Browsing by Author "Gilch, Sabine"
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Item Open Access A Bioinformatics Analysis to Identify Parasite Proteins that Mimic Host Immune Proteins(2016) Srivastava, Shruti; Wasmuth, James; McKay, Derek; Melin, Amanda; Gilch, Sabine; de Koning, JasonParasitic helminths (worms) are major parasites of humans and livestock around the world. These helminths are usually long lived and cause chronic infections, which invoke immune responses in the host leading to pathological changes. These changes are not always considered negative. For example, a helminth infection often leads to a shift in the host immune system away from inflammation. As such, some people with severe auto-immune diseases have experienced remission when infected with helminths. Examples include inflammatory bowel diseases. This `helminth-therapy' is controversial, but motivates a better understanding of helminth-host interactions. Previous work has shown that parasites encode proteins within their genomes which are secreted into their host, which modulate the host immune system. However, proteomic based studies have not identified the proteins involved. Here, we are taking a bioinformatics and genomics approach to discover the key proteins.Item Open Access Cellulose ether treatment inhibits amyloid beta aggregation, neuroinflammation and cognitive deficits in transgenic mouse model of Alzheimer’s disease(2023-07-28) Ali, Tahir; Klein, Antonia N.; McDonald, Keegan; Johansson, Lovisa; Mukherjee, Priyanka G.; Hallbeck, Martin; Doh-ura, Katsumi; Schatzl, Hermann M.; Gilch, SabineAbstract Alzheimer’s disease (AD) is an incurable, progressive and devastating neurodegenerative disease. Pathogenesis of AD is associated with the aggregation and accumulation of amyloid beta (Aβ), a major neurotoxic mediator that triggers neuroinflammation and memory impairment. Recently, we found that cellulose ether compounds (CEs) have beneficial effects against prion diseases by inhibiting protein misfolding and replication of prions, which share their replication mechanism with Aβ. CEs are FDA-approved safe additives in foods and pharmaceuticals. Herein, for the first time we determined the therapeutic effects of the representative CE (TC-5RW) in AD using in vitro and in vivo models. Our in vitro studies showed that TC-5RW inhibits Aβ aggregation, as well as neurotoxicity and immunoreactivity in Aβ-exposed human and murine neuroblastoma cells. In in vivo studies, for the first time we observed that single and weekly TC-5RW administration, respectively, improved memory functions of transgenic 5XFAD mouse model of AD. We further demonstrate that TC-5RW treatment of 5XFAD mice significantly inhibited Aβ oligomer and plaque burden and its associated neuroinflammation via regulating astrogliosis, microgliosis and proinflammatory mediator glial maturation factor beta (GMFβ). Additionally, we determined that TC-5RW reduced lipopolysaccharide-induced activated gliosis and GMFβ in vitro. In conclusion, our results demonstrate that CEs have therapeutic effects against Aβ pathologies and cognitive impairments, and direct, potent anti-inflammatory activity to rescue neuroinflammation. Therefore, these FDA-approved compounds are effective candidates for developing therapeutics for AD and related neurodegenerative diseases associated with protein misfolding.Item Open Access Chronic Wasting Disease: Investigations on Prion Shedding and Risk of Transmission to Caribou(2016) Cheng, Yo Ching; Gilch, Sabine; Jirik, Frank Robert; Kutz, Susan J.Chronic wasting disease (CWD) is a transmissible prion disease affecting cervids mainly in North America. The responsible agent for this disease is the protease-resistant and infectious prion protein isoform (PrPSc) which is converted from a normal host-encoded glycoprotein (PrPC). In this study, I employed a novel in vitro amplification assay; the real-time quaking-induced conversion assay (RT-QuIC) to detect CWD prions in feces. I demonstrated the use of optimized RT-QuIC to define the shedding pattern of CWD prions in feces during the course of CWD. Additionally, I analyzed the distribution of PrP polymorphism among caribou populations in Alberta. The results permit predictions on the probability of CWD transmission to caribou populations. My studies provide a new tool to improve CWD surveillance and transmission risk assessment, and may be useful for CWD management in the future.Item Open Access Conditionally immortalizing porcine hematopoietic stem cells using a Hox-B8 retrovirus and characterizing the porcine bone marrow niche(2022-05-24) Chiriboga Proano, Jose