Biology subjects

Stuart, E.

Publications and source records attributed to Stuart, E..

10 recordsLinked to original sources

bioRxiv2026

PRNP mutations initially generate an alternatively misfolded PrP species that dissuades prion replication

Mutations in the PRNP gene, which encodes the prion protein (PrP), cause genetic prion disease. However, how PRNP mutations lead to spontaneous prion formation remains poorly understood. Building off the observation that expression of mutant bank vole PrP (BVPrP) in mice causes spontaneous prion disease, we sought to identify early events in prion formation by expressing mutant BVPrP in cultured cells lacking endogenous PrP. Expression of D178N- and E200K-mutant, but not wild-type BVPrP, in CAD5-PrP-/- and N2a-PrP-/- cells generates an alternatively misfolded PrP species, which we term PrPAM, that is detergent-insoluble and resistant to digestion with the protease thermolysin. PrPAM is also present in the brains of young, asymptomatic knock-in mice expressing mutant BVPrP. However, PrPAM does not appear to be a direct precursor of prions since 1) the prion disease-protective G127V substitution enhances PrPAM levels when coupled with the D178N or E200K mutations; 2) small molecule anti-prion drugs fail to reduce PrPAM accumulation in cells; 3) unlike cells expressing wild-type BVPrP, cells expressing D178N- or E200K-mutant BVPrP are largely resistant to infection with several prion strains; and 4) PrPAM-containing cell homogenate does not contain seeds that induce misfolding of wild-type BVPrP in either cells or the RT-QuIC assay. These results raise the possibility that prion disease-causing PRNP mutations initially generate a protective misfolded PrP species that is refractory to prion formation, which provides a potential explanation for why genetic prion diseases manifest later in life despite the presence of a mutation from birth.

neuroscience↗

bioRxiv2026

α-Synuclein strain homogeneity in multiple system atrophy clinical subtypes

Conformationally distinct strains of -synuclein aggregates are believed to contribute to the clinical and pathological diversity observed among synucleinopathies such as multiple system atrophy (MSA) and Parkinsons disease. Cases of MSA can be classified into two distinct clinical subtypes: the cerebellar variant, MSA-C, and the parkinsonian variant, MSA-P. To assess whether distinct -synuclein strains may be present in individuals with MSA-C versus MSA-P, we characterized the conformational and seeding properties of -synuclein aggregates in various brain regions from MSA-C and MSA-P patients and performed propagation studies in M83 transgenic mice. Biochemical fingerprinting of -synuclein aggregates using limited proteolysis and a conformational stability assay failed to reveal differences between MSA-C and MSA-P either before or after propagation in mice. Similarly, using brain extracts from either MSA patients or MSA-inoculated mice, MSA-C and MSA-P -synuclein aggregates exhibited indistinguishable seeding attributes in a seed amplification assay. Finally, no differences were observed in either the kinetics of disease progression or the extent of cerebral -synuclein deposition in M83 mice inoculated with either MSA-C or MSA-P, regardless of the brain region from which the injected -synuclein aggregates were derived. These results suggest that MSA clinical subtypes are unlikely to arise due to distinct -synuclein strains. Instead, our findings support a model in which the same -synuclein strain initially forms in different brain regions, leading to differences in disease manifestation.

neuroscience↗

bioRxiv2026

A self-complementary recombinant adeno-associated virus vector coding for an anchorless prion protein carrying the G127V mutation extends survival in a rodent prion disease model

