Browsing by Author "Zabel, Mark, advisor"
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Item Open Access Chronic wasting disease strain diversity, distribution and transmission(Colorado State University. Libraries, 2021) Wagner, Kaitlyn, author; Zabel, Mark, advisor; Avery, Anne, committee member; MacNeill, Amy, committee member; Moreno, Julie, committee memberChronic wasting disease (CWD) is an invariably fatal prion disease affecting captive and free-ranging cervids, including white-tailed deer, mule deer, moose, elk and reindeer. Since the initial discovery of the disease in the 1960's, CWD has spread across the US and Canada, South Korea, and, most recently, Europe. While some outbreaks of CWD were caused by transport of infected animals from endemic regions, the origin of CWD in other epizootics is unclear and not all outbreaks have been characterized. Previous studies have shown that there are multiple strains of CWD; however, the continuous spread and the unclear origin of several outbreaks warrant continued surveillance and further characterization of strain diversity. Moreover, studies implicating extraneural prions as more zoonotic motivated us to examine within-host prion strain diversity. The overarching goal of the work presented here was threefold: 1) address CWD strain differences between lymphoid and brain tissue from the same animal, 2) assess if there are any differences in CWD from either within or between contiguous and non-contiguous outbreaks and 3) address aspects of plant-vectored CWD transmission. The work presented here has important implications for understanding strain diversity within and between deer, as well as identifies samples that appear to be novel strains that warrant follow up assessment. Finally, we show how plants may be playing a role in vectoring infectious prions shortly after exposure. This research has important implications for our understanding of prion strain diversity and distribution as well as adds insight to plant-vectored prion transmission. First, we assessed differences between lymph node-derived and brain-derived prions from within the same animal to characterize strain differences within a single animal. To do this, we assessed isolates using biochemical techniques including electrophoretic mobility, glycoform ratio and conformational stability. Interestingly, we found that there were biochemical differences between lymph node and brain isolates, novel intermediate conformations of the prions in the brain (but not the lymph node) and increased variability in the lymph node-derived prions. Collectively, these results suggest that there are more diverse prion strains in the periphery and are distinct from neurological prions. The research discussed here advances our understanding of the differences between lymph node-derived and brain-derived prions. In addition to within-host strain comparisons, we also wanted to assess biochemical strain differences from naturally infected cervid species. Numerous studies have examined CWD strains upon passage into transgenic mouse models. For the purposes of our research, we wanted to examine CWD strains from the natural host for a number of reasons: 1) bioassay is expensive and time consuming, making strain characterization challenging, 2) research indicating that host factors other than PrPC may be influencing strain characteristics and 3) to determine if we could detect dramatic biochemical differences in strains, thereby providing an easier method to determine CWD strain prevalence in cervid populations without bioassay. Because the origin of CWD is unknown and some outbreaks of CWD have no clear exposure/connection to ongoing CWD outbreaks, this research would provide insight into the evolution and origin of CWD. Here, we show that there are some cases of CWD that present with novel biochemical characteristics that distinguish them from other CWD isolates. These instances suggest a new strain has emerged or that there is differential evolution in these subpopulations. Importantly, this work highlights that there is a lot more variability CWD biochemical characteristics than previously described. As a part of the strain typing project, two samples were received from captive white-tailed deer in Texas. These samples immediately proved to be a challenge to work with because they were behaving in an unusual way in our biochemical strain typing assays. In short, these isolates behaved in strange ways depending on the detergent class with which they were being digested. Because there was no known introduction of CWD to this captive herd, we were suspicious that we were seeing a novel strain of CWD. Isolates were passaged into cervid and human PrP mice. Upon passage, these isolates looked like