A; Warren, Amy L; Gilch, Sabine; DeVinney, Rebekah; Galezowski, Angelica; Jenne, CraigStudies in the field of veterinary immunology have been hampered by the availability of species-specific research tools that yield more reliable and accurate experimental planning and results. Limited resources exist to study and understand immune cells, such as macrophages, in pigs and other domestic species. Porcine immune cells are often derived in vitro and require isolation from the blood or bone marrow of live animals. The goal of this research project was to conditionally immortalized hematopoietic stem cells in pigs that can produce all immune cells by using an estrogen-controlled Hox-B8 lentivirus. In the presence of estrogen and expression of HoxB8, hematopoietic stem cells would remain in a conditionally immortalized progenitor stage. With removal of estrogen and addition of appropriate cytokines, hematopoietic stem cells would differentiate into a spectrum of common porcine immune cells. To be able to conditionally immortalize the hematopoietic stem cells and prime these cells to differentiate into in vitro, we first had to develop mammalian cell lines capable of expressing and secreting porcine recombinant cytokines - the second aim of this thesis. Methods to isolate porcine bone marrow and create media and conditions to culture bone marrow in the lab were also created in this project, which further allowed us to gain insight into hematopoiesis and the niche microenvironment in pigs.Item Open Access Development of a Diagnostic Platform to Detect Protein Biomarkers of Infectious Diseases(2020-04-28) Mukherjee, Sonia; De Buck, Jeroen M.; Schryvers, Anthony Bernard; Gilch, Sabine; Storey, Douglas G.Production limiting diseases are a major cause of economic losses to the dairy industry and often affect animal welfare. The implementation of robust diagnostic tests that can detect diseases at an early stage is essential to decrease the high costs associated with the treatment of chronic cases and lost productivity. In this study, we focus on the development of diagnostic tests for two important production limiting diseases, mastitis and enzootic bovine leukosis (EBL). Somatic cell count is the gold standard test for the diagnosis of subclinical mastitis, but an increase in somatic cell count may not always correlate to infection of the udder as it is influenced by multiple factors. On the other hand, the most common methods to diagnose EBL are agar gel immunodiffusion or ELISA. A major limitation of these tests is the requirement of manual handling of samples and the long processing time, which makes them unsuitable as on-farm diagnostic tests. Therefore, the development of reliable point-of-care technology to detect these diseases in a convenient and cost-effective manner is required. In this regard, biomarkers of infection and disease can be detected with a novel biosensor, based on a novel split trehalase (TreA) enzyme, developed in our lab. Glucose is the output signal for this biosensor platform which can be detected by a colorimetric enzymatic assay or a handheld glucometer. In this thesis, we have applied the existing biosensor platform to detect BLV antibodies in clinical serum by using bacterial surface complementation of fusion proteins, containing split TreA fragments fused to antigens expressed on the outer membrane of E. coli. Our results indicate that using this assay, it is possible to detect anti-BLV antibodies in clinical serum and distinguish between healthy and BLV positive samples. We also applied the split TreA biosensor platform to detect lactoferrin, which is the biomarker of subclinical mastitis in cattle. A phage display library approach was used to identify the peptides with affinity for LF. In conclusion, we expanded the existing split TreA biosensor platform with tests for the detection of production diseases like EBL and mastitis that are compatible with on-farm use.Item Open Access Identifying Genetic Factors of BSE Susceptibility(2021-09) Dudas, Sandor; Cross, James; Gilch, Sabine; Crowley, John; Janzen, EugeneBovine spongiform encephalopathy (BSE) is a transmissible, neurologic disease of cattle that decimated the cattle industry in many countries when it was discovered to be zoonotic. This disease is caused by a unique pathogen believed to be entirely composed of a misfolded, degradation-resistant form of a host-encoded protein. No therapies or cure currently exist for BSE, and so understanding the pathogenesis and host genes involved in this process could guide work to find ways to prevent or treat this invariably fatal disease. To identify