The replacement of a single codon in the human prion gene, causing the substitution of glycine with valine at position 127 (G127V) of the prion protein (PrP), prevents development of prion disease. We set out to explore if prion disease survival extension manifests in mice if the V127 mutant is delivered through a recombinant adeno-associated virus (rAAV) packaged as a self-complementary DNA. The notorious delivery limitations of rAAVs were overcome using a cross-correction approach that relied on the expression of the mutation in the context of glycosylphosphatidylinositoI-anchorless ({Delta}GPI) PrP. In this proof-of-concept study, we inoculated Rocky Mountain Laboratory (RML) prions into knock-in mice, in which the endogenous murine prion protein gene (Prnp) was replaced with the bank vole prion protein gene (BvPrnp). Prion-inoculated mice that were retro-orbitally transduced with a protective rAAV vector encoding BvPrnpV127{Delta}GPI survived [~]50 days longer than control mice that were unprotected. A deep proteomic analysis revealed that BvPrnpV127{Delta}GPI was protective by slowing perturbations to the proteome observed in late-stage RML prion disease. In addition to capturing details of synaptic decay and depletion of proteins in proximity to PrP, the proteomic dataset revealed the identity of proteins of potential diagnostic value that may be central to the brains attempt to fight prion disease by contributing to astrocytosis or microgliosis, by coping with calcium influx, or by enhancing the endoplasmic reticulum processing of essential proteins. Taken together, our results demonstrate that a gene therapy based on a GPI-anchorless PrP containing the G127V mutation can delay the onset of prion disease in mice, providing a framework for development of a corresponding therapy in humans. AUTHOR SUMMARYA rare change in the human prion protein, involving a single building block, has been linked to strong protection against prion diseases--fatal neurodegenerative disorders. This study tested whether that protective effect could be reproduced using gene therapy in mice. To this end, we exposed the animals to infectious prions and then delivered the protective version of the protein into mice using a viral carrier. Treated mice survived about seven weeks longer than untreated animals, showing that the approach can meaningfully slow disease progression. To understand why, we examined changes in brain proteins during disease and found that treatment helped preserve the normal protein levels of cellular proteins, particularly those involved in communication between nerve cells. The analysis also identified proteins altered in the disease that are linked to the brains defense responses, including inflammation, stress handling, and protein processing, some of which may serve as future disease markers. Importantly, the limited protection observed was not due to poor delivery of the therapy but likely reflects biological limits of the model used. Overall, the findings support the idea that gene therapies based on naturally protective human variants could help slow prion diseases and improve understanding of how the brain responds to them.

neuroscience↗

bioRxiv2025

Stochastic Misfolding Drives the Emergence of Distinct α-Synuclein Strains

The existence of -synuclein conformational strains provides a potential explanation for the clinical and pathological differences among synucleinopathies such as Parkinsons disease and multiple system atrophy. However, how distinct -synuclein strains are formed in vivo remains unknown. Here, we examined whether unique strains of self-propagating -synuclein aggregates can arise within a consistent molecular environment. Unexpectedly, we observed conformational heterogeneity between individual preparations of -synuclein pre-formed fibrils (PFFs) generated by polymerizing recombinant wild-type or A53T-mutant human -synuclein under identical conditions. Moreover, we found that -synuclein aggregates formed spontaneously in the brains of a transgenic synucleinopathy mouse model were conformationally diverse, leading to the identification of three distinct disease subtypes. Propagation of putative PFF- and brain-derived -synuclein strains in mice initiated several distinct synucleinopathies, characterized by differences in disease onset times, cerebral -synuclein deposition patterns, and the conformational attributes of -synuclein aggregates. The conformational diversity of -synuclein aggregates across PFF preparations and between the brains of individual transgenic mice demonstrates that -synuclein can spontaneously form multiple self-propagating strains within an identical environment both in vitro and in vivo. This suggests that stochastic misfolding into distinct aggregate structures drives the emergence of -synuclein strains and implies that the intrinsic variability of common synucleinopathy research tools must be considered when designing and interpreting experiments.

neuroscience↗

bioRxiv2024

The Brain Interactome of a Permissive Prion Replication Substrate

Bank voles are susceptible to prion strains from many different species, yet the molecular mechanisms underlying the ability of bank vole prion protein (BVPrP) to function as a universal prion acceptor remain unclear. Potential differences in molecular environments and protein interaction networks on the cell surface of brain cells may contribute to BVPrPs unusual behavior. To test this hypothesis, we generated knock-in mice that express physiological levels of BVPrP (M109 isoform) and employed mass spectrometry to compare the interactomes of mouse (Mo) PrP and BVPrP following mild in vivo crosslinking of brain tissue. Substantial overlap was observed between the top interactors for BVPrP and MoPrP, with established PrP-interactors such as neural cell adhesion molecules, subunits of Na+/K+-ATPases, and contactin-1 being equally present in the two interactomes. We conclude that the molecular environments of BVPrP and MoPrP in the brains of mice are very similar. This suggests that the unorthodox properties of BVPrP are unlikely to be mediated by differential interactions with other proteins.

neuroscience↗

bioRxiv2024

α-Synuclein strain propagation is independent of cellular prion protein expression in transgenic mice

The cellular prion protein, PrPC, has been postulated to function as a receptor for -synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of -synuclein aggregates in neurodegenerative disorders such as Parkinsons disease. To test this hypothesis, we compared the propagation behavior of two different -synuclein aggregate strains in M83 transgenic mice that either expressed or did not express PrPC. Following intracerebral inoculation with the S or NS strain, the presence of PrPC had minimal influence on -synuclein strain-specified attributes such as the kinetics of disease progression, the extent of cerebral -synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced -synuclein deposits, and the structural fingerprints of protease-resistant -synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of -synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of -synuclein strains. Thus, other putative receptors or cell-to-cell propagation mechanisms may play a larger role in the spread of -synuclein aggregates during disease.