classical CWD in Tg33 mice and, fortunately, don't appear to have any zoonotic transmission potential into human PrP mice. Importantly, this work highlights that CWD can present in a unique way in a cervid host but cause a classical-type disease in transgenic animals. Finally, we examined the role of plants to transmit CWD. Previous research implicated plants as having a possible role as a vector in prion transmission. We built upon this previous research by using CWD prions rather than hamster prions and a different plant model. The research presented here will show that plants are able to uptake prions shortly after exposure, but that these prions are no longer detected by 72 h. The work presented here implicates plants as potential CWD vectors in the short term.Item Open Access Crossing the blood-brain barrier: siRNA treatment for prion diseases(Colorado State University. Libraries, 2017) Bender, Heather Rose, author; Zabel, Mark, advisor; Telling, Glenn, committee member; Gustafson, Daniel, committee member; Bamburg, James, committee member; Dow, Steve, committee memberProtein misfolding diseases such as prion diseases, Alzheimer's disease, and Parkinson's disease, are fatal neurodegenerative diseases caused by a misfolded protein. There are no known therapeutics that extend survival times of afflicted individuals with these diseases. An attractive therapeutic option for protein misfolding disorder is RNA interference, which uses either short hairpin RNA or small interfering RNA (siRNA) to target a specific mRNA for degradation that results in a reduction of protein levels. The reduction of a target mRNA/protein can result in a decrease of misfolded protein in the central nervous system (CNS). However, crossing the blood-brain barrier remains the main challenge for developing RNA interference therapeutics to the CNS. Liposomes are commonly utilized to deliver siRNA to peripheral sites and are being investigated for their ability to deliver siRNA to the brain. We have previously reported on a new liposome delivery system that delivered siRNA targeted towards the cellular prion protein, PrPC, to mouse neuroblastoma cells. PrPC is a normal host cellular protein that misfolds into a protease resistant isomer, PrPRes, which leads to the development of prion diseases. We call these siRNA delivery vehicles: liposome-siRNA-peptide complexes (LSPCs). LSPCs are targeted towards the CNS using a small peptide from the rabies virus glycoprotein, called RVG-9r. In the second chapter of this dissertation, we show that an intravenous injection of LSPCs results in a 40-50% reduction of neuronal PrPC. Upon injection of LSPCs, we observed that half of all treated mice had PrPC siRNA targeted towards the area of the brain several hours after injection. However, we also observed a clearance of PrPC siRNA by the kidneys in the other half of LSPCs-treated mice. Therefore, we designed two other liposomal delivery vehicles that would allow us to encapsulate the siRNA in the liposome and covalently link RVG-9r to the outside of the liposome. We also added PEG lipids to these new delivery vehicles to extend the circulation half-life of the liposomes. We call these additional delivery vehicles peptide-addressed liposome-encapsulated therapeutic siRNA (PALETS). The two PALETS formulations include one cationic (DOTAP [1,2-dioleoyl-3-trimethylammonium-propane]) PALETS and one anionic (DSPE [1,2-Distearoyl-sn-glycero-3-phosphoethanolamine]) PALETS. We have utilized the cation protamine sulfate to encapsulate the siRNA within the anionic PALETS. The addition of protamine sulfate to the siRNA resulted in an encapsulation efficiency of 80-90% in DSPE PALETS. Four days after treatment with LSPCs and PALETS, LSPCs have the biggest decrease in neuronal PrPC on the cellular surface, while DOTAP PALETS have the greatest reduction of PrPC-positive cells. DSPE PALETS showed no statistical difference between the treated and untreated mice at this time point; however, two of the three treated mice did have a decrease in their neuronal PrPC, indicating that this delivery vehicle is able to deliver PrPC siRNA to the brain. There was no reduction in mRNA levels of any of the treated mice in the brain but the DOTAP LSPCs and DOTAP PALETS resulted in a 2-fold decrease of PrPC mRNA levels in the kidney, while DSPE PALETS resulted in a 2-fold increase of PrPC mRNA levels in the same organ. The first therapeutics for prion diseases targeted the mechanism of conversion between PrPC and PrPRes. These therapeutics were successful in decreasing the amount of PrPRes in vitro but they had limited success in vivo. Challenges of these therapeutics included toxicity, inability to cross the blood-brain barrier, strain specificity, and/or failure