striatal adaptions underlying repetitive behaviors, such as excessive grooming in SAPAP3 deficient mice. Spinophilin, a major striatal postsynaptic phosphatase targeting protein, regulates striatal function, mGluR5 signaling, and forms a protein-protein interaction with SAPAP3 that is increased by mGluR5 co-expression. Therefore, we hypothesized that spinophilin expression in striatal medium spiny neurons mediates mGluR5-dependent excessive grooming. To test this, we used a novel conditional spinophilin mouse line combined with functional, behavioral, and molecular approaches to elucidate spinophilin's MSN subtype-specific contributions to rodent excessive grooming behavior associated with increased mGluR5 function. We found that loss of spinophilin in either MSN subtype abrogated plasticity in the sensorimotor striatum associated with increased mGluR5 function and decreased two models of excessive grooming associated with increased mGluR5 function—SAPAP3 deficient mice and global administration of a mGluR5-specific positive allosteric modulator (VU0360172). Additionally, we found that spinophilin’s protein interaction with mGluR5 correlates with grooming behavior and loss of spinophilin shifts mGluR5 interactions from lipid-raft associated proteins toward postsynaptic density proteins implicated in psychiatric disorders. Collectively, these results identify spinophilin as a novel striatal signaling hub molecule in MSNs that MSN subtype-specifically mediates striatal adaptations associated with repetitive motor dysfunction in psychiatric disorders.Item Cell-Specific Spinophilin Function Underlying Striatal Motor Adaptations Associated with Amphetamine-Induced Behavioral Sensitization(2022-07) Watkins, Darryl Shumon; Yamamoto, Bryan K.; Atwood, Brady K.; Baucum, Anthony J. II; Hudmon, Andy; Logrip, Marian L.Striatal-mediated pathological disease-states such as Obsessive-Compulsive Disorder (OCD), Parkinson’s Disease (PD), and psychostimulant drug addiction/abuse are coupled with distinct motor movement abnormalities. In addition, these disorders are associated with perturbed synaptic transmission. Proper synaptic transmission is critical for maintaining neuronal communication. Furthermore, in many striatal-dependent disease-states, the principle striatal neurons, medium spiny neurons (MSNs), exhibit differential perturbations in downstream signaling. Signal transduction pathways that are localized to the glutamatergic post-synaptic density (PSD) of GABAergic MSNs regulate protein phosphorylation in a tightly controlled manner. Alterations in the control of this phosphorylation in striatal MSNs are observed in myriad striatal pathological diseasestates and can give rise to perturbations in synaptic transmission. While serine/threonine kinases obtain substrate specificity, in part, by phosphorylating specific consensus sites, serine/threonine phosphatases such as protein phosphatase 1 (PP1) are much more promiscuous. To obtain substrate selectivity, PP1 associates with targeting proteins. The major targeting protein for PP1 in the PSD of striatal dendritic spines is spinophilin. Spinophilin not only binds PP1, but also concurrently interacts with myriad synaptic proteins. Interestingly, dopamine depletion, an animal model of PD, modulates spinophilin protein-protein interactions in the striatum. However, spinophilin function on basal striatal-mediated motor behaviors such as the rotarod or under hyperdopaminergic states such as those observed following psychostimulant-induced behavioral sensitization are less well characterized. To elucidate spinophilin function more specifically, we have generated multiple transgenic animals that allow for cell type-specific loss of spinophilin as well as cell-specific interrogation of spinophilin protein interactions. Here, I report the functional role of spinophilin in regulating striatal mediated motor behaviors and functional changes associated with amphetamine-induced locomotor sensitization. In addition, we define changes in spinophilin protein-protein interactions that may mediate these behavioral changes. Furthermore, global loss of spinophilin abrogates amphetamine-induced sensitization and plays a critical role in striatal motor learning and performance. The data suggest that the striatal spinophilin protein interactome is upregulated in MSNs following psychostimulant administration. In addition, loss of spinophilin changes protein expression in myriad psychostimulant-mediated striatal adaptations. Taken together the data suggests that spinophilin’s protein-protein interactions in the striatum are obligate for appropriate striatal mediated motor function.Item Defining Inner Ear Cell Type Specification at Single-Cell Resolution in a Model of Human Cranial Development(2022-07) Steinhart, Matthew Reed; Meyer, Jason S.; Koehler, Karl R.; Herbert, Brittney-Shea; Landreth, Gary E.; Shearer, A. Eliot; Yates, Charles W.Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue.Item MicroRNA Regulation of Key Proteins Involved in Alzheimer's Disease Pathogenesis(2022-06) Wang, Ruizhi; Du, Yansheng; Lahiri, Debomoy K.; Kim, Jungsu; Reeves, Cristian A. Lasagna; Zhou, Feng C.Alzheimer’s disease (AD) is a neurodegenerative disease histopathologically characterized by the coexistence of amyloid plaques and neurofibrillary tangles, mainly consisting of amyloid β peptides hyperphosphorylated tau proteins, respectively. Multiple proteins and pathways are involved in the pathogenesis of AD, including Aβ precursor protein (APP), β-site APP-cleaving enzyme (BACE1), neprilysin, endothelin converting enzyme (ECE), repressor element-1 silencing transcription factor (REST), microtubule-associated protein tau, glycogen synthase kinase, and pro-inflammatory cytokines. However, how these proteins and pathways are dysregulated and converge in AD pathogenesis remains unclear. Genetic, epigenetic and environmental factors play important roles in disease progression. MicroRNAs (miRNAs), a group of small noncoding RNAs, are important epigenetic regulators that participate in AD development. We have identified three miRNAs capable of targeting several proteins in different AD-related pathways: miR-181-5p, miR-153-3p and miR-101-3p. We tested miR-181 activity with recombinant reporter gene- MME 3’-UTR constructs. All four miR-181-5p (miR-181a, miR-181b, miR-181c and miR-181d) sequences downregulated the reporter signal. Human differentiated neural cells were transfected with miR-181d-5p mimics. miR-181d-5p treatment significantly reduced MME mRNA levels, protein levels and enzyme activity. In addition, miR-181d-5p increased tau and phosphorylated tau levels proportionally. We further demonstrate that miR-153-3p reduced REST 3’-UTR activities, mRNA and protein levels in multiple human cell lines. Moreover, we show that miR-153-3p, by knocking down REST protein, induces apoptosis in HeLa cells but not differentiated neural cells. In addition, miR-153-3p regulates neuronal differentiation in neuronal stem cells, potentially via REST knockdown. We further found that miR-153 levels were correlated with a reduced likelihood of developing AD. Last, we demonstrated that miR-101-3p reduced ECE1 and GSK3β protein levels in multiple cell lines. miR-101-3p increased REST and pro-inflammatory cytokine secretion in microglia cells. In sum, we tested the hypothesis that miRNAs can serve as the master regulator of AD pathogenesis.Item Aberrant Neural Activity in Cortico-Striatal-Limbic Circuitry Underlies Behavioral Deficits in a Mouse Model of Neurofibromatosis Type 1(2022-05) Drozd, Hayley Paulina; McKinzie, David L.; Clapp, D. Wade; Shekhar, Anantha; Lukkes, Jodi L.; Lapish, Christopher L.; Block, Michelle L.Nearly 18% of children are diagnosed with developmental disabilities. Autism spectrum disorders (ASDs) and attention deficit hyperactivity disorder (ADHD) are increasingly common developmental disabilities, but neither is well understood. ADHD and ASD are both prevalent in the genetic disorder Neurofibromatosis type 1 (NF1) which impairs the Ras-MAPK/ERK pathway through mutation of the neurofibromin gene (NF1+/−). More broadly, syndromic forms of developmental disorders are often caused by mutations of proteins in pathways interconnected with Ras including TSC1/2, FMR1, and SynGAP. Of NF1 patients, around 30-50% are diagnosed with ASDs and more than 60% with ADHD. These studies are the first to show that male mice haploinsufficient for the Nf1 gene (Nf1+/−) exhibit deficits in behavioral inhibition in multiple contexts, a key feature of ADHD. They exhibit