neuroscience↗

bioRxiv2023

Anti-prion drugs do not improve survival in knock-in models of inherited prion disease

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Paradoxically, the combination of Anle138b and IND24 appeared to accelerate disease by 16% and 26% in kiBVIE200K and kiBVID178N mice, respectively, and accelerated the aggregation of mutant PrP molecules in vitro. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions.

pharmacology and toxicology↗

bioRxiv2023

Convergent generation of atypical prions in knock-in mouse models of genetic prion disease

Most cases of human prion disease arise due to spontaneous misfolding of wild-type or mutant prion protein. Though recapitulating spontaneous prion conversion in animal models has proven challenging, transgenic mice expressing the misfolding-prone bank vole prion protein (BVPrP) recreate certain key aspects of sporadic and genetic prion disease. However, it remains unclear whether spontaneous prion generation can occur in the absence of protein over-expression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knock-in mice expressing physiological levels of either wild-type or mutant BVPrP with isoleucine at codon 109. While mice expressing wild-type BVPrP remained free from neurological disease, a subset of knock-in mice expressing BVPrP with mutations that cause either fatal familial insomnia (D178N) or familial Creutzfeldt-Jakob disease (E200K) developed progressive neurological illness. Brains from spontaneously ill knock-in mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant prion protein. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP knock-in mice transmitted disease to mice expressing wild-type BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical both pre- and post-transmission, suggesting that both mutations guide the formation of a highly similar atypical prion strain. These findings imply that knock-in mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. Therefore, these mice represent useful tools for studying the early stages of genetic prion diseases.

neuroscience↗

bioRxiv2019

Is useful research data usually shared? An investigation of genome-wide association study summary statistics

Primary data collected during a research study is increasingly shared and may be re-used for new studies. To assess the extent of data sharing in favourable circumstances and whether such checks can be automated, this article investigates the summary statistics of primary human genome-wide association studies (GWAS). This type of data is highly suitable for sharing because it is a standard research output, is straightforward to use in future studies (e.g., for secondary analysis), and may be already stored in a standard format for internal sharing within multi-site research projects. Manual checks of 1799 articles from 2010 and 2017 matching a simple PubMed query for molecular epidemiology GWAS were used to identify 330 primary human GWAS papers. Of these, only 10.6% reported the location of a complete set of GWAS summary data, increasing from 4.3% in 2010 to 16.8% in 2017. Whilst information about whether data was shared was usually located clearly within a data availability statement, the exact nature of the shared data was usually unspecified. Thus, data sharing is the exception even in suitable research fields with relatively strong norms regarding data sharing. Moreover, the lack of clear data descriptions within data sharing statements greatly complicates the task of automatically characterising shared data sets.

scientific communication and education↗

bioRxiv2017

Gradual Cerebral Hypoperfusion Impair Fear Conditioning and Object Recognition Learning and Memory in Mice: Potential Roles of Neurodegeneration and Cholinergic Dysfunction

In the present study, male C57BL/6J mice were subjected to gradual cerebral hypoperfusion by implanting an ameroid constrictor (AC) on the left common carotid artery (CCA) and a stenosis on the right CCA. In the sham group, all surgical procedures were kept the same except no AC was implanted and stenosis was not performed. One month following the surgical procedures, fear conditioning and object recognition tests were conducted to evaluate learning and memory functions and motor functions were assessed using a balance beam test. At the experimental endpoint, mice were perfused and brains were collected for immunostaining and histology. Learning and memory as well as motor functions were significantly impaired in the hypoperfusion group. The immunoreactivity to choline acetyltransferase was decreased in dorsal striatum and basal forebrain of the hypoperfusion group indicating that cholinergic tone in these brain regions was compromised. In addition, an increased number of Fluoro-Jade positive neurons was also found in cerebral cortex, dorsal striatum and hippocampus indicating neurodegeneration in these brain regions. Based on this pattern of data, we argued that this mouse model would be a useful tool to investigate the therapeutic interventions for the treatment of vascular dementia. Additionally, this model could be employed to exploit the effect of microvascular occlusions on cognitive impairment in the absence and presence of Alzheimer pathology.

neuroscience↗