The channel activities of the full-length prion and truncated proteins

Prion disease is a fatal neurodegenerative disorder, in which the cellular prion protein PrPC is converted to a misfolded prion which in turn is hypothesized to permeabilize cellular membranes. The pathways leading to toxicity in prion disease are not yet completely elucidated and whether it also includes formation of membrane pores remains to be answered. Prion protein consists of two domains: a globular domain (GD) and a flexible N-terminus (FT) domain. Although a proximal nine polybasic amino acid (FT(23-31)) sequence of FT is a prerequisite for cellular membrane permeabilization, other functional domain regions may influence FT(23-31) and its permeabilization. By using single-channel electrical recordings, we reveal that FT(23-50) dominates the membrane permeabilization within the full-length mouse PrP (mPrP(23-230)). The other domain of FT(51-110) or C-terminal domain down-regulates the channel activity of FT(23-50) and the full-length mouse PrP (mPrP(23-230)). The addition of prion mimetic antibody, POM1 significantly enhances mPrP(23-230) membrane permeabilization, whereas POM1-Y104A, a POM1 mutant that binds to PrP but cannot elicit toxicity has negligible effect on membrane permeabilization. Additionally, anti-N-terminal antibody POM2 or Cu2+ stabilizes FT domain, thus provoking FT(23-110) channel activity. Furthermore, our setup provides a more direct method without an external fused protein to study the channel activity of truncated PrP in the lipid membranes. We therefore hypothesize that the primary N-terminal residues are essential for membranes permeabilization and other functional segments play a vital role to modulate the pathological effects of PrP-medicated neurotoxicity. This may yield essential insights into molecular mechanisms of prion neurotoxicity to cellular membranes in prion disease.

Apparent stability masks underlying change in a mule deer herd with unmanaged chronic wasting disease

The contagious prion disease “chronic wasting disease” (CWD) infects mule deer (Odocoileus hemionus) and related species. Unchecked epidemics raise ecological, socioeconomic, and public health concerns. Prion infection shortens a deer’s lifespan, and when prevalence (proportion of adults infected) becomes sufficiently high CWD can affect herd dynamics. Understanding population responses over time is key to forecasting long-term impacts. Here we describe unexpected stability in prevalence and abundance in a mule deer herd where CWD has been left unmanaged. High apparent prevalence (~30%) since at least 2005 likely drove observed changes in the proportion and age distribution of wild-type native prion protein (PRNP) gene homozygotes among deer sampled. Predation by mountain lions (Puma concolor) may be helping keep CWD in check. Despite stable appearances, prion disease nonetheless impairs adult survival and likely resilience in this deer herd, limiting its potential for growth despite refuge from hunter harvest and favorable habitat and winter conditions.

Effects of the Pathological E200K Mutation on Human Prion Protein: A Computational screening and Molecular Dynamic approach

The Human Prion protein gene (PRNP) is mapped to short arm of chromosome 20 (20pter-12). Prion disease is associated with mutations in the Prion Protein encoding gene sequence. The mutations that occur in the prion protein could be divided into two types based on their influence on pathogenic potential: 1. Mutations that cause disease. 2. Disease-resistance mutations. Earlier studies found that the mutation G127V in the PRNP increases protein stability, whereas the mutation E200K, which has the highest mutation rate in the Prion protein, causes Creutzfeldt–Jakob disease (CJD) in humans and induces protein aggregation. We used a variety of bioinformatic algorithms, including SIFT, PolyPhen, I-Mutant, PhD-SNP, and SNP&GO, to predict the association of the E200K mutation with Prion disease. MD simulation is performed and graphs for RMSD, RMSF, Rg, DSSP, PCA, porcupine and FEL are generated to confirm and prove the stability of the wild type and mutant protein structures. The protein is analyzed for aggregation, and the results indicates more fluctuations in the protein structure during the simulation by the E200K mutation, however the G127V mutation makes protein structure stable against aggregation during the simulation.