genes playing a role in BSE susceptibility in cattle, genetic analysis was carried out on cattle that were experimentally-infected with BSE and showed variable disease outcomes. Chapter 2 explores potential genetic reasons for an abnormal outcome following oral BSE challenge. This analysis found that breed composition contributes to BSE outcomes in these experimental animals as well as in Canadian BSE field case cattle. In Chapter 3, an in-depth single nucleotide polymorphism (SNP) chip was used to genotype more than 200 experimental BSE challenged cattle with variable disease outcomes. Several genes with the potential to impact cellular calcium levels were identified as contributing to BSE susceptibility. The results also indicated that relatedness of the animals and breed composition are playing a role in the disease status. With several genomic targets identified as important, the research in Chapter 4 explored these genomic regions with targeted next generation sequencing. This analysis flagged additional SNPs in or near genes that can potentially alter the expression of genes involved in BSE pathogenesis. Experimental BSE susceptibility appears to be polygenic, with several genes each contributing a small amount, and this seems to be linked to relatedness and breed. The genes identified are highly expressed in the central nervous system and play a role in the function, maintenance, and survival of cells critical to this tissue. Several genes are linked to intracellular calcium homeostasis, a critical process playing a role in prion and other protein misfolding, neurodegenerative diseases. Additional work to decipher the contribution of the host breed and the breed of the BSE inoculum is warranted to understand the observed breed effects.Item Open Access Increased Microglial Reactivity Alters Morphine Analgesia(2017) Leduc-Pessah, Heather; Trang, Tuan; Pittman, Quentin; Altier, Christophe; Gilch, Sabine; Gendron, LouisOpioids are a potent class of analgesics in the management of both moderate to severe acute pain and chronic pain. Although neuronal response to opioids is well described, we are lacking a complete characterization of the diverse interplay of neurons with other cells types in response to opioids. Microglia, the immune cells of the CNS, are key targets of opioids and their response to repeated opioid exposure is implicated in the severe side effects associated with prolonged opioid use: opioid analgesic tolerance, opioid-induced hyperalgesia and opioid use disorder. In addition, increased microglial reactivity can alter analgesic response to opioids. Thus, understanding the role of microglia in response to opioids is both critical for improving the analgesic efficacy of opioids and for interfering with the negative side effects associated with prolonged opioid use. This thesis explores the contributions of the microglial P2X7 receptor and the microglial transcription factor Runx1 in morphine analgesia and in the development of adverse effects. My over-arching hypothesis is that increased microglial reactivity diminishes the analgesic potential of opioids, such as morphine. Here I show that repeated morphine causes a potentiation in microglial P2X7R function mediated by µ-receptor activation of Src kinase. Specifically, I identified tyrosine residues 382-384 on the P2X7R C-terminal domain as a critical site of phosphorylation and found that interfering with this site attenuated the development of tolerance in rats. I also show that the microglial transcription factor Runx1 regulates microglial reactivity in vitro and in vivo and that inhibition of Runx1 causes a decline in morphine analgesia. I characterized a novel strain of microglial-specific Runx1 knock-out mice and show that Runx1 deficiency causes a reduction in acute morphine analgesia and an exacerbation of opioid tolerance, hyperalgesia, and naloxone-precipitated withdrawal. Collectively, in this thesis I found that increased microglial reactivity, through variable mechanisms causes a reduction in the antinociceptive response to morphine in rodents. In conclusion, the work of this thesis has identified novel targets (P2X7R and Runx1) for interfering with the side effects associated with prolonged opioid use and in the acute analgesic response.Item Open Access Interaction of Porcine circovirus 2 with the Swine Immune System(2017) Solis Worsfold, Cristina Marie; Czub, Markus; Yates, Robin; Jirik, Frank; Gilch, SabinePorcine circovirus 2 (PCV2) is a virus with a single-stranded, DNA circular genome that is ubiquitous in pig populations worldwide. PCV2 is the causative agent of the post-weaning