to affect survival times. PrPC became an attractive therapeutic option when it was shown that PrP-null mice did not develop any outward phenotypic differences from the removal of PrPC. Our LSPCs, with PrPC siRNA, reduced the amount of PrPC protein and PrPC mRNA levels in mouse neuroblastoma cells. This reduction in PrPC resulted in a concomitant decrease of PrPRes and a 'curing' of the prion-infected cells. In the third chapter of this dissertation, we have treated two different mouse models with our LSPCs at different time points to assess the pharmacodynamics of the treatment. In vivo live imaging followed by flow cytometry revealed delivery of PrPC siRNA to the brain one hour after intravenous injection. The LSPCs resulted in a decrease of neuronal PrPC in a C57Bl/6 mouse model at 24, 48 hours, and 4 days after treatment. A decrease in neuronal PrPC was also observed in a CD1 mouse model at 4 and 15 days after treatment. Surprisingly, mRNA levels did not always concur with the protein level data. At certain time points, the mice with the biggest decline in PrPC protein had the greatest increase of PrPC mRNA. Off-target effects were observed in the kidney, which might have been caused non-specifically by LSPCs treatment and not by the PrPC siRNA. We also show that PrPC protein levels decrease by 70% in prion-infected mice after three consecutive LSPCs treatments spaced two weeks apart. Analysis of mRNA levels of these mice after three treatments revealed a simultaneous reduction in PrPC mRNA levels. Several researchers have shown a reversal in prion neuropathology that results after decreasing the amount of PrPC, either by a Cre/loxP system or short hairpin RNA. Therefore, we treated prion-infected mice with our LSPCs treatment targeting PrPC. Two treatment studies were conducted to determine the optimal dosing regimen of LSPCs treatment. The first study treated prion-infected mice with LSPCs every two weeks starting at 120 days post inoculation and the second study treated mice with LSPCs every 3-5 weeks starting at 120 days post inoculation. The mice were intraperitoneally inoculated with a low dose of RML-5 prions to simulate a more natural prion infection. Unfortunately, in the fourth chapter of this dissertation we show that neither of the dosing regimens resulted in an increase in survival times of prion-infected mice. The mice in these two dosing studies were also subjected to burrowing and nesting behavioral tests to determine if LSPCs treatment improves behavioral outcomes. We show that LSPCs treatment every two weeks improves behavior scores at 141 and 169 days post inoculation in some treated groups. This improvement in behavior indicates that, while the LSPCs treatment are not affecting survival times, they are improving behavioral outcomes of prion-infected mice. Surprisingly, three of the uninfected, treated controls died immediately after LSPCs treatment of an apparent Type III hypersensitivity. Therefore, we performed ELISAs to measure the immune response towards the RVG-9r peptide. Several groups of treated mice in the terminal dosing studies had increased levels of IgG against RVG-9r compared to the infected, untreated control. In another study, it was revealed that three total IgG levels against RVG-9r increased after three subsequent LSPCs treatments spaced two weeks apart. We also assessed the amount of PrPRes in the brains and spleens of LSPCs-treated mice. Using the protein misfolding cyclic amplification assay, we determined that LSPCs treatment causes an increase in PrPRes levels in the brain after one to six LSPCs treatments. No trends can be seen in the spleen. Taken together these results indicate that the current LSPCs formulation using RVG-9r and PrPC siRNA result in an immune response that may interfere with any benefits of the treatment. Another explanation for these results is that PrPC may be tightly regulated at the transcriptional level, so the cell may try to return the mRNA/protein levels to normal by increasing PrPC mRNA when it detects a decrease in PrPC mRNA or protein levels. The increase in PrPC may be the cause of the increase of PrPRes observed in these studies. Therefore, transiently decreasing PrPC via siRNA may not be the best therapeutic option available. It is recommended that more studies are undertaken to further elucidate the transcriptional regulation and immune response towards the LSPCs treatment. LSPCs will need to be further modified to become a viable therapeutic option for prion diseases.Item Open Access Early peripheral immunological events dictate chronic wasting disease(Colorado State University. Libraries, 2013) Michel, Brady, author; Zabel, Mark, advisor; Hoover, Ed, committee member; Dow, Steve, committee