Browsing by Author "Chanda, Soham, committee member"
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Item Open Access A retinal contribution to chronic opioid-induced sleep/wake dysfunction(Colorado State University. Libraries, 2023) Bergum, Nikolas, author; Vigh, Jozsef, advisor; Myers, Brent, committee member; Hentges, Shane, committee member; Chanda, Soham, committee memberLight is among the most important environmental factors that regulate mammalian sleep/circadian behaviors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit environmental light information to key sleep/circadian centers in the brain through a process known as photoentrainment. Interestingly, past studies have revealed that ipRGCs express µ-opioid receptors (MORs), the primary molecular target for opioid analgesics. Furthermore, MOR agonists can directly inhibit ipRGC firing. Therefore, we hypothesize that opioid drugs acting on MORs expressed by ipRGCs could disrupt ipRGC-mediated regulation sleep/wake rhythms in response to environmental light/dark cycles. To test this idea, we need to confirm that morphine reaches the mouse retina following systemic delivery. To accomplish this, tissue (retina, brain and serum) was collected from mice following intraperitoneal morphine administration. Importantly, results from this study show that systemically administered morphine selectively accumulates in the mouse retina, but not the serum or the brain. To test the role that MORs expressed by ipRGCs play in opioid-induced dysregulation of sleep/circadian behaviors, we used mini-telemetry devices to assess how chronic morphine alters their sleep/wake behavior in mice. Importantly, we performed these experiments in wildtype mice along with mice lacking MORs exclusively in ipRGCs (McKO). Results from these studies reveal that McKO animals exhibit decreased morphine-induced locomotion compared to controls, which implicates MORs expressed by ipRGCs as a mediator of opioid-induced sleep-wake alterations. Finally, we tested whether ipRGCs developed cellular tolerance to MOR agonists following chronic exposure to morphine. The lack of cellular tolerance development at the level of solitary ipRGCs provides a potential cellular correlate for the persistent sleep/wake dysfunction commonly attributed to chronic opioid exposure. Taken together, these findings support the idea that opioid accumulation in the eye persistently activate MORs on ipRGCs, continuously altering the ability of ipRGCs to transmit light information to the brain's sleep/wake circuitry. This alteration in photic input to the brain could underlie some of the sleep/wake problems associated with long-term opioid use.Item Embargo Gene-targeted mouse models provide novel insights into strain diversity and interspecies transmission of chronic wasting disease(Colorado State University. Libraries, 2022) Sun, Julianna, author; Telling, Glenn, advisor; Ross, Eric, committee member; Chanda, Soham, committee member; Perera, Rushika, committee memberPrion diseases are fatal, transmissible neurodegenerative diseases that affect humans and other animals and are caused by the aberrant misfolding of the prion protein (PrP) to a disease-causing form. The term 'prion' was coined in 1982 by Stanley Prusiner to denote a small proteinaceous infectious particle which is now known to be the cause of scrapie in sheep, transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in cervids such as deer and elk. Additionally, humans can develop prion diseases via multiple routes – spontaneously in the case of sporadic Creutzfeldt-Jakob disease (CJD), inherited in the cases of fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker (GSS) syndrome, or acquired in the cases of variant CJD and Kuru. In addition to classical prion diseases, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Frontotemporal dementia have recently been classified as prion-like diseases due to similar protein misfolding mechanisms critical to these disease pathogeneses. Thus, the exploration of prion disease mechanisms has implications for a variety of neurodegenerative diseases. The main focus of this thesis will be the characterization of CWD strains and pathogenesis using mouse models of CWD. The disease was first described in Colorado in the 1960s in mule deer and rocky mountain elk and since then has expanded in has expanded in both geographical range and host species range including white-tailed deer, moose, red deer and reindeer. In North America, CWD has now been documented in 30 American states and three provinces in Canada. In addition to cases in North America, CWD has been identified in South Korea as a result of accidental transmission of subclinically infected