Allen Herbst

Prion Protein Polymorphisms Affect Chronic Wasting Disease Progression

Analysis of the PRNP gene in cervids naturally infected with chronic wasting disease (CWD) suggested that PRNP polymorphisms affect the susceptibility of deer to infection. To test this effect, we orally inoculated 12 white-tailed deer with CWD agent. Three different PRNP alleles, wild-type (wt; glutamine at amino acid 95 and glycine at 96), Q95H (glutamine to histidine at amino acid position 95) and G96S (glycine to serine at position 96) were represented in the study cohort with 5 wt/wt, 3 wt/G96S, and 1 each wt/Q95H and Q95H/G96S. Two animals were lost to follow-up due to intercurrent disease. The inoculum was prepared from Wisconsin hunter-harvested homozygous wt/wt animals. All infected deer presented with clinical signs of CWD; the orally infected wt/wt had an average survival period of 693 days post inoculation (dpi) and G96S/wt deer had an average survival period of 956 dpi. The Q95H/wt and Q95H/G96S deer succumbed to CWD at 1,508 and 1,596 dpi respectively. These data show that polymorphisms in the PRNP gene affect CWD incubation period. Deer heterozygous for the PRNP alleles had extended incubation periods with the Q95H allele having the greatest effect.

Down-Regulation of Shadoo in Prion Infections Traces a Pre-Clinical Event Inversely Related to PrPSc Accumulation

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp a and Prnp b mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

A Quantitative Proteomic Approach to Prion Disease Biomarker Research: Delving into the Glycoproteome

Mass spectrometry (MS) -- based proteomic approaches have evolved as powerful tools for the discovery of biomarkers. However, the identification of potential protein biomarkers from biofluid samples is challenging because of the limited dynamic range of detection. Currently there is a lack of sensitive and reliable premortem diagnostic test for prion diseases. Here, we describe the use of a combined MS-based approach for biomarker discovery in prion diseases from mouse plasma samples. To overcome the limited dynamic range of detection and sample complexity of plasma samples, we used lectin affinity chromatography and multidimensional separations to enrich and isolate glycoproteins at low abundance. Relative quantitation of a panel of proteins was obtained by a combination of isotopic labeling and validated by spectral counting. Overall 708 proteins were identified, 53 of which showed more than 2-fold increase in concentration whereas 58 exhibited more than 2-fold decrease. A few of the potential candidate markers were previously associated with prion or other neurodegenerative diseases.

Apoptosis and necrosis mediate skeletal muscle fiber loss in age-induced mitochondrial enzymatic abnormalities.

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.

Prion disease diagnosis by proteomic profiling.

Definitive prion disease diagnosis is currently limited to postmortem assay for the presence of the disease-associated proteinase K-resistant prion protein. Using cerebrospinal fluid (CSF) from prion-infected hamsters, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines (SVM), we have identified peptide profiles characteristic of disease state. Using 10-fold leave-one-out cross-validation, we report a predictive accuracy of 72% with a true positive rate of 73% and a false positive rate of 27% demonstrating the suitability of using proteomic profiling and CSF for the development of multiple marker diagnostics of prion disease.

Deer Prion Proteins Modulate the Emergence and Adaptation of Chronic Wasting Disease Strains

ABSTRACT Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrPC). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrPC modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95+) with distinct biological properties. Transmission of first-passage tg60CWD-H95+ isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrPC primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrPC molecules results in the generation of novel CWD strains. IMPORTANCE CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrPC) to the emergence of a novel CWD strain (H95+). We show that, upon infection, deer expressing H95-PrPC molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95+ CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD.

Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone

Identification of genes expressed in response to prion infection may elucidate biomarkers for disease, identify factors involved in agent replication, mechanisms of neuropathology and therapeutic targets. Although several groups have sought to identify gene expression changes specific to prion disease, expression profiles rife with cell population changes have consistently been identified. Cuprizone, a neurotoxicant, qualitatively mimics the cell population changes observed in prion disease, resulting in both spongiform change and astrocytosis. The use of cuprizone-treated animals as an experimental control during comparative expression profiling allows for the identification of transcripts whose expression increases during prion disease and remains unchanged during cuprizone-triggered neuropathology. In this study, expression profiles from the brains of mice preclinically and clinically infected with Rocky Mountain Laboratory (RML) mouse-adapted scrapie agent and age-matched controls were profiled using Affymetrix gene arrays. In total, 164 genes were differentially regulated during prion infection. Eighty-three of these transcripts have been previously undescribed as differentially regulated during prion disease. A 0.4% cuprizone diet was utilized as a control for comparative expression profiling. Cuprizone treatment induced spongiosis and astrocyte proliferation as indicated by glial fibrillary acidic protein (Gfap) transcriptional activation and immunohistochemistry. Gene expression profiles from brain tissue obtained from cuprizone-treated mice identified 307 differentially regulated transcript changes. After comparative analysis, 17 transcripts unaffected by cuprizone treatment but increasing in expression from preclinical to clinical prion infection were identified. Here we describe the novel use of the prion disease mimetic, cuprizone, to control for cell population changes in the brain during prion infection.

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age.

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.

Mitochondrial Biogenesis Drives a Vicious Cycle of Metabolic Insufficiency and Mitochondrial DNA Deletion Mutation Accumulation in Aged Rat Skeletal Muscle Fibers

Aged muscles possess dysfunctional fibers that contain intracellular expansions of somatically derived mitochondrial DNA deletion mutations. At high abundance, these mutations disrupt the expression of mitochondrially-encoded protein subunits of the electron transport chain resulting in aerobic respiration deficient muscle fiber segments. These fiber segments atrophy and break contributing to the loss of muscle mass and function that occurs with age. By combining micro-dissection of individual muscle fibers with microarray analysis, we observed the response induced within these abnormal muscle fibers and detected an increase in many genes affecting metabolism and metabolic regulation. The transcriptional profile and subsequent protein validation suggested that a non-compensatory program of mitochondrial biogenesis was initiated. We hypothesized that this non-adaptive program of mitochondrial biogenesis was driving mtDNA deletion mutation accumulation. We tested this hypothesis by treating aged rats with β-Guanidinopropionic acid, a compound that stimulates mitochondrial biogenesis. β-Guanidinopropionic acid treatment increased muscle mitochondrial genome copy number and resulted in a 3.7 fold increase in the abundance of electron transport chain negative muscle fiber segments. We conclude that in electron transport system abnormal muscle fiber segments, a vicious cycle of metabolic insufficiency and non-compensatory mitochondrial biogenesis drive mtDNA deletion mutation accumulation.

Infectious Prions Accumulate to High Levels in Non Proliferative C2C12 Myotubes

Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrPC) into a disease specific isoform PrPSc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrPSc and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrPSc and that the level of infectivity produced in these post-mitotic cells, 105.5 L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.