hyperactivity and impulsivity in an open field, delay discounting task, and cliff avoidance reaction test, rescuable through treatment with the clinically effective ADHD drug, guanfacine (α2A adrenergic receptor agonist). Previous experiments in our lab identified social deficits including deficits in consolidation of social memory. Using optogenetics and awake behaving electrode recordings, we explored the role of the cortico-striatal-limbic circuitry in impulsivity and in social deficits in male Nf1+/− mice. Manipulation of the prefrontal cortex, nucleus accumbens, or basolateral amygdala through optogenetics rescued social deficits. These studies are the first to record brain activity in a preclinical model of NF1 during impulsive behavior, finding broad spectrum changes across slow, delta, theta, and gamma oscillatory frequencies and decreased synchrony of the prefrontal cortex and nucleus accumbens during a delay discounting task. Overall, Nf1+/− male mice with deletion of a single NF1 gene recapitulate cognitive phenotypes of NF1 patients and are a useful model system to identify alterations in neural circuitry associated with ASD and ADHD.Item Characterization of Cerebral Blood Flow in Older Adults: A Potential Early Biomarker for Alzheimer's Disease(2022-04) Swinford, Cecily Gwinn; Risacher, Shannon L.; Saykin, Andrew J.; Apostolova, Liana G.; Wu, Yu-Chien; Gao, SujuanOver 5 million older adults have Alzheimer's disease (AD) in the US, and this number is projected to double by 2050. Clinical trials of potential pharmacological treatments for AD have largely shown that once cognitive decline has occurred, targeting AD pathology in the brain does not improve cognition. Therefore, it is likely that the most effective treatments for AD will need to be administered before cognitive symptoms occur, necessitating a biomarker for the early, preclinical stages of AD. Cerebral blood flow (CBF) is a promising early biomarker for AD. CBF is decreased in individuals with AD compared to their normally aging counterparts, and it has been shown that CBF is altered in mild cognitive impairment (MCI) and earlier stages and may occur prior to amyloid or tau aggregation. In addition, CBF can be measured using arterial spin labeled (ASL) MRI, a noninvasive imaging technique that can be safely repeated over time to track prognosis or treatment efficacy. The complex temporal and spatial patterns of altered CBF over the course of AD, as well as the relationships between CBF and AD-specific and -nonspecific factors, will be critical to elucidate in order for CBF to be an effective early biomarker of AD. Here, we begin to characterize the relationships between CBF and risk factors, pathologies, and symptoms of AD. Chapter 1 is a systematic review of published literature that compares CBF in individuals with AD and MCI to CBF in cognitively normal (CN) controls and assesses the relationship between CBF and cognitive function. Chapter 2 reports our original research assessing the relationships between CBF, hypertension, and race/ethnicity in older adults without dementia from the the Indiana Alzheimer’s Disease Research Center (IADRC) and Alzheimer’s Disease Neuroimaging Initiative (ADNI). Chapter 3 reports our original research assessing the relationships between CBF and amyloid beta and tau aggregation measured with PET, as well as whether hypertension or APOEε4 positivity affects these relationships, in older adults without dementia from the IADRC. Chapter 4 reports our original research assessing the relationship between the spatial distribution of tau and subjective memory concerns.Item Prostaglandin-Mediated Reinstatement of Drug Taking After Alcohol Drinking by Female Adolescent Rats(2022-04) Kline, Hannah L.; Engleman, Eric A.; Atwood, Brady K.; McKinzie, David L.; Truitt, William A.; Yamamoto, Bryan K.Adolescent alcohol abuse is a global problem that initiates lifelong addiction. Alcohol use during adolescence is associated with subsequent Meth dependence in humans. Specifically, female adolescents are particularly vulnerable to serial alcohol and Meth use. However, it is unknown if prior voluntary alcohol drinking impacts subsequent Meth-taking in female adolescent rats. Both alcohol and Meth increase the prostaglandin synthesis enzyme