cervids from Canada. In 2015, Norway reported a case of CWD in a herd of reindeer, and shortly after also reported cases of CWD in three free-ranging moose, marking the first cases of CWD in Europe. As a result, surrounding countries increased CWD surveillance, and Finland reported two cases of CWD in moose, and Sweden reported four cases of CWD in moose. At the time of writing, 20 reindeer, 11 moose, and two red deer in Norway, four moose in Sweden, and two moose in Finland have been diagnosed as CWD positive in Europe. The persistent spread of CWD raises both ecological and economical concerns thus the characterization of CWD pathogenesis is of utmost importance. Prions are unlike viral and bacterial pathogens in that their infectious component is entirely proteinaceous. The templated conversion of PrPC to PrPSc is driven by PrPSc imposing its infectious conformation onto PrPC. In other words, there are no primary structural differences between PrPC and PrPSc and thus higher order structural differences between PrPC and PrPSc must account for infectivity of PrPSc. This is confirmed by recently solved cryogenic-electron microscopy structures of PrPSc which show an insoluble, β-sheet rich protein structure, as opposed to the soluble, α-helical rich PrPC conformation. Though all heritable information is encoded in protein conformation, prions can exhibit strain characteristics similar to other pathogens. Strains are operationally defined by characteristics such as time to disease onset, clinical signs, and neuropathology. While these characteristics can be defined in the natural disease host, the use of the mouse bioassay has facilitated the ease of strain typing. Since the primary structure of mouse PrP is slightly different than cervid PrP, transmission of CWD to mice is generally inefficient. Our and other labs combat this by the design of transgenic mice expressing cervid-PrP. Specifically, the Telling lab designed prototype transgenic overexpressing cervid-PrP mice, expressing either glutamate (E) or glutamine (Q) at residue 226 of PrP. This is the only primary structural difference between CWD susceptible cervid species: North American elk express E226, while deer, moose, and reindeer express Q226. Our lab then designed gene-targeted mice which express endogenous levels of cervid-PrP, either E226 or Q226 expressing. These mice serve as a proxy to characterize CWD strain characteristics and lend insight into the pathogenesis of CWD. The work included in this thesis largely utilizes these mice to answer fundamental questions pertaining to CWD. These questions include: 1. What effect does the polymorphism at residue 226 of cervid PrP have on CWD pathogenesis? 2. How do the strain profiles of emergent cases of Nordic CWD compare to well-characterized cases of North American CWD? 3. Did CWD originate from a cross species transmission, and what is the potential for further cross species transmission of CWD?Item Open Access Glial inflammation as a key regulator and therapeutic target for prion disease(Colorado State University. Libraries, 2023) Hay, Arielle, author; Zabel, Mark, advisor; Moreno, Julie, advisor; Tjalkens, Ronald, committee member; Chanda, Soham, committee member; Santangelo, Kelly, committee memberPrion diseases are lethal neurodegenerative diseases characterized by the misfolding of the cellular prion protein, PrPC, into the infectious PrPSc. PrPSc accumulation in the brain contributes to the activation of microglia and the subsequent increase in reactive astrocytes, which together contribute to neuroinflammation. PrPSc aggregation triggers and leads to the dysregulation of a variety of cellular stress pathways, including the oxidative stress response, unfolded protein response, ubiquitin-proteosome system, autophagy and lysosomal degradation. Most critically, PrPSc contributes to neuronal toxicity and death, but the mechanism behind this is poorly understood. Prion diseases affect humans and a variety of mammalian species, with no available treatments. The majority of therapeutics developed to combat these diseases have targeted the prion protein itself. As these have been unsuccessful, it is time to turn our attention to treatments that target the cellular pathways and neuroinflammation caused by PrPSc accumulation in the brain. The overarching goal of this work is to identify glial-induced inflammation as a candidate for therapeutic intervention of prion diseases. We assessed the use of mesenchymal stromal cells (MSCs), which are potent regulators of inflammatory signaling and glial polarization, in cell culture and animal models of prion disease. Additionally, we investigate the role of a key inflammatory signaling pathway, Nuclear