A Drug-Based Cellular Assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: A practical guide

Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrP(C)) in this process, remain enigmatic. A window into the normal function of PrP(C), and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines. We have used this drug-sensitizing effect to develop a simple Drug-Based Cell Assay (DBCA) that reproduces several features of mutant PrP toxicity observed in vivo, including the rescuing activity of wild-type PrP. In this paper, we present a detailed guide for executing the DBCA in several, different experimental settings, including a new slot blot-based format. This assay provides a unique tool for studying PrP cytotoxic and cytoprotective activities in cell culture.     

Ion channels induced by the prion protein

Prion diseases comprise a group of rapidly progressive and invariably fatal neurodegenerative disorders for which there are no effective treatments. While conversion of the cellular prion protein (PrP^C) to a β-sheet rich isoform (PrP^Sc) is known to be a critical event in propagation of infectious prions, the identity of the neurotoxic form of PrP and its mechanism of action remain unclear. Insights into this mechanism have been provided by studying PrP molecules harboring deletions and point mutations in the conserved central region, encompassing residues 105–125. When expressed in transgenic mice, PrP deleted for these residues (Δ105–125) causes a spontaneous neurodegenerative illness that is reversed by co-expression of wild-type PrP. In cultured cells, Δ105–125 PrP confers hypersensitivity to certain cationic antibiotics and induces spontaneous ion channel activity that can be recorded by electrophysiological techniques. We have utilized these drug-hypersensitization and current-inducing activities to identify which PrP domains and subcellular locations are required for toxicity. We present an ion channel model for the toxicity of Δ105–125 PrP and related mutants and speculate how a similar mechanism could mediate PrP^Sc-associated toxicity. Therapeutic regimens designed to inhibit prion-induced toxicity, as well as formation of PrP^Sc, may prove to be the most clinically beneficial.     

The N-terminal, polybasic region is critical for prion protein neuroprotective activity

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.     

A deleted prion protein that is neurotoxic in vivo is localized normally in cultured cells

The prion protein (PrP) possesses sequence-specific domains that endow the molecule with neuroprotective and neurotoxic activities, and that may contribute to the pathogenesis of prion diseases. To further define critical neurotoxic determinants within PrP, we previously generated Tg(ΔCR) mice that express a form of PrP harboring a deletion of 21 amino acids within the central domain of the protein [ Li et al., EMBO J . 26 (2007), 548 ]. These animals exhibit a neonatal lethal phenotype that is dose-dependently rescued by co-expression of wild-type PrP. In this study, we examined the localization and cell biological properties of the PrP(ΔCR) protein in cultured cells to further understand the mechanism of PrP(ΔCR) neurotoxicity. We found that the distribution of PrP(ΔCR) was identical to that of wild-type PrP in multiple cell lines of both neuronal and non-neuronal origin, and that co-expression of the two proteins did not alter the localization of either one. Both proteins were found in lipid rafts, and both were localized to the apical surface in polarized epithelial cells. Taken together, our results suggest that PrP(ΔCR) toxicity is not a result of mislocalization or aggregation of the protein, and more likely stems from altered binding interactions leading to the activation of deleterious signaling pathways.     

Ion channels induced by the prion protein: Mediators of neurotoxicity

Prion diseases comprise a group of rapidly progressive and invariably fatal neurodegenerative disorders for which there are no effective treatments. While conversion of the cellular prion protein (PrP^C) to a β-sheet rich isoform (PrP^Sc) is known to be a critical event in propagation of infectious prions, the identity of the neurotoxic form of PrP and its mechanism of action remain unclear. Insights into this mechanism have been provided by studying PrP molecules harboring deletions and point mutations in the conserved central region, encompassing residues 105–125. When expressed in transgenic mice, PrP deleted for these residues (Δ105–125) causes a spontaneous neurodegenerative illness that is reversed by co-expression of wild-type PrP. In cultured cells, Δ105–125 PrP confers hypersensitivity to certain cationic antibiotics and induces spontaneous ion channel activity that can be recorded by electrophysiological techniques. We have utilized these drug-hypersensitization and current-inducing activities to identify which PrP domains and subcellular locations are required for toxicity. We present an ion channel model for the toxicity of Δ105–125 PrP and related mutants and speculate how a similar mechanism could mediate PrP^Sc-associated toxicity. Therapeutic regimens designed to inhibit prion-induced toxicity, as well as formation of PrP^Sc, may prove to be the most clinically beneficial.Key words: prion, toxicity, ion channel, neurodegeneration, model     

Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105-125

To identify sequence domains important for the neurotoxic and neuroprotective activities of the prion protein (PrP), we have engineered transgenic mice that express a form of murine PrP deleted for a conserved block of 21 amino acids (residues 105-125) in the unstructured, N-terminal tail of the protein. These mice spontaneously developed a severe neurodegenerative illness that was lethal within 1 week of birth in the absence of endogenous PrP. This phenotype was reversed in a dose-dependent fashion by coexpression of wild-type PrP, with five-fold overexpression delaying death beyond 1 year. The phenotype of Tg(PrPDelta105-125) mice is reminiscent of, but much more severe than, those described in mice that express PrP harboring larger deletions of the N-terminus, and in mice that ectopically express Doppel, a PrP paralog, in the CNS. The dramatically increased toxicity of PrPDelta105-125 is most consistent with a model in which this protein has greatly enhanced affinity for a hypothetical receptor that serves to transduce the toxic signal. We speculate that altered binding interactions involving the 105-125 region of PrP may also play a role in generating neurotoxic signals during prion infection.     

Antagonistic roles of the N-terminal domain of prion protein to doppel

Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23-88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23-88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25-50 or 51-90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl .     

A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

In prion diseases, the infectious isoform of the prion protein (PrP^Sc) may subvert a normal, physiological activity of the cellular isoform (PrP^C). A deletion mutant of the prion protein (Δ105–125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP^C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.     

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.     

Toxicity of novel C-terminal prion protein fragments and peptides harbouring disease-related C-terminal mutations.

Mice expressing a C-terminal fragment of the prion protein instead of wild-type prion protein die from massive neuronal degeneration within weeks of birth. The C-terminal region of PrPc (PrP121-231) expressed in these mice has an intrinsic neurotoxicity to cultured neurones. Unlike PrPSc, which is not neurotoxic to neurones lacking PrPc expression, PrP121-231 was more neurotoxic to PrPc-deficient cells. Human mutations E200K and F198S were found to enhance toxicity of PrP121-231 to PrP-knockout neurones and E200K enhanced toxicity to wild-type neurones. The normal metabolic cleavage point of PrPc is approximately amino-acid residue 113. A fragment of PrPc corresponding to the whole C-terminus of PrPc (PrP113-231), which is eight amino acids longer than PrP121-231, lacked any toxicity. This suggests the first eight amino residues of PrP113-121 suppress toxicity of the toxic domain in PrP121-231. Addition to cultures of a peptide (PrP112-125) corresponding to this region, in parallel with PrP121-231, suppressed the toxicity of PrP121-231. These results suggest that the prion protein contains two domains that are toxic on their own but which neutralize each other's toxicity in the intact protein. Point mutations in the inherited forms of disease might have their effects by diminishing this inhibition.     

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.     

Stress-protective signalling of prion protein is corrupted by scrapie prions

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.     

An N-terminal polybasic domain and cell surface localization are required for mutant prion protein toxicity

There is evidence that alterations in the normal physiological activity of PrP(C) contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP(C) has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of Δ105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP(Sc) formation, suggesting that common structural features may govern both the functional activity of PrP(C) and its conversion to PrP(Sc).     

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.     

Mammalian prion protein suppresses Bax-induced cell death in yeast

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).     

GFP-tagged PrP supports compromised prion replication in transgenic mice

The ability of green fluorescent protein (GFP)-prion protein (PrP) fusions to support prion propagation has not been demonstrated. Here, we show that while transgenic mice expressing PrP tagged at its amino terminus with enhanced GFP, referred to as EGFPrP-N, supported prion replication, disease onset was prolonged, the brains of diseased mice did not exhibit typical disease neuropathology and disease-associated EGFPrP-N lacked the full spectrum of biochemical properties normally associated with PrP(Sc). Co-expression of wild-type PrP and EGFPrP-N substantially reduced prion incubation times and resulted in accumulation of protease-resistant EGFPrP(Sc)-N in the brains of transgenic mice as well as chronically infected cultured cells, suggesting that wild-type PrP rescued a compromised amino terminal function in EGFPrP-N. While our results show that EGFPrP(C)-N adopts a conformation necessary for the production of infectious prions, the synergistic interaction of wild-type and EGFPrP-N underscores the importance of the amino terminus in modulating prion pathogenesis.     

Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.     

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.     

Cytoplasmic prion protein induces forebrain neurotoxicity

The prion protein (PrP) is essential for the pathogenesis of prion disease. PrP has been detected in the cytosol of neurons and transgenic mice expressing PrP in the cytosol (cyPrP) under a pan-neuronal promoter developed rapid cerebellar granule neuron degeneration. Yet, it remains unclear whether cyPrP is capable to cause toxicity in other neuronal populations. Here, we report that transgenic mice expressing cyPrP in the forebrain neurons developed behavioral abnormalities including clasping and hyperactivity. These mice had reduced thickness in cortex and developed astrogliosis in hippocampal and cortical regions. Moreover, cyPrP in these mice was recognized by the A11 anti-oligomer antibody and was associated with the hydrophobic lipid core of membranes, indicating that cyPrP oligomer caused membrane perturbation contributes to cyPrP neurotoxicity. Together, our results clearly revealed that cyPrP is able to cause toxicity in different neuronal populations, supporting a role of cyPrP in PrP-mediated neurodegenerative disorders.     

Context dependent neuroprotective properties of prion protein (PrP)

Although it has been known for more than twenty years that an aberrant conformation of the prion protein (PrP) is the causative agent in prion diseases, the role of PrP in normal biology is undetermined. Numerous studies have suggested a protective function for PrP, including protection from ischemic and excitotoxic lesions and several apoptotic insults. On the other hand, many observations have suggested the contrary, linking changes in PrP localization or domain structure—independent of infectious prion conformation—to severe neuronal damage. Surprisingly, a recent report suggests that PrP is a receptor for toxic oligomeric species of a-β, a pathogenic fragment of the amyloid precursor protein, and likely contributes to disease pathogenesis of Alzheimer’s disease. We sought to access the role of PrP in diverse neurological disorders. First, we confirmed that PrP confers protection against ischemic damage using an acute stroke model, a well characterized association. After ischemic insult, PrP knockouts had dramatically increased infarct volumes and decreased behavioral performance compared to controls. To examine the potential of PrP’s neuroprotective or neurotoxic properties in the context of other pathologies, we deleted PrP from several transgenic models of neurodegenerative disease. Deletion of PrP did not substantially alter the disease phenotypes of mouse models of Parkinson’s disease or tauopathy. Deletion of PrP in one of two Huntington’s disease models tested, R6/2, modestly slowed motor deterioration as measured on an accelerating rotarod but otherwise did not alter other major features of the disease. Finally, transgenic overexpression of PrP did not exacerbate the Huntington’s motor phenotype. These results suggest that PrP has a context-dependent neuroprotective function and does not broadly contribute to the disease models tested herein.