member; Ross, Eric, committee memberChronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, like scrapie, has been shown to involve the immune system, which most likely contributes to their relatively proficient horizontal and environmental transmission. While CWD prions probably interact with the innate immune system immediately following peripheral exposure, little is known about this initial encounter. In the first chapter of this dissertation we examined initial events in lymphotropic and intranodal prion trafficking by tracking highly enriched, fluorescent CWD prions from infection sites to draining lymph nodes. We observed biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. CWD prions rapidly reached draining lymph nodes in a cell autonomous manner within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) showed a strong dependence on Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells comprised the majority of prion-bearing cells in the mediastinal lymph node by six hours. As most B cells are mainly located in the follicles, acquisition of prions by these cells most likely occurred through interaction with resident DCs, subcapsulary sinus macrophages, or directly from the follicular conduit system. These data highlight a novel mechanism of cell autonomous prion transport, and a vital role for B cells in intranodal prion trafficking. Upon entry into the draining lymph nodes, prion accumulation and replication on follicular dendrtic cells (FDCs) is greatly facilitated by the complement system. Complete elimination of CD21/35 significantly delays splenic prion accumulation and terminal prion disease in mice inoculated intraperitoneally with mouse-adapted scrapie prions. In the second chapter of this thesis we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. Ablation of complement receptors CD21/35 greatly diminished splenic prion accumulation and replication throughout the course of disease, similar to CD21/35 deficient murine PrP mice infected with mouse scrapie. Mice with deficiencies in CD21/35 showed a reduction in severity of neuropathology and deposition of misfolded, protease-resistant PrP associated with CWD. Prion infection resulted in translocation of CD21/35 to lipid rafts in B cells, and FDC expression of CD21/35 mediated a strong germinal center response that may be conducive to prion amplification. Complement component C3 is a central protein in the complement system whose activation is essential for the elimination of infectious pathogens. C3 is the most abundant complement protein, being found in the blood at physiological concentrations of 1 mg/ml. Among the complement proteins, C3 is perhaps the most adaptable and multifunctional protein identified to date, having evolved structural characteristics that allow it to associate with over 25 different proteins. Previous experiments suggest a vital role of C3 in scrapie prion pathogenesis. In the last chapter of my thesis we showed that lack of C3 expression by 5037 mice either transiently or genetically leads to delays in prion pathogenesis. C3 impacts disease progression in the early stages of disease by slowing the kinetic rate of accumulation and/or replication of PrPRES. This slower kinetic increase in PrPRES correlates with an increase in survival time in mice deficient in C3. This delay in disease is in sharp contrast to the complete rescue we saw in CWD infected Tg 5037;CD21-/- mice. This suggests a role for CD21/35 in peripheral prion pathogenesis independent of their endogenous ligands. Taken together we show that the innate immune system dictates the course of CWD. We have discovered novel immune cells, trafficking pathways, and complement components important in CWD pathogenesis. These data not only highlight the key role of the innate immune system in CWD, but also provide a strong foundation for future immunological studies of prion diseases.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 Immune modulatory and antimicrobial properties of mesenchymal stromal cells delivered systemically(Colorado State University. Libraries, 2020) Johnson, Valerie, author; Dow, Steve, advisor; Zabel, Mark, advisor; Avery, Anne, committee member; Tjalkens, Ron, committee memberTo view the abstract, please see the full text of the document.Item Open Access Longitudinal analysis of cytokine profiles during prion infection(Colorado State University. Libraries, 2015) Hill, Dana, author; Zabel, Mark, advisor; Spraker, Terry, committee member; Callan, Robert, committee member; Nichols, Tracy, committee memberTo view the abstract, please see the full text of the document.Item Open Access Microglial innate and adaptive immune function modulates disease pathology in and environmental pesticide model of Parkinson's