Factor-Kappa B (NF-κB) in microglial response to prion infection. Our findings both characterize contributions of specific glial cells to prion-induced inflammation, as well as uncovering novel targets for the treatment of prion diseases. First, we assessed the therapeutic potential of adipose-derived mesenchymal stromal cells (AdMSCs) in a cell culture model of glial prion infection. MSCs are known for their ability to migrate to sites of inflammation and produce immunomodulators. We evaluated the ability of cultured AdMSCs to respond to molecular factors present in brain homogenates from prion-infected animals. We found that these cells upregulate anti-inflammatory genes in response to both specific inflammatory cytokines and crude prion brain homogenates. Moreover, AdMSCs migrate towards prion brain homogenates in an in vitro model. Co-culturing AdMSCs with prion-treated BV2 cells or infected primary mixed glial cultures resulted in a significant decrease in markers of inflammation and disease-associated microglia and reactive astrocyte markers. These findings were independent of PrPSc, as AdMSCs had no effect on prion accumulation in mixed glial cultures. Collectively, these findings highlight AdMSCs as an intriguing candidate for modulating glial-induced inflammation in prion disease. Next, we evaluated AdMSCs in a mouse model of prion disease. Prior to delivery into prion-infected mice, AdMSCs were stimulated with TNFα, which we show increases their upregulation of anti-inflammatory molecules and growth factors. Stimulated AdMSCs were delivered intranasally to prion-infected mice every two weeks beginning from early in infection (10 weeks post-infection (wpi)) and ending late in infection (20 wpi). A cohort of mice was euthanized at various stages in infection, at 14 wpi, 16 wpi and 18 wpi. We show that AdMSCs are able to migrate throughout the brain when delivered intranasally, with the most cells being found in the hippocampus and thalamus. Although AdMSCs were not successful in improving behavior or increasing survival in prion-infected mice, they did induce changes in prion pathology at early time points in disease. A decrease was seen in inflammatory cytokines and markers of glial activation. No changes were seen in PrPSc accumulation or neuronal loss compared to untreated controls. However, at both 16- and 18 wpi, we identified significant changes in both glial numbers as well as morphology, indicating that AdMSCs attenuate reactivity in microglia and astrocytes. Together, these findings highlight AdMSCs as potent regulators of prion-induced glial inflammation, and warrants further investigation to optimize these cells as a treatment for prion disease. In addition to assessing therapeutics that decrease inflammation and reprogram glial cells to a homeostatic phenotype, we wanted to better characterize specific inflammatory pathways and understand how these were being regulated in glial cells in response to prion infection. NF-κB-related genes have long been identified in the brains of animal models with prion disease, but studies that have investigated its role in prion pathogenesis have focused on neurons and astrocytes. Microglia are critical innate immune regulators in the brain, and interact closely with both neurons and astrocytes to regulate inflammation and cell survival. Therefore, we saw an immediate need to characterize NF-κB signaling in microglia, and its contribution to prion-induced neuroinflammation. IKβ kinase (IKK) is a complex that responds to cell stressors and is critical for NF-κB signaling to occur. We utilized a primary mixed glial model containing wild-type (WT) astrocytes and IKK KO microglia. Upon infecting these mixed glial cultures with prions, we saw a drastic decrease in NF-κB-related genes compared to cultures containing WT astrocytes and WT microglia. Despite this, cultures containing IKK KO microglia still contribute neurotoxic signals that induce neuronal cell death. Moreover, we found that cultures with IKK KO microglia showed significantly more PrPSc accumulation, suggesting that these cells may have impaired autophagy. This work implicates microglial NF-κB-signaling and IKK as a potent inducer of inflammation and regulator of autophagy in prion disease.Item Open Access Interruption of neuron-microglia bidirectional communication to modulate cofilin:actin rod formation(Colorado State University. Libraries, 2022) Zoller, Maia, author; Bamburg, James, advisor; Chanda, Soham, committee member; Zabel, Mark, committee memberImmune responses in the central nervous system are mediated by microglia, whose responses to CNS threats can be replicated in vitro to study the role of microglia in the onset, progression, and