cyclooxygenase-2 (COX-2) in the brain but the effect of serial exposure to alcohol and Meth on COX-2 has not been determined. The first study uses a novel method of serial voluntary alcohol drinking and Meth self-administration in female adolescent rats to model human patterns of co-abuse. Prior alcohol drinking did not affect subsequent Meth self-administration, but it reduced the cue-primed reinstatement of Methseeking after abstinence from Meth. Rats with a history of adolescent alcohol drinking also had increased COX-2 in the dorsal striatum, regardless of subsequent Meth selfadministration. These findings demonstrate that a history of adolescent alcohol drinking does not alter Meth self-administration but persistently reduces cue-primed Meth seeking and increases COX-2 after prolonged abstinence from alcohol. To further examine the role of COX-2 in alcohol drinking, the second study found that adolescent alcohol drinking not only increased COX-2 after four weeks of alcohol abstinence, but also increased endothelin-1 (ET-1) and prostaglandin E2 (PGE2) in the dorsal striatum. Furthermore, adolescent alcohol drinking increased alcohol drinking after abstinence, and this increase was attenuated by treatment with the COX-2 inhibitor nimesulide during abstinence. Antagonism of the interaction between PGE2 and its receptor 1 (EP1) also attenuated the increase in relapse drinking and restored alcohol drinking to the rate of alcohol naïve rats. Overall, these experiments identified a prostaglandin-mediated mechanism that is a putative target for the treatment of alcohol relapse following abstinence in individuals with a history of adolescent alcohol abuse.Item Air Pollution Exposure and the Lung-Brain Axis: Implications for Alzheimer's Disease(2022-03) Greve, Hendrik Jacob; Oblak, Adrian; Block, Michelle; Nass, Richard; Landreth, GaryAlzheimer’s disease (AD) is a devastating neurodegenerative disease that is expected to affect approximately 6.2 million Americans. Despite its high prevalence, the mechanisms underlying AD remain poorly understood. In recent years, increasing reports indicate that exposure to urban air pollution is a risk factor for the development of AD. However, the mechanistic underpinnings of this association are not well studied. Rats exposed to diesel exhaust (DE) showed neuroinflammation and impaired expression of TREM2 and disease-associated microglia (DAM), a cell subtype hypothesized to play beneficial roles during neurodegeneration, markers. Microglia in the cortex of rats exposed to DE, also showed decreased association with the vasculature, providing a novel link between the microglia and the brain vasculature. Examining the functional role of TREM2 during DE exposures, Trem2-/- mice showed an altered pro-inflammatory profile in both the brain and lungs in response to DE particles as well as altered phagocytic oxidase related gene expression. Examining another prominent component of air pollution, ozone (O3), in a mouse model of AD, it was discovered that subchronic O3 exposure exacerbates amyloid pathology through impaired microglial-plaque association in 5xFAD mice. 5xFAD mice exposed to O3 also showed increased expression of pro-inflammatory cytokines, increased markers of dystrophic neurites, and decreased expression of key acetylcholinergic pathway components. Examining the peri-plaque microenvironment, it was discovered that O3 dysregulates key DAM proteins and amyloid processing proteins. In the lung, it was found that O3 exacerbated immune cell infiltration in 5xFAD mice compared to WT controls, suggesting that ongoing amyloid pathology regulates pulmonary immune response to air pollution. To examine how O3-induced pulmonary immune responses may be signaling to the CNS, we examined the serum of 5xFAD mice, where HMGB1, VEGF, and IL-9 were upregulated. Injection of rHMGB1 into mice showed similar gene changes to 5xFAD mice exposed to O3, along with impaired Trem2 expression. Using a peripheral myeloid specific knock-out model of HMGB1, we also show that HMGB1 regulates O3-induced Trem2 expression impairment. Taken together, these data support that air pollution exposure impairs TREM2, DAM cells, and the microglial plaque response through a bidirectional lung-brain axis to exacerbate AD-like pathology.