multisystemic wasting syndrome (PMWS), a multifactorial disease that affects six to twelve-week-old pigs, and which is characterized by weight loss and immunosuppression. PCV2 vaccination has diminished the presentation of PMWS in the field, although PCV2 infection is not prevented. PCV2 infects lymphocytes and it depends on the host enzymes to replicate. Enhanced PCV2 infection rates are associated with increased mitotic activity, although the effect of PCV2 replication on lymphoid cell function is unknown. The main goal of this thesis was to understand the interactions of PCV2 with the swine immune system, by determining the effect of PCV2 on the antibody response in pigs under field conditions, and studying the impact of the primary PCV2 infection on lymphocyte activation, proliferation, and viability. As shown in Chapter 2, a PCV2 persistent infection was detected in farmed pigs of all age groups. Furthermore, a great variability in their neutralizing antibody titers was observed, regardless of their vaccination status. Chapter 3 provides details about the establishment of an in vitro cell model to study the effect of primary PCV2 infection on the immune cells, by using snatched-farrowed, porcine colostrum deprived (SF-pCD) PCV2-free pigs as blood donors of PCV2-naïve peripheral blood mononuclear cells (PBMCs). These cells were exposed to the polyclonal mitogens ionomycin/PMA and PCV2 infection. As shown in chapters 4 & 5, enhanced PCV2 infection rates in ionomycin/PMA-stimulated PBMCs, decreased proliferation in PCV2 infected cells, and high bystander cell death rates in PCV2-exposed PBMCs were observed. This thesis contributes to the current knowledge on PCV2 immunology by bringing insight into factors that contribute to viral pathogenesis and immune modulation and emphasizes the need of developing new vaccines that prevent PCV2 infection.Item Open Access Investigations into Macrophage-Infectious Bronchitis Virus (IBV) Interaction and Shell-less Egg Syndrome(2018-08-29) Amarasinghe, Aruna; Abdul-Careem, Mohamed Faizal; van der Meer, Frank; Cork, S. C.; Gilch, Sabine; Gomis, Susantha MuhandiramgeInfectious bronchitis virus (IBV) is a coronavirus and infects chickens globally causing economic losses. The disease caused by IBV is known as infectious bronchitis (IB) and is prevalent in commercial broiler and layer chickens and breeder flocks in Canada. The control of IB relies on vaccination done on the day of hatch and then several times during the grower period depending on the purpose of rearing the chickens. Although the vaccine-induced immunity protects chickens from production losses induced by IBV infection, vaccine failures are frequent. Given the issues in current IB control measures, sustainable control measures developed understanding the host-IBV interaction is required. The studies conducted in the thesis focused in two major areas; 1) understanding the interaction between IBV and host immune system mainly macrophages and 2) investigating the role of IBV in a recently emerged concern of Western Canadian table-egg layer industry, shell-less egg syndrome (SES). The work described in chapter 2 of the thesis led to the finding that IBV replicates in avian macrophages in vivo and in vitro. In vitro, we showed that IBV not only targets macrophages leading to productive infection but also affects selected functions of macrophages, particularly the production of nitric oxide (NO). As shown in chapter 3, IBV infection upregulates the expression of interleukin (IL)-1β in both tracheal and lung tissues. An additional observation made was that there was a significant association between the IBV genome load and macrophage recruitment in lungs. Overall, we found that macrophages can act as a source of cytokines, which is beneficial against IBV infection. However, the ability of IBV to replicate within macrophage by decreasing selected immune functions can be detrimental to the host. Chapter 4 provides details of our work leading to the elucidation of the etiology of SES. First, molecular characterization showed that about 70% of the IBV strains isolated from layer flocks affected with SES in Western Canada were Massachusetts (Mass) genotype. Infection of layer chickens with one of the Mass IBV isolate induced shell-less eggs. The work of chapter 5 compared two Mass IBV isolates recovered from Western Canadian layer flocks for whole genome variations and documented the differences in pathogenicity, tissue distribution, and macrophage response. The knowledge generated in the thesis increased the understanding of IBV-macrophage interaction, documented the IBV genotypes observable