treatment of neurodegenerative diseases. Previous work has identified a pathway common to neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, and HIV-Associated Neurocognitive Dementia in which the actin-severing protein, cofilin, forms a 1:1 bundle with actin making rod-shaped inclusions (rods) that can be found in the dendrites and axons of neuronal cells. This thesis focuses on developing methods for examining the role of primary microglia, activated by different factors, to secrete rod-inducing chemokines/cytokines or directly attacking neurons leading to neuronal death. Understanding both of these mechanisms is important in study of neuroinflammation and disease progression. Hemin, a hemoglobin metabolite, and alarmin, S100B, a astrocyte secreted, calcium binding protein, protein, were used to model the environment of intracerebral hemorrhage and general neuroinflammation respectively. Preliminary experimental results suggest blockage of actin-rod inducing signaling pathways via CXCR4/CCR5 receptor antagonist improves neuronal survival to both microglia conditioned medium and direct exposure the microglia-activating hemin or S100B. Further studies are in progress to obtain sufficient statistical data to verify these results.Item Open Access Sex dependent regulation of immune responses in ex vivo lung slices(Colorado State University. Libraries, 2023) Patlin, Brielle Honor, author; Snow, Chris, advisor; Tobet, Stuart, advisor; Chanda, Soham, committee memberSex differences in respiratory disease have been increasingly obvious over the last several decades ranging from asthma, to interstitial lung disease, to the common cold. One way that lung functions are dependent on sex is in their immune responses to disease. While there are many factors that contribute to the severity of immune responses and recovery from illness, neuroimmune signaling is an understudied aspect. This could be partially caused by the difficulty of studying individual neuronal circuits in the periphery in live animals and the lack of necessary cell types in organoid or cell line models. To study these processes in the lung, an organotypic model, known as a precision cut lung slice (PCLS) can be utilized to maintain intracellular and extracellular signaling. The first study herein addresses the role of the most prevalently produced neuropeptide in the lung on immune, neuronal, and epithelial populations in the lung. This abundant neuropeptide, calcitonin gene related peptide (CGRP), generates several sex dependent responses in PCLS. With CGRP treatment, number of B cells, size of neuroendocrine bodies (NEB), and surfactant protein C (SPC) granule are higher in female PCLS. However, the number of CGRP immunoreactive fibers in female PCLS is lower than in male PCLS. These sex related changes of lung cell behavior may partially explain some disease susceptibilities and are important factors to consider in pharmaceutical development for respiratory diseases. PCLS can be used to test pharmaceutically relevant substances and drug delivery systems. The recent pandemic has made it evident that better ways to deliver pharmaceuticals to the lungs are required. Regarding this problem, a focus on nasal drug delivery is important. The current leading technology in this type of delivery mechanism involves utilizing lipid nanoparticles and nasal administration. However, this is not the most efficient way to treat the lungs. Crystallized protein structures have begun to be used for purposes other than determining protein structure. In the second study included here, protein crystals were loaded with biologically relevant molecules to purposefully induce immune responses, without causing an immune response by themselves. This functionality has a variety of benefits, because a primary problem in respiratory disease is over activation of the immune response. In this study, crystals were customized by loading different molecules (e.g., lipopolysaccharide (LPS)) and the immune modulatory affects were observable in PCLS. This generated a sex dependent immune response in PCLS, which was less over time than slices treated with pure LPS indicating a differential response over 48 hours. Using protein nanocrystals for pharmaceuticals may provide new ways to target respiratory disease by nasal delivery with benefits over lipid nanoparticles.Item Open Access Stress during pregnancy leads to long-term consequences in the offspring(Colorado State University. Libraries, 2024) Sheng, Julietta Angelina, author; Tobet, Stuart, advisor; Smith, Bret, advisor; Myers, Brent, committee member; Hale, Taben, committee member; Chanda, Soham, committee memberNeuropsychiatric disorders encompass a wide range of