High titers of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrP in vivo

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrPSc) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrPSc. This work questions the correlation between PrPSc level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrPSc as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.https://doi.org/10.1074/jbc.M704329200282pubpub4

Investigating early cellular and molecular responses to misfolded PrP protein

The generation and spread of misfolded protein aggregates is common to all Protein Misfolding Diseases (PMDs) including but not limited to Alzheimer’s, Parkinson’s and Prion disease. Yet despite this common feature, the early mechanisms underpinning the apparent perturbation in protein handling and processing in such conditions remain poorly understood. Research in our lab has previously shown protein misfolding may be seeded and supported in different ways in the brain. Mice homozygous for the P101L mutation in murine PrP (101LL) are capable of supporting protein misfolding and amyloid plaque formation after challenge with pre-formed PrP fibrils and wild type mice of the same genetic background do not, and appear to be capable of curtailing this accumulation of abnormal protein in vivo. Thus, both mouse lines provided ideal tools to investigate early cellular and molecular responses to abnormal PrP fibril challenge. PMDs occur in vivo with a regional dependant progression however; such investigations are more amenable to controlled initiation and visualisation in vitro. Therefore, primary neuronal hippocampal cell cultures were generated from each line (101LL and WT) and were thoroughly assessed at the cellular level. Comparative molecular analysis of these cultures and in vivo hippocampal tissue from both WT and 101LL lines was carried out and transcriptome analysis confirmed a broadly comparable global expression profile at a basal level between in vivo, in vitro and genotype. Interestingly, this suggests that the genotype specific differential protein misfolding responses are therefore likely to be the result of regulatory cascades initiated after inoculation. To examine this at the cellular level, cultures were then challenged with fluorescently labelled recombinant PrP fibrils. Fibrils were directly interacting with PrPC on neuronal surfaces but did not cause neurotoxicity. However, post-synaptic marker PSD-95 was significantly reduced in 101LL cultures suggestive that 101LL neurons were more susceptible to fibril insult than WT. Microglial cells were activated post fibril challenge in both genotypes. This generic response was possibly to limit neuronal damage and promote fibril degradation through phagocytosis. Transcriptome analyses supported these observations and showed an increase in expression of genes associated with phagocytosis was occurring after fibril challenge in both genotypes. Hypertrophic astrocytes and an increase in expression of genes associated with astrocyte differentiation and development was only evident in WT fibril-challenged cultures indicating differential glial responses were occurring between genotypes. Intracellular labelling showed localisation with endosomes and lysosomes in WT cells indicating an endolysosomal pathway was operative however; localisation in 101LL cells was primarily in endosomes indicating an impairment in abnormal protein degradation pathways was occurring in 101LL fibril-challenged cultures. Transcriptomic analysis identified reduced gene expression of lysosomal associated genes Laptm5 and Ctsz in 101LL fibril-challenged cultures, supporting endolysosomal processing was reduced in this genotype. Additional transcriptomic profiles obtained post-fibril challenge showed an increase in gene expression in WT cultures associated with endocytosis, macrophage engulfment, immune and defence response and ER-associated misfolded protein catabolic processes none of which were induced in 101LL fibril-challenged cultures. These analyses suggest a dysfunction of multiple mechanisms may be associated with an inability to clear misfolded protein. These data provide key information on pathways and cellular mechanisms involved in either clearance of misfolded protein or initiation of amyloid formation. Identifying these pathways will provide a number of possible test targets with the potential of impacting diagnosis and/or intervention in PMD’s such as AD, PD and prions

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures" for publication in Brain Communications.

From Crossref journal articles via Jisc Publications RouterHistory: epub 2021-07-07, issued 2021-07-07Article version: AMPublication status: PublishedAbstract Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures in an on-going question. Here we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines

Transmission of murine scrapie to P101L transgenic mice

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Item is not available in this repository.The PrP protein is central to the transmissible spongiform encephalopathies (TSEs), and the amino acid sequence of this protein in the host can influence both incubation time of disease and targeting of disease pathology. The N terminus of murine PrP has been proposed to be important in the replication of TSE agents, as mutations or deletions in that region can alter the efficiency of agent replication. To address this hypothesis and to investigate the mechanisms by which host PrP sequence controls the outcome of disease, we have assessed the influence of a single amino acid alteration in the N-terminal region of murine PrP (P101L) on the transmission of TSE agents between mice. Mice homozygous for the mutation (101LL) were inoculated with TSE strains 139A and 79A derived from mice carrying a Prnpa allele, and 79V and 301V derived from mice carrying a Prnpb allele. Incubation times in 101LL mice were extended with all four strains of agent when compared with those in the corresponding mouse genotype from which the infectivity was derived. However, the degree to which the incubation period was increased showed considerable variation between each strain of agent. Moreover, the presence of this single amino acid alteration resulted in a 70 day reduction in incubation time of the 301V strain in Prnpa mice. The effect of the 101L mutation on murine scrapie incubation time appears therefore to be strain specific.https://doi.org/10.1099/vir.0.19147-084pubpub1

