Non-neuronal functions of the prion protein : insights from a unique animal model

Abstract

The physiological function of the cellular prion protein (PrPC) remains enigmatic. A misfolded, infectious conformer of the protein, known as the scrapie isoform PrPSc, is able to aggregate in brain tissue and cause a group of fatal transmissible neurodegenerative disorders in humans and animals. Amongst them are Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in cervids. PrPC is evolutionary conserved and abundantly expressed in neurons, but also widely in other non-neuronal tissues, depicting a central role in cellular physiology. Several methods have been used for studies of PrPC function and the development of Prnp knockout mice during the 1990s, provided researchers with a new and valuable tool for studies of in vivo consequences of PrPC ablation. Interestingly, apart from being completely resistant to prion disease, Prnp knockout mice displayed no major aberrant phenotypes; rather they developed normally and exhibited normal lifespans. Further studies have however attributed several phenotypes to the loss of PrPC, including inhibition of apoptosis, protection against oxidative stress, a role in synapses, NMDA receptor signaling and modulation of behavior, amongst others. Recently, a line of Norwegian Dairy Goats was found to carry a nonsense mutation at codon 32 of the PRNP reading frame that completely blocks PrPC synthesis. A non-transgenic, non-rodent mammalian PrPC-null model represents a new and valuable resource for prion research that could, in combination with other model systems, shed light on PrPC physiology. The studies going into this thesis represent the first sets of analysis and experiments characterizing this unique line of goats.

Firstly, we set out to characterize goats with (PRNP+/+) and without (PRNPTer/Ter) PrPC with regard to standard health parameters. Hematological analyses revealed that PRNPTer/Ter goats presented with an increased number of red blood cells (RBCs) with a smaller mean cell volume (MCV) as well as a tendency for increased levels of neutrophils, an alteration we collectively coined a “hematological shift”. This observation suggests that PrPC may serve a role in bone marrow hematopoiesis. The bone marrow morphology, however, did not deviate between the genotypes and further studies are needed to clarify PrPC’s role in RBC development and/or physiology. Based on the moderate, but dynamic expression of PrPC in mononuclear cells and previous research showing that PrPC might play a role in modulating basic immune cell functions and immune responses, we pursued this by investigating basal immune cell traits. Numbers of peripheral blood mononuclear cells (PBMCs) were similar between the PRNP genotypes. Moreover, basal immune cells functions such as monocyte phagocytosis and lymphocyte proliferation were also similar between the genotypes, suggesting that PrPC deficiency had no major effects on these important processes, which is contrast to some observations in mouse models. A deeper analysis of PrPC loss was undertaken by investigating the transcriptome of PBMCs from PRNP+/+ and PRNPTer/Ter animals. Analysis of differentially expressed genes revealed a significant upregulation of type I interferon (IFN)-responsive genes in PRNPTer/Ter cells, which could not be attributed to differences in cell populations or altered expression of genes encoding major components of the type I IFN signaling pathway, indicating that PrPC somehow downregulates tonic type I INF signaling. By using several clones of human neuroblastoma SH-SY5Y cells, stably expressing different levels of PrPC, we were able to demonstrate that mock-transfected cells with very low levels of PrPC responded with increased transcription of the IFN-responsive gene MX2 after treatment with IFN-α, compared with clones expressing moderate or high levels of PrPC. Although, providing support to the observations from the PBMCs, the data from the SH-SY5Y cells did not reveal a simple dose-response relationship between PrPC levels and apparent sensitivity to INF-α. However, an independent dataset from an in vivo lipopolysaccharide (LPS) challenge of goats with and without PrPC, showed a similar gene expression signature in circulating leukocytes both at basal level and after LPS exposure, demonstrating that the phenotype is also present and functional in vivo. Further studies are needed to reveal the molecular mechanisms behind these observations and to clarify at which level PrPC impacts type I IFN signaling. Goats naturally devoid of PrPC can have significant breeding value. Knowing that PrPC is normally present at high levels in the male genital tract, including ejaculated spermatozoa, we wanted to investigate if lack of PrPC could influence semen quality and spermatozoa stress resilience, as suggested by mouse studies. Our data confirmed the prominent presence of PrPC in testicle and epididymis as well as lower levels in spermatozoa in PRNP+/+ animals. However, analysis of freeze tolerance, DNA integrity, viability, motility, ATP levels and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of ROS after exposure to FeSO4 and H2O2 revealed no differences between the PRNP genotypes. Since cytoprotective roles have been assigned to PrPC, we wanted to broaden our analysis by inclusion of PBMCs with and without PrPC in our in vitro analysis of oxidative and genotoxic stress. Similar to the observations from spermatozoa, PrPC appeared dispensable for in vitro stress resilience of PBMCs. Expression levels of genes involved in DNA damage repair and ROS scavenging in PBMCs were also unaffected by PrPC loss. In conclusion, no PRNP genotype related differences in stress resilience were detected concerning viability and global accumulation of DNA damage in PBMCs after treatment with H2O2, doxorubicin or MMS. These observations were corroborated by data from SH-SY5Y cells expressing very low or moderate levels of PrPC, again revealing no direct cyto-protective function of PrPC, under these in vitro conditions. Altogether, the degree to which PrPC is able to confer cytoprotection during stressful situations in vitro is questioned, as we were unable to detect any differences between cells with and without PrPC in our studies. However, PrPC could still execute functions in neuro-immune crosstalk at rest and during inflammatory stress, possibly protecting immune-privileged tissues.

Taken together, this thesis has provided unique new knowledge concerning PrPC’s normal physiological function by the use of a non-transgenic animal model. Animals without PrPC displayed a hematological shift, but no effects were observed in circulating leukocytes or basal immune cell functions. The finding of an immunological signature dominated by increased level of type I IFN-responsive genes in PBMCs is a previously unrecognized phenotype in cells without PrPC, which can function as a gateway for further studies needed to dissect the pathway in which PrPC might be involved. No stress-protective properties could be found in vitro in the cells with PrPC assessed in this work, however it remains to be investigated if these effects are better explored in vivo.

Det cellulære prionproteinets (PrPC) fysiologiske funksjon er, til tross for årevis med forskning, fortsatt ukjent. En feilfoldet, infeksiøs isoform av prionproteinet, bedre kjent som scrapievarianten, PrPSc, har evne til å aggregere i hjernevev og forårsake en rekke fatale, overførbare nevrodegenerative sjukdommer hos mennesker og dyr. Blant disse er Creutzfeldt Jakobs sjukdom hos menneske, skrapesjuke hos sau og geit, bovin spongiform encefalopati eller kugalskap hos storfe, og skrantesjuke hos hjortedyr.