conditions that affect neurological health and brain function and lead to disabilities worldwide. Such disorders include, but are not limited to, Major Depressive Disorder, schizophrenia, and anxiety disorders. Risk factors for developing neuropsychiatric disorders are multifaceted and can range from genetic predisposition, lifestyle, and environmental influences. Exposure to maternal stress is one type of environmental factor that can lead to changes in brain function and signaling pathways and increase susceptibility for related diseases. Maternal stress encompasses a diverse array of environmental stimuli, ranging from acute traumatic events to chronic or day-to-day life stressors. Maternal stressors, experienced by pregnant women, lead to overexposure of stress hormones in the developing fetus and impact short- and long-term neurological health the offspring. These studies evaluated developmental, neuroendocrine, and behavioral outcomes in offspring exposed to different models of maternal stress. Chapter 1 provided a brief history of stress, the development of the hypothalamic-pituitary-adrenal axis that regulates the stress response, and maternal-fetal interactions in stress regulatory systems and related behaviors. Chapter 2 evaluated several models of maternal stress, maternal high fat diet, maternal caloric restriction, maternal exposure to synthetic glucocorticoids. Although there were vast discrepancies between each type of maternal stress, one similarity was an activated immune response with elevated maternal cytokines. Therefore, Chapter 3 characterized a model of maternal immune activation using a toll-like receptor agonist, Resiquimod, that increased maternal and fetal cytokines, produced delayed developmental milestones and stress-related behavioral impairments in prepubertal (social-like) and adult (social-like, depressive-like, anxiety-like) offspring. Because these behavioral phenotypes are partially regulated by the paraventricular nucleus of the hypothalamus (PVN), Chapter 4 examined the neuroendocrine stress response and blood-brain barrier of the PVN. Data showed altered stress response accompanied by impaired blood-brain barrier integrity in the PVN of the adult offspring exposed to maternal injection of Resiquimod. Taken together, Chapters 2, 3, and 4 suggest maternal stress led to negative developmental, behavioral, and cellular pathologies indicative of neuropsychiatric-like disease. By teasing apart these specific programming mechanisms, we can better diagnose and treat progression of neuro-related disorders.Item Open Access The autism-associated loss of δ-catenin function disrupts social behavior(Colorado State University. Libraries, 2023) Mendez-Vazquez, Hadassah, author; Kim, Seonil, advisor; Tamkun, Michael, committee member; Myers, Brent, committee member; Chanda, Soham, committee memberSocial impairment is a key symptom of several neuropsychiatric disorders, including autism spectrum disorder (ASD), anxiety, depression, and schizophrenia. Despite the increasing prevalence of these disorders the physiological, cellular, and molecular factors underlying social dysfunction are still poorly understood. In humans, mutations in the δ-catenin gene have been linked to severe forms of ASD. δ-catenin is a post-synaptic scaffolding protein that is expressed in excitatory synapses and functions as an anchor for N-cadherin and the AMPA receptor (AMPAR) subunit GluA2 at the postsynaptic density. A glycine 34 to serine (G34S) mutation in the δ-catenin gene was identified in ASD patients and induces a loss of δ-catenin function, which may mediate ASD pathogenesis. The mechanism by which this G34S mutation causes loss of δ-catenin function to induce ASD remains unclear. Initial findings revealed that the G34S mutation increases glycogen synthase kinase 3β (GSK3β)-dependent δ-catenin degradation to reduce δ-catenin levels. Moreover, we found that mice possessing the G34S δ-catenin mutation have significantly reduced synaptic cortical δ-catenin and GluA2 levels. The G34S mutation was also found to differentially alter glutamatergic activity in cortical excitatory and inhibitory cells. Furthermore, G34S δ-catenin mutant mice exhibit markedly impaired social behavior, which is a characteristic feature of ASD. Most significantly, we found that inhibition of GSK3β is sufficient to reverse the G34S-induced loss of δ-catenin function in cells and mice. Altogether, our study reveals that the loss of δ-catenin function arising from the ASD-associated G34S mutation induces social dysfunction via disruptions in glutamatergic activity, and that GSK3β inhibition can reverse abnormal δ-catenin G34S-induced glutamatergic activity and social deficits.