Development of an Automated Capillary Immunoassay to Detect Prion Glycotypes in Creutzfeldt-Jakob Disease.

Creutzfeldt-Jakob disease (CJD) comprises a group of transmissible neurodegenerative diseases with vast phenotypic diversity. Sporadic CJD heterogeneity is predominantly influenced by the genotype at codon 129 of the prion-encoding gene and the molecular weight of PrPSc fragments after protease digestion, resulting in a classification of 6 subtypes of CJD (MM1, MM2, MV1, MV2, VV1, and VV2). The majority of cases with CJD can be distinguished using this classification system. However, a number of reported CJD cases are phenotypically unique from others within their same subtype, such as variably protease-sensitive prionopathies, or exist as a mixture of subtypes within the same patient. Western blotting of brain tissue, along with the genotyping of codon 129 of the prion-encoding gene, is considered the "gold standard" for the biochemical characterization of CJD. Western blotting requires a significant amount of prion protein for detection, is labor-