disease(Colorado State University. Libraries, 2022) Rocha, Savannah M., author; Zabel, Mark, advisor; Tjalkens, Ronald B., advisor; Bouma, Jerry, committee member; Kading, Rebekah, committee member; Moreno, Julie, committee memberParkinson's Disease (PD) is the world's foremost movement disorder with pathological features including loss of dopaminergic neurons (DAn) within the substantia nigra pars compacta (SNpc), chronic activation of glial cells, and the misfolding and aggregation of a-synuclein (a-syn). Compounding evidence gathered over the past two centuries suggests environmental exposures, genetics, and aging can induce complicated cell-to-cell interactions that evoke and facilitate chronic inflammatory states; but the role that individual glial cells, in particular microglia, have in the progression of disease remains unknown. Difficulties in recapitulating the three pathological hallmarks of PD underscore the need for better animal models. To address this gap in functional investigation, the studies herein provide, for the first time, an optimized environmental exposure model with the pesticide rotenone (2.5mg/kg/day) in murine, which has proven effective at mirroring DAn degeneration, gliosis and misfolded a-syn accumulation. The pathology observed was region-, time- and dose-dependent, emphasizing the importance of environmental exposure and associated PD diagnosis. The successful optimization of this exposure model has allowed for its implementation in transgenic mice, which was previously unfeasible. To determine microglial specific innate inflammatory reactions in the progression of PD, we targeted the inflammatory transcriptional regulator NF-kB by use of transgenic CX3CR1-CreItem Open Access Prion strain adaptation: breaking and building species barriers(Colorado State University. Libraries, 2014) Reid, Crystal Meyerett, author; Zabel, Mark, advisor; Hoover, Edward, committee member; Spraker, Terry, committee member; Fails, Anna, committee memberPrions have been an enigma to researchers and agricultural producers alike since their inception. The timing and order of prion disease discovery can be attributed to the scrutiny of the prion protein-only hypothesis. The characterization of bacteria, viruses, and the infectious qualities encoded by their genomes only confounded the hypothetical notion of protein as an infectious agent. Perhaps viral etiology theories could have been disregarded earlier if genetic prion diseases were not quickly overshadowed by experimental transmissibility of the putative infectious protein. Despite the discordant journey, mounting evidence suggests that prion pathogenesis is caused by the conversion of the normal cellular host protein, (PrPC) into a protease-resistant, abnormal disease-causing isoform devoid of nucleic acid (PrPRES). Importantly, no differences are observed in the primary sequence of PrPC as compared to PrPRES indicating that observable differences between the normal and disease-causing proteins must be conformational. Additionally, even in the absence of nucleic acid, prions are able to infect various hosts differently, suggesting the phenomenon of prion strains. Characteristically long incubation periods and incomplete attack rates, as consequence of primary passage of prion infected material between differing species, but often even within the same species, have been defined as the species and transmission barrier respectively. Conversion efficiency of infectious prions is most efficient when host and donor PrPC are identical leading some researchers to believe that heterologous PrP blocks conversion, extending the days to onset of clinical disease. Evidence also suggests that prion protein primary sequence predisposes PrPC to fold in an un-infectious normal conformation but interaction with a PrPRES conformer, enciphering biological strain characteristics, provides a template for misfolding PrPC into an infectious conformation. Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRES acts as a template that can convert normal prion protein (PrPC) into the infectious misfolded PrPRES isoform. PMCA utilizes sonication to break up PK resistant aggregates into smaller prion seeds that may interact and template PrPC substrate present in the uninfected brain homogenate. Uniquely, prion disease can be inherited, transmitted, or occur spontaneously. Recently, several investigators have reported spontaneous generation of infectious prions using in vitro methods such as PMCA. Additional investigations into host factors needed for efficient conversion and replication has led to the discovery of differences in the propensity of PrPC misfolding among different species. Several groups have recently suggested that cervid prion protein has a higher