in Western Canada layer flocks and elucidated the etiology of SES observed in layer operations in Canada.Item Open Access Investigations on the role of rab7 in prion infection(2022-06) Cherry, Pearl; Gilch, Sabine; Kar, Satyabrata; Braun, Janice; Haigh, Cathryn; Tsutsui, ShigekiPrion diseases are fatal and infectious neurodegenerative diseases caused by the misfolding of the cellular prion protein PrPc into its infectious isoform PrPSc, which comprises the main if not the only constituent of prions. In response to prion infection certain cellular impairments such as reduced membrane association of Rab7, compromised lysosomal acidification and elevated cholesterol levels are observed. Here, we found that prion infected primary neurons are marked by an increased expression of active Rab7 (Rab7.GTP) levels during the initial stages of the infection, followed by a loss in its levels as the infection progresses. However, astrocytes in prion infected terminal mice upregulate the active Rab7 expression. The reduced Rab7 activation is linked to its reduced ubiquitination status. In neurons, the loss in active Rab7 leads to a delay in the Rab7 mediated trafficking of LDL to the lysosomes and Golgi, resulting in a defective feedback regulation of cholesterol metabolism. This in turn triggers de novo cholesterol synthesis. Over-expression of a constitutively active mutant of Rab7 in prion infected neuronal cell-lines rescues the delay in LDL trafficking, restores the normal cholesterol levels and reduces prion propagation. Inspired by the differential kinetics of Rab7 activation during prion infection and the cell-type specific differences of Rab7 activation in the prion infected terminal mouse brain, we studied the Rab7 interactome using quantitative proteomics. We identified a loss in the Rab7 interactome associated with ubiquitination in prion infected cells and synaptic signalling in prion infected brains, whereas proteins in the mitochondrial respiratory chain were observed to be lost in both cases. The enriched pathways identified in Rab7 interactome were linked to mRNA transport and degradation to name some. In the prion-infected brain, we identified the cholesterol esterification pathway to be enriched in the Rab7 interactome in the brain. These studies indicate a possible involvement of Rab7 in the above-mentioned pathways and suggest candidate proteins to be evaluated for their potential to ubiquitinate Rab7, as a functional impairment of the Rab7 ubiquitin ligase can cause reduced activation observed in prion infections.Item Open Access Modulation of Prion Protein Aggregate Size Composition to Study Effects on Cellular Transport and Propagation(2018-01-26) Kay, Peter; Gilch, Sabine; Braun, Janice; Schaetzl, HermannPrion diseases are a group of detrimental neurodegenerative diseases that arise when the cellular prion glycoprotein is misfolded into a beta-sheet rich amyloidogenic isoform known as “PrPSc”. The accumulation and aggregation of this PrPSc in neuronal cells of the central nervous system leads to severe neurodegeneration and is fatal. With any given type of prion disease there is a vast array of different PrPSc aggregate sizes present, and it was previously found that the size of a PrPSc aggregate can influence certain properties, such as cellular uptake and transport. We were able to elucidate and compare the aggregate size profiles of the ME7 and 22L mouse-adapted scrapie strains using a sedimentation centrifugation technique. To better understand the effects of altering the prion protein aggregate size profile, we successfully utilized the chemical compound known as O4 to stabilize larger prion aggregates and therefore shifted the equilibrium towards the larger sizes. We showed that O4 was able to increase the proteolytic resistance of prion aggregates after stabilization, and reduced PrPSc levels in neuroblastoma cell models persistently infected with prions. In addition, we attempted to study the transport of prions across cellular barriers, through transcytosis, and how this might be related to aggregate size. Overall our work has furthered the understanding of the role that the aggregate size profile plays in prion diseases, and has given important insights as to how prion infection may be reduced or prevented.Item Open Access Novel Approaches to Fight Prion Diseases(2020-04-29) Thapa, Simrika; Schaetzl, Hermann M.; Gilch, Sabine; Trang, Tuan; van Marle, Guido; Coffin, Carla S.; Telling, Glenn C.Prion diseases are fatal neurodegenerative disorders caused by PrPSc, the misfolded and infectious isoform of the cellular prion protein (PrPC). Currently, no preventive or therapeutic measures are available. In this work, we focused on therapeutic and prophylactic strategies against prion infections. In the therapeutic approach, we targeted cellular pathways and investigated the role of the quality control (QC) proteins, ERp57 and VIP36, on prion propagation. We found that the overexpression of ERp57 or VIP36 significantly reduced PrPSc levels in persistently prion-infected cells and decreased the susceptibility of uninfected cells to de novo prion infection. Moreover, lentiviral-mediated overexpression of ERp57 prolonged the survival of prion-infected mice. Mechanistically, we found that ERp57 overexpression reduced endoplasmic reticulum (ER) stress. To translate this proof-of-concept into potential drug therapy, we investigated the anti-prion effect of Sephin1, shown to prolong the phosphorylation of eIF2α and lower ER stress in the cells. In persistently prion-infected neuronal cells, we found that treatment with Sephin1 markedly reduced PrPSc levels. Moreover, Sephin1 reduced ER stress-induced PrP aggregates in cells and significantly extended the survival of prion-infected mice. These data provide the basis for targeting these cellular pathways as novel anti-prion therapy. In our prophylactic approach, we hypothesized that active vaccination is useful to contain chronic wasting disease (CWD), a contagious and expanding prion disease of cervids. Here, we vaccinated transgenic mice expressing elk prion protein with adjuvant CpG alone, or one of four recombinant PrP (rPrP) immunogens: deer dimer (Ddi), deer monomer (Dmo), mouse dimer (Mdi), and mouse monomer (Mmo). After challenging the animals with CWD prions intraperitoneally, we found that all vaccinated groups had longer survival times than the CpG control group. Interestingly, the Mmo-immunized group revealed that survival was extended by 60%. We also observed 28.4% and 24.1% prolongation in Dmo and Ddi groups, respectively. Our preliminary study in reindeer showed substantial humoral immune response induced by Mdi and Ddi, and the sera from the Ddi-vaccinated reindeer significantly reduced CWD prions in a cell culture model. Taken together, this study describes potential vaccine candidates against CWD. However, their protective effect in the natural cervid host needs further investigation.Item Open Access Oral administration of repurposed drug targeting Cyp46A1 increases survival times of prion infected mice(2021-04-01) Ali, Tahir; Hannaoui, Samia; Nemani, Satish; Tahir, Waqas; Zemlyankina, Irina; Cherry, Pearl; Shim, Su Y; Sim, Valerie; Schaetzl, Hermann M; Gilch, SabineAbstract Prion diseases are fatal, infectious, and incurable neurodegenerative disorders caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform (PrPSc). In humans, there are sporadic, genetic and infectious etiologies, with sporadic Creutzfeldt-Jakob disease (sCJD) being the most common form. Currently, no treatment is available for prion diseases. Cellular cholesterol is known to impact prion conversion, which in turn results in an accumulation of cholesterol in prion-infected neurons. The major elimination of brain cholesterol is achieved by the brain specific enzyme, cholesterol 24-hydroxylase (CYP46A1). Cyp46A1 converts cholesterol into 24(S)-hydroxycholesterol, a membrane-permeable molecule that exits the brain. We have demonstrated for the first time that Cyp46A1 levels are reduced in the brains of prion-infected mice at advanced disease stage, in prion-infected neuronal cells and in post-mortem brains of sCJD patients. We have employed the Cyp46A1 activator efavirenz (EFV) for treatment of prion-infected neuronal cells and mice. EFV is an FDA approved anti-HIV medication effectively crossing the blood brain barrier and has been used for decades to chronically treat HIV patients. EFV significantly mitigated PrPSc propagation in prion-infected cells while preserving physiological PrPC and lipid raft integrity. Notably, oral administration of EFV treatment chronically at very low dosage starting weeks to months after intracerebral prion inoculation of mice significantly prolonged the lifespan of animals. In summary, our results suggest that Cyp46A1 as a novel therapeutic target and that its activation through repurposing the anti-retroviral medication EFV might be valuable treatment approach for prion diseases.Item Embargo PDGF Signaling in Sertoli Cell Cilia and Seminiferous Tubule Morphogenesis(2023-07) Ahmadi Jeyhoonabadi, Maryam; Dobrinski, Ina; Gilch, Sabine; Jiami GuoImmature porcine testicular Sertoli cells possess primary cilia, organelles that transmit extracellular signals into the cell. A yet unidentified ciliary signal in Sertoli cells has been found necessary for in vitro morphogenesis of tubules and testicular organoids. Herein, we hypothesized that platelet-derived growth factor receptor alpha (PDGFRα) signaling through the cilia of the Sertoli cells modifies cell migration and extracellular matrix (ECM) production, consequently altering in vitro formation of testicular organoids and seminiferous tubules. Immunocytochemistry revealed PDGFRα was present on Sertoli cell cilia as well as the rest of the cell membrane and cytoplasm. Inhibiting PDGFRα signaling did not prevent organoid and tubule formation. However, it appeared to correlate with ablation of cytoplasmic extensions from the surface of the in vitro formed tubules. Inhibition of PDGFRα signaling resulted in reduction in cell area, increase in the percentage of cells with prominent peripheral paxillin expression, and increase in the number of ciliated cells and cilia length. Immunohistochemistry revealed that cilia, which colocalized with PDGFRα, were positioned away from the basement membrane of the seminiferous tubules, close to the Sertoli cell nuclei, in an area that was occupied with Sertoli cell cytoplasm. When observed via transmission electron microscopy, Sertoli cell cilia exhibited ciliary pockets around their axoneme. These results demonstrate that PDGFRα activity in Sertoli cells can influence in vitro tubule morphogenesis, possibly by altering cell cytoskeleton, cell-ECM interaction, and cilia. However, the role of cilia-specific signaling remains to be determined.Item Open Access A Prion Protein Gene Polymorphism at Codon 138 Modulates Chronic Wasting Disease Pathogenesis(2021-08) Arifin, Maria Immaculata; Gilch, Sabine; Schätzl, Hermann; Czub, Markus; Jirik, Frank; Mathiason, Candace; Musiani, MarcoPrion diseases are fatal and infectious neurodegenerative diseases caused by prions. Chronic wasting disease (CWD) is a prion disease of cervids found in North America (NA), Scandinavia and South Korea. Although there are no reports of CWD in caribou (Rangifer tarandus spp.) in NA so far, previous findings show that reindeer (R. t. tarandus) are susceptible to CWD. Single amino acid substitutions (SAAS) within the cervid prion protein (PrP) sequence have been shown to prolong survival times and produce incomplete attack rates upon CWD infection. Prion protein SAAS have been found in caribou populations in NA, including a serine to asparagine substitution at codon 138 (S138N). Previous studies reported that animals harboring the N variant at this codon were either resistant or less susceptible to natural CWD prion exposure. Based on these reports, we hypothesized that the S138N PrP amino acid substitution modulates CWD pathogenesis. We report that the 138N allele frequency is rare among caribou in areas with high risk of contact with CWD-infected species, particularly in woodland caribou (R. t. caribou) herds in Saskatchewan and Alberta. We also report that the barren-ground caribou (R. t. groenlandicus) herds have higher frequencies of the 138N allele. We found that the S138N SAAS did not alter endogenous PrP properties, but rather impairs the prion conversion process. Transgenic knock-in (KI) mice expressing the 138NN PrP genotype did not develop clinical disease up to 700 days post-inoculation (dpi), whilst their wild-type deer (138SS) counter parts succumbed to CWD between ~450-580 dpi. The 138NN KI mice did, however, harbor prions capable of inducing conversion in an in vitro prion conversion assay. Remarkably, even upon intracerebral prion inoculation, seeding activity was first detected in the spleens of these KI mice. Our findings provide new insights into the role of PrP genotype in tissue tropism of prion replication. Caribou in NA are a Threatened species and an essential resource for Indigenous people. Thus, determining the mechanisms by which the 138N allele modulates CWD pathogenesis is important for future CWD management strategies, especially in areas where caribou are at a high risk of contracting the disease.Item Open Access A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture(2022-07-08) Thiageswaran, Shiama; Dobrinski, Ina; Klein, Claudia; Rancourt, Derrick; Gilch, SabineSpermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, necessitating in vitro culture systems to increase the numbers of SSCs available for downstream applications. Using the pig as a non-rodent model, we developed spermatogonial culture systems to expand spermatogonia from 1- and 8-week-old porcine testes, comparing feeder layers consisting of populations enriched for Sertoli cells, peritubular myoid cells (PMCs), pig fetal fibroblasts (PFFs), and testicular endothelial cells (TECs). As previously developed porcine spermatogonial culture systems relied exclusively on Sertoli cell feeder layers, we explored whether constituent cells of the SSC niche, such as PMCs and TECs, or fibroblastic cells like PFFs, may also support SSC expansion. Spermatogonia co-cultured with PMCs and PFFs had comparable rates of proliferation and apoptosis to spermatogonia co-cultured with Sertoli cells. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in the dynamic crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes were found to be taken up by spermatogonia, and the inhibition of exosomal release reduced spermatogonial proliferation.Item Open Access The Role of Microglial P2X7 Receptor Phosphorylation in Neuropathic Pain(2016) Pilapil, Alexandra; Trang, Tuan; Zamponi, Gerald; Pang, Daniel; Gilch, SabineNeuropathic pain is a debilitating chronic pain condition that can arise because of injury to a peripheral nerve. The lack of effective therapies for neuropathic pain demands a better understanding of the underlying mechanisms. Converging evidence suggests that neuropathic pain is caused by aberrant cellular and molecular changes within the central nervous system. Microglia and P2X7 receptors (P2X7Rs) are spinal targets implicated in neuropathic pain, but the core mechanisms that modulate microglial P2X7R activity remain obscure. We identified Y382-384 within the P2X7R C-terminal domain as a putative phosphorylation site that underlies nerve injury-induced mechanical allodynia in a sex-dependent mechanism. Thus, site-specific tyrosine phosphorylation of P2X7R in microglia is a novel spinal determinant involved in neuropathic pain for male animals. The findings of this study provide evidence for microglial-mediated sex differences in neuropathic pain, which further reinforces the importance of including male and female subjects in preclinical pain research.Item Open Access The Role of Quaternary Structure Organization in Prion Strain Selection and Adaptation(2023-08) Chang, Sheng Chun; Gilch, Sabine; Braun, Janice; Gilleard, John; Tsutsui, Shigeki; Booth, StephaniePrion diseases are fatal and infectious neurodegenerative diseases caused by the misfolded, protease-resistant, and aggregated prion protein isoform, PrPSc. Unlike conventional pathogens, the propagation of prions does not depend on the replication of genetic material, but different cases of infection can lead to PrPSc adopting specific conformations resulting in different and stably transmissible phenotypic properties that are, by definition, the determinants of different strains. The distribution of PrPSc quaternary structure has previously been determined to vary across prion strains, and different PrPSc aggregate assemblies in turn harbour varying levels of infectivity. We were interested in determining how PrPSc aggregate size modulate strain properties and how these properties are affected by different inoculation routes, specifically in comparing peripheral versus intracerebral infection. Using in vivo murine models, we explored prion disease pathogenesis and strain selection upon inoculation of different PrPSc protein quaternary structures or prion strains. Transgenic mice overexpressing cervid prion protein inoculated with different elk chronic wasting disease PrPSc fractions exhibited strikingly different survival times and disease phenotypes, including variations in the prion biochemical, neuropathological, and biophysical profiles; interestingly, these properties converged upon passaging, though not the differing clinical signs. Gene-targeted mice expressing the cervid prion protein at physiological levels infected with CWD prions either directly into the brain or from the periphery resulted in different biochemical and neuropathological properties, notably with a converging conformational profile among the peripherally inoculated group. Similar observations were recorded where animals peripherally inoculated with CWD prion aggregates exhibited markedly reduced variations in survival times compared to infection directly into the brain. These results indicate that the direct selection of PrPSc conformers, or PrPSc conformational mutation, is likely to occur in the presence of a barrier interfering with the ideal replication setting as adaptation under a more restrictive set of circumstances takes place. This research demonstrates the intricacies of prion strain selection and adaptation that would provide novel insight into future investigations of the evolution and emergence of prion strains.