Biology subjects

Schmitt-Ulms, G.

Publications and source records attributed to Schmitt-Ulms, G..

9 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

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

The Protein Disulfide Isomerase P4HB/PDIA1 Modulates PrPC Levels and Prion Replication

Prions are misfolded, self-propagating versions of the cellular prion protein (PrPC) that cause invariably fatal transmissible neurodegenerative diseases in humans and animals. Little is known about how prions replicate in the brain, including whether other proteins participate in prion replication in vivo. Several members of the protein disulfide isomerase family have been shown to reside in close spatial proximity to PrPC in cells and mice, implying that they could be involved in prion biogenesis. Here, we show that stable knock-down of the protein disulfide isomerase P4HB (also called PDIA1) in prion-susceptible CAD5 cells reduces PrPC levels and results in lower levels of protease-resistant PrP (PrPres), a marker of infectious prions, following infection with two different prion strains. Partial reduction of P4HB activity using the P4HB-selective inhibitor KSC-34 also decreases PrPC levels in uninfected CAD5 cells whereas treatment of prion-infected CAD5 cells with KSC-34 results in higher levels of PrPres. A proportion of P4HB reaches the cell surface where PrPC is located, and a secreted P4HB variant increases PrPres levels in cells. Collectively, these results suggest that P4HB facilitates prion replication in cells by stabilizing PrPC and potentially acting as a chaperone that directly modulates prion conversion. Thus, targeting P4HB during prion disease may have therapeutic benefit.

neuroscience↗

bioRxiv2025

Tau deletion results in sex-dependent modulation of synaptic weakening in rat hippocampus

Tangles, a defining characteristic of Alzheimers disease (AD), are composed principally of hyperphosphorylated and misfolded tau species. However, dysregulation of tau precedes tangle formation by many years and is associated with cognitive decline, the best functional correlate of which is synaptic weakening and eventual synaptic loss. Although tau is present at synapses, its normal synaptic function is poorly understood - information vital to the rational development of tau-modifying therapies. Since rats have a greater cognitive repertoire than mice and display more robust signs of tau pathology in AD models, we developed a tau knockout (Mapt-/-) rat to investigate the impact of tau elimination on synaptic function. We observed that long-term depression (LTD), a form of synaptic weakening, is enhanced in the hippocampus of male, but not female, rats. This enhanced LTD was dependent on the synaptic activation of group I metabotropic glutamate receptors and involved altered synaptic actin polymerization. These studies therefore provide mechanistic insights into how tau modulates synaptic function via regulation of the actin cytoskeleton. Our findings are relevant to the understanding of the physiological roles of synaptic tau, the functional consequences of tau-lowering therapies, and the influence of sex in AD susceptibility.

neuroscience↗

bioRxiv2024

Transient receptor potential vanilloid channel 2 contributes to multi-modal endoplasmic reticulum and perinuclear space dilations that can also be observed in prion-infected mice

Our recent work on the prion protein and Na+,K+-ATPases (NKAs) led us to revisit data from over 50 years ago, which suggested a similarity between vacuolation phenotypes in rodents poisoned with cardiac glycosides (CGs) and spongiform degeneration in prion disease. At that time, this hypothesis was dismissed because the vacuolation observed in prion diseases affects neurons, whereas CG poisoning in rodent brains led to swellings of the endoplasmic reticulum (ER) in astrocytes. We speculated that this difference might be specific to rodents and document here that the vacuolation shifts to neurons in mice expressing a humanized NKA 1 subunit. Next, we investigated the molecular mechanisms that could cause similar ER vacuolation in human cells in vitro. We found that certain stressors--such as overexpression of NKA subunits and exposure to specific toxins known to trigger the unfolded protein response--can induce a phenotype characterized by profound ER dilation that is most strikingly observed for the perinuclear space (PNS). The ion imbalance typically caused by functional NKAs does not contribute to this phenotype. In fact, it can occur even with the overexpression of catalytically inactive NKAs. Several lines of evidence, generated with pharmacological agents, ion-specific dyes, antagonists, and truncated expression constructs, suggest a calcium leak channel in the ER, known as transient receptor potential vanilloid 2 (TRPV2), plays a role in this ER and PNS dilation. Additionally, we observed that the formation of these vacuoles coincides with a decrease in steady-state levels of the lipid kinase PIKFYVE, which is recognized for its role in endolysosomal fission and fusion processes. Finally, we found evidence of vacuoles in cryo-sectioned brains of prion-infected mice that can be filled with a fluorescent marker targeted to the ER and PNS. This raises the possibility that this vacuolation phenomenon contributes to spongiform degeneration seen in prion diseases.