propensity for misfolding in vitro and in vivo as a result of a unique rigid loop identifiable in cervid PrPC secondary structure. It has been proposed that increased transmission efficiency of cervid prions can be attributed to the presence of this rigid loop. The principle interest in the current research of this dissertation is to gain deeper knowledge about what fundamental factors play a role in prion strain adaptation, to challenge current theories about prion strain fidelity and to assess species barriers and prion strain dynamics with the aid of differential mouse models of prion disease. The comprehensive hypothesis of this dissertation is that host factors, including but not solely PrPC, mediate prion strain adaptation and determine host range and strength of species barriers. We used PMCA, bioassay using transgenic mice expressing variable amounts of PrPC from mouse and cervid species, and cell culture lines expressing different host PrPC to address these questions. We challenged the efficiency and congruency of PMCA by characterizing strain properties of amplified material in parallel with mouse bioassay by: incubation period, PK resistance, glycoform ratios, lesion profiles, and conformational stability. We further wanted to test if PMCA de novo generated prions were infectious and what strain properties they would emulate. We hypothesized that the PK resistant material generated with PMCA was infectious and transmissible and possess strain properties reminiscent of other cervid prion strains. Finally, our lab hypothesized that PrPRES conformation enciphers prion strain properties by acting as a template for nascent PrPRES but that host factors also play a role in adapting prion strains derived from a different host and that species barriers can be overcome through this adaptation.Item Open Access Prions in the environment: from the host to the environment and back again(Colorado State University. Libraries, 2013) Wyckoff, A. Christy, author; Zabel, Mark, advisor; VerCauteren, Kurt, advisor; Spraker, Terry, committee member; Wallenstein, Matthew, committee memberTo view the abstract, please see the full text of the document.Item Open Access Serial protein misfolding cyclic amplification (sPMCA) to detect surrogate markers for chronic wasting disease in surface water, municipal water and soil(Colorado State University. Libraries, 2008) Nichols, Tracy A., author; Zabel, Mark, advisorChronic wasting disease (CWD) is a transmissible spongiform encephalopathy of deer and elk. Research has indicated that CWD is transmitted horizontally, and that both blood and saliva can transmit disease. Environmental exposure to pens where infected animals have been kept has resulted in disease transmission to deer. However, examination of environmental components such as soil and water for prions has been hampered by sensitivity limitations of conventional western blotting and inoculation limitations of bioassays. In this study we evaluated the ability of protein misfolding cyclic amplification (PMCA) to detect protease-resistant prion protein (PrPres) in environmental samples such as water and soil. Serial protein misfolding cyclic amplification (sPMCA) of PrPres, the misfolded proteinase-resistant protein associated with prion disease, was used to detect prion-infected brain homogenate spiked into soil and water to determine detection limits of this assay in environmental samples. The PrPres detection limit for water after 6 rounds of PMCA was 1:26 x 106. Detection of a CWD spike in soil with our current methodology was not possible. We next evaluated surface and drinking water from a CWD endemic region of Colorado for PrPres by sPMCA. PrPres was detected in Cache la Poudre River and flocculant samples at a time of high snowmelt runoff, suggesting that sPMCA can be a useful tool in evaluating water for PrP res in CWD-endemic areas.Item Open Access The role of plants as an environmental reservoir of chronic wasting disease prions(Colorado State University. Libraries, 2016) Ortega, Aimee Elise, author; Zabel, Mark, advisor; Mathiason, Candace, committee member; Leach, Jan, committee member; Wilusz, Jeffrey, committee memberTransmissible Spongiform Encephalopathies (TSEs) are a group of diseases caused by an abnormal version, PrPRES, of the normal cellular host protein prion protein (Prnp) termed PrPC. Disease is fatal resulting in amyloid deposits and spongiform degeneration in the brain in most but not all cases. Clinical signs can include wasting, increases in salivation, and general motor impairment but many other clinical signs exist and can vary between TSEs. PrPRES is incredibly resistant to inactivation and can withstand radiation, formalin treatment, and autoclaving to name a few tried decontamination methods whereas PrPC is degraded normally. This difference in degradation allows for differentiation between the two protein forms as PrPRES is resistant to degradation by Proteinase K. In the early 1980s this abnormal protein was discovered to be the sole causative agent of the various TSEs which at the time was a novel finding and a novel method of disease transmission. It is thought that slightly misfolded forms of PrPC occur which can then misfold further eventually forming PrPRES. PrPRES then has the ability to act as a template for conversion, converting PrPC. Numerous TSEs exist that affect both humans and a variety of animals. One of the animal TSEs is Chronic Wasting Disease (CWD) which affects cervids such as elk, deer, and moose (Cervus candensis, Odocoileus hemionus, Alces alces) and has become endemic in both free-ranging and captive herds. The exact mechanisms behind spread of CWD are unknown but research has shown that environmental reservoirs play a role in transmission dynamics. We chose to explore whether PrPRES can be detected on or inside grasses and plants naturally exposed to prions in CWD endemic areas by use of Protein Misfolding Cyclic Amplification (PMCA). Here we present novel environmental evidence showing that PrPRES can be found on the surface of multiple plants from Rocky Mountain National Park and mice inoculated with these samples are showing clinical signs of disease.Item Open Access Unraveling prions: the complexities between the prion protein, complement, and B cells in diverse pathogenic settings(Colorado State University. Libraries, 2017) Kane, Sarah, author; Zabel, Mark, advisor; Bamburg, James, committee member; Tjalkens, Ronald, committee member; Avery, Anne, committee memberPrions diseases affect numerous mammalian species and may arise spontaneously, from genetic predisposition of the prion protein PrPC to misfold and aggregate, or from contacted with prion-contaminated materials. The first described prion disease, Scrapie, manifests in sheep, and records date back to the 18th century. Other mammalian species susceptible to prion diseases include humans, cats, mink, cervids (deer, elk, and moose), and cattle. The term Transmissible Spongiform Encephalopathy (TSE) arose to describe this new class of infectious diseases which exhibit spongiform degeneration in the central nervous system (CNS). TSEs are invariably fatal diseases, and only herd culling or breeding resistance mitigate disease spreading. However, chronic wasting disease (CWD) in cervids represents the first known TSE to occur in free-ranging wildlife, and the apparent facile spread demands strategies to halt its spread and prevent species eradication. Human prion disease characterization dates back to the 1920s. However, the bovine spongiform encephalopathy (BSE or mad cow disease) outbreak and subsequent transmission into a small number of humans in the 1980s and 90s pressed the need to understand the TSE agent. Many researchers since the 1960s postulated protein at least partially comprised the agent, but Stanley Prusiner provided the first scientific evidence of protein composition correlating with infectivity. Further, he coined the term proteinaceous particle, or prion. Follow-up research elegantly highlighted a host protein requisite to cause disease. Researchers now broadly accept the disease mechanism involves prions perverting the cellular prion protein to alter its conformation and join the highly stable growing prion aggregate. Upon peripheral exposure, most prion strains propagate in the lymphoreticular system prior to invading the CNS. Many elegant studies reveal the Complement system promotes initial prion trafficking and propagation in spleen and lymph nodes because mice deficient in various Complement proteins or receptors exhibit delayed or no disease. Once in the LRS, many postulate prions retrogradely infect the brain via sympathetic nerve fibers and the spinal cord. Once in the brain, prions provoke astrogliosis, neurodegeneration, and invariable death. While prion researchers made great strides in characterizing TSEs within a short few decades, many fundamental questions remain unaddressed. For example: what additional host factors foster prion pathogenesis? What is the normal function of the properly-folded, cellular prion protein? Lastly, do prion binding partners provide therapeutic targets? Data presented in this dissertation highlight crucial roles for Complement regulatory protein Factor H and Complement receptor CD21 in Scrapie pathogenesis, suggest C1q may strain-specifically impact prion disease, highlight PrPC as a crucial mediator in the adaptive immune system, and provide potential therapeutic tools and targets to combat prion disease.