Mapping prion pathology in mice using quantitative imaging: an MRI study

Human prion diseases are fatal neurodegenerative disorders which cause cognitive impairment and neurological deficits. Additional measures of tissue status are necessary for improving the sensitivity and specificity of clinical diagnosis as in many cases clinical forms of prion disease are commonly mistaken for other forms of dementia. To that effect, we developed a set of quantitative magnetic resonance-based tools, including magnetic resonance spectroscopy (MRS), magnetization transfer ratio (MTR) and quantitative T1 and T2 imaging to study the course of the disease in an animal model of prion disease. Using in vivo MTR, significant changes were detected in the cortex and thalamus of late-stage prion -infected mice as compared to littermates. In addition, we found a significant increase of MTR in thalamus and cortex of 80 dpi healthy mice when compared with 160 dpi healthy mice suggestive of changes occurring during the development of the brain. Using quantitative T2 mapping, significantly higher values were measured in thalamus of prion mice at all stages of the disease (T2=40ms) while T1 was found to be significantly higher in cortex (T1=1.89s) and hippocampus, albeit only in late-stage prion mice as compared to aged-matched controls (T1=1.67s). Using quantitative MRS significant changes were detected in glutamate (Glu) and myo-inositol (Ins) at all stages of prion disease when compared with the control group. NAA, Cr, Lactate and Lipids were only found to be significantly different at early and late stages of the disease while Taurine (Tau) was only significantly increased in the asymptomatic stage without any significant change at early and late stages of the disease. These changes in MRI and MRS signals, which precede clinical signs of disease, could provide insights into the pathogenesis of this disease and may enable early detection of pathology.

Validation and Application of Skin RT-QuIC to Patients in China with Probable CJD

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrPSc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test the diagnostic potential of RT-QuIC of skin specimens for probable sCJD, we collected the paired skin and CSF samples from 51 recruited living patients referred to the Chinese CJD surveillance center, including 34 probable sCJD, 14 non-CJD, and 3 genetic prion disease (gPrD). The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate. Using skin RT-QuIC assay, 91.2% (31/34) probable sCJD patients, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while 85.7% (12/14) non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 14 probable sCJD patients. Analysis of the reactivity of 38 positive skin RT-QuIC tests revealed that the positive rates in the preparations of 10−2, 10−3 and 10−4 diluted skin samples were 88.6% (39/44), 63.6% (28/44), and 25.0% (11/44), respectively. Eleven probable sCJD patients donated two skin specimens collected at different sites simultaneously. Although 95.5% (21/22) skin RT-QuIC elicited positive reaction, the reactivity varied. Our preliminary data indicate high sensitivity and specificity of skin RT-QuIC in prion detection for Chinese probable sCJD and highlight that skin prion-seeding activity is a reliable biomarker for premortem diagnosis of human prion disease.

Can Chemical Exchange Saturation Transfer (CEST MRI) be used as biomarker of disease progression in Prion Disease?

Human prion diseases are fatal neurodegenerative disorders that may have prolonged asymptomatic incubation periods. However, the underlying mechanism by which prions cause brain damage remains unclear. In turn, characterization of early pathological aspects would be of benefit for the diagnosis and potential treatment of these progressive neurodegenerative disorders. We investigated chemical exchange saturation transfer (CEST) MRI based on its exquisite sensitivity to cytosol protein content as a surrogate for prion disease pathology. Three groups of prion-infected mice at different stages of the disease underwent conventional magnetic resonance imaging and CEST MRI at 9.4T. For each mouse, chemical exchange contrasts were measured by applying five RF powers at various frequency offsets using magnetization transfer asymmetries. Relayed Nuclear Overhauser effects (NOE*) and amide proton transfer (APT*) were also assessed. For comparison, CEST MRI measurements were also made in healthy control mice brains. Here we show that alterations in CEST signal were detected before structural modifications or any clinical signs of prion disease. The detected CEST signal displayed different patterns at different stages of the disease indicating its potential for use as a longitudinal marker of disease progression. Highly significant correlations were found between CEST metrics and histopathological findings. A decline in NOE signal was positively correlated with abnormal prion protein deposition (R2 = 0.91) in the thalami of prion infected mice. Moreover, the NOE signal was negatively correlated with astrogliosis (R2 = 0.71) in the thalamus. No significant correlations were detected between NOE signals and spongiosis. MTR asymmetry at 3.5 ppm was also correlated with astrogliosis (R2 = 0.59), and prion protein deposition (R2 = 0.63) in thalamus. No significant changes were detected in APT* between prion-infected and control mice at all stages of the disease. Finally, MTR asymmetry between 2.8 and 3.2 ppm was correlated with prion protein deposition (R2 = 0.47) in the thalamus of prion -infected mice. To conclude, CEST MRI has potential utility as a biomarker of neurodegenerative processes in prion disease

Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.