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

The Molecular Determinants of a Universal Prion Acceptor

In prion diseases, the species barrier limits the transmission of prions from one species to another. However, cross-species prion transmission is remarkably efficient in bank voles, and this phenomenon can be recapitulated in mice by expression of the bank vole prion protein (BVPrP). The molecular determinants of BVPrPs ability to function as a universal or near-universal acceptor for prions remain incompletely defined. Building on our finding that cultured cells expressing BVPrP can replicate both mouse and hamster prion strains, we conducted a systematic analysis to identify key residues in BVPrP that permit cross-species prion replication. Consistent with previous findings, we demonstrate that residues N155 and N170 of BVPrP, which are absent in mouse PrP but present in hamster PrP, are critical for cross-species prion replication. Additionally, BVPrP residues V112, I139, and M205, which are absent in hamster PrP but present in mouse PrP, are also required to enable replication of both mouse and hamster prions. Unexpectedly, we found that residues E227 and S230 near the C-terminus of BVPrP severely restrict the accumulation of prions following cross-species prion challenge, suggesting that they may have evolved to counteract the inherent propensity of BVPrP to misfold. PrP variants with an enhanced ability to replicate both mouse and hamster prions displayed accelerated spontaneous aggregation kinetics in vitro. These findings suggest that BVPrPs unusual properties are governed by a key set of amino acids and that the enhanced misfolding propensity of BVPrP may enable cross-species prion replication.

neuroscience↗

bioRxiv2022

Somatostatin slows Aβ plaque deposition in aged APPNL-F/NL-F mice by blocking Aβ aggregation in a neprilysin-independent manner

The molecular underpinnings that govern the endoproteolytic release of the amyloid beta peptide (A{beta}) from the amyloid precursor protein (APP) are now quite well understood. The same cannot be said for the events that precipitate the aggregation and amyloid deposition of A{beta} in Alzheimers disease (AD). The 14-amino-acid cyclic neuroendocrine peptide somatostatin (SST-14) has long been thought of as playing a role, foremost by controlling the expression of the A{beta} clearing enzyme neprilysin, and more recently by directly interacting with A{beta} oligomers. Missing have been in vivo data in a relevant A{beta} amyloidosis model. Here we addressed this shortcoming by crossing AppNL-F/NL-F mice with Sst-deficient mice of identical genetic background to assess if and how the presence of Sst influences key pathological hallmarks of A{beta} amyloidosis that develop in AppNL-F/NL-F mice after 10 months of age. Surprisingly, we found that Sst had no influence on whole brain neprilysin transcript, protein or activity levels, an observation that cannot be accounted for by a compensatory upregulation of the Sst paralog, cortistatin (Cort), that we observed in 15-month-old Sst-deficient mice. The absence of Sst did lead to a subtle but significant increase in the density of cortical A{beta} amyloid plaques. Follow-on western blot analyses of whole brain extracts indicated that Sst interferes with early steps of A{beta} assembly that manifest in Sst null brains through the appearance of SDS-stable smears of 55- 150 kDa. As expected, no effect of Sst on tau steady-state levels or its phosphorylation were observed. Results from this study are easier reconciled with an emerging body of data that point toward Sst affecting A{beta} amyloid plaque formation through direct interference with A{beta} aggregation rather than through its effects on neprilysin expression.

neuroscience↗

bioRxiv2022

Identification of a cardiac glycoside exhibiting favorable brain bioavailability and potency for reducing levels of the cellular prion protein

Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4-dehydro-oleandrin to engage with its cognate binding pocket within the NKA subunit. The improved brain bioavailability of C4-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases. AUTHOR SUMMARYPrion diseases are fatal neurodegenerative diseases for which there is no effective treatment. An abundance of data indicates that reducing the levels of a specific protein, termed the cellular prion protein (PrPC), would not only be safe but would delay disease onset and extend prion disease survival. This project builds on our recent discovery that PrPC binds to NKAs, specific cellular transport proteins that use energy to electrify cellular membranes by pumping charged potassium and sodium metals in and out of cells. We showed that targeting NKAs with their natural inhibitors, cardiac glycosides (CGs), causes brain cells to internalize and degrade NKAs, and that PrPC, on account of residing next to NKAs, gets co-degraded. Natural CGs act primarily on the heart. Here, we used computational modeling to identify a CG, termed KDC203, that is predicted to have favorable characteristics for brain applications. We show that KDC203 reduces PrPC levels by 84% in immortalized human brain-like cells grown in the dish. Moreover, we show that KDC203 exhibits relatively low toxicity, predominantly targets the brain when subcutaneously injected into mice, and has other promising pharmacological characteristics that recommend it for further evaluation for the treatment of prion diseases.

neuroscience↗