Variable tau accumulation in murine models with abnormal prion protein deposits

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177The conversion of cellular prion protein (PrP) into a misfolded isoform is central to the development of prion diseases. However, the heterogeneous phenotypes observed in prion disease may be linked with the presence of other misfolded proteins in the brain. While hyperphosphorylated tau (p.tau) is characteristic of Alzheimer's disease (AD), p.tau is also observed in human prion diseases. To explore this association in the absence of potential effects due to aging, drug treatment, agonal stage and postmortem delay we analyzed p.tau and PrP immunopositivity in mouse models. Analyses were performed on mice inoculated with prion agents, and mice with PrP amyloid in the absence of prion disease. We observed that p.tau was consistently present in animals with prion infectivity (models that transmit disease upon serial passage). In contrast, p.tau was very rarely observed or absent in mice with PrP amyloid plaques in the absence of prion replication. These data indicate that the formation of p.tau is not linked to deposition of misfolded PrP, but suggest that the interaction between replication of infectivity and host factors regulate the formation of p.tau and may contribute to the heterogeneous phenotype of prion diseases.https://doi.org/10.1016/j.jns.2017.10.040383pubpubDecember 201

Influence of COVID-19 Building Restrictions on Physical Activity Promotion Through Increased Stair Use and Limited Elevator Access: A Quasi-Experimental Study—Sport and Physical Activity Group Active Campus Project

Background: The aim of this study was to determine the effectiveness of UK Government Covid-19 safe offices policy to increase stair-use in a higher education setting during the Covid-19 pandemic. Methods: Automated counts at three Ground Floor staircases and the elevator entrances were used to estimate stair to elevator use ratio for ascent and descent from/to the Ground Floor of a University building at Baseline (January to March 2020), First and Second Intervention Months (October 2020, November 2020, respectively). Stair promoting signage and a one-way system was implemented, in line with Government policy. Results: At Baseline, stair to elevator use ratio for ascent from and descent to the Ground Floor was 1.36 ± 0.02 and 1.88 ± 0.02 people, respectively. The ratio significantly increased in the first intervention month to 2.64 ± 0.09 and 3.96 ± 0.22 people for ascent and descent, respectively. However, the ratio decreased between the First and Second Intervention Months to 1.63 ± 0.06 and 3.05 ± 0.52 people for ascent and descent respectively. Conclusion: UK Government Covid-19 policy was effective at increasing stair use in a higher education setting

Associations between cerebrospinal fluid markers and cognition in ageing and dementia: A systematic review

A biomarker associated with cognition in neurodegenerative dementias would aid in the early detection of disease progression, complement clinical staging and act as a surrogate endpoint in clinical trials. The current systematic review evaluates the association between cerebrospinal fluid protein markers of synapse loss and neuronal injury and cognition. We performed a systematic search which revealed 67 studies reporting an association between cerebrospinal fluid markers of interest and neuropsychological performance. Despite the substantial heterogeneity between studies, we found some evidence for an association between neurofilament‐light and worse cognition in Alzheimer's diseases, frontotemporal dementia and typical cognitive ageing. Moreover, there was an association between cerebrospinal fluid neurogranin and cognition in those with an Alzheimer's‐like cerebrospinal fluid biomarker profile. Some evidence was found for cerebrospinal fluid neuronal pentraxin‐2 as a correlate of cognition across dementia syndromes. Due to the substantial heterogeneity of the field, no firm conclusions can be drawn from this review. Future research should focus on improving standardization and reporting as well as establishing the importance of novel markers such as neuronal pentraxin‐2 and whether such markers can predict longitudinal cognitive decline

Neurogranin in Alzheimer’s disease and ageing: a human post-mortem study

Neurogranin (Ng), a post-synaptic protein involved in memory formation, has been investigated as a biomarker in the cerebrospinal fluid (CSF) in Alzheimer's disease (AD) and ageing. CSF Ng levels are elevated in AD relative to healthy controls and correlate with cognition; however, few studies have focused on Ng abundance in the brain. Synapse loss in the brain correlates closely with cognitive decline in AD making synaptic biomarkers potentially important for tracking disease progression, but the links between synaptic protein changes in CSF and brain remain incompletely understood. In the current study, Ng abundance was examined in post-mortem human brain tissue across AD, healthy ageing (HA), and mid-life (ML) cohorts. Ng levels were quantified in three brain regions associated with cognitive change found during ageing and neurodegenerative diseases, namely the middle temporal gyrus, primary visual cortex and the posterior hippocampus using immunohistochemistry. To support immunohistochemical analysis, total homogenate and biochemically enriched synaptic fractions from available temporal gyrus tissues were examined by immunoblot. Finally, we examined whether Ng is associated with lifetime cognitive ageing. Ng levels were significantly reduced in AD relative to HA and ML cases across all regions. Additionally Ng was significantly reduced in HA in comparison to ML in the primary visual cortex. Immunoblotting confirms reduced Ng levels in AD cases supporting immunohistochemical results. Interestingly, there was also a significant reduction of synapse-associated Ng in our group who had lifetime cognitive decline in comparison to the group with lifetime cognitive resilience indicating loss of neurogranin in remaining synapses during ageing is associated with cognitive decline. Our findings indicate that increases in CSF Ng reflect loss of brain neurogranin and support the use of CSF Ng as a biomarker of AD and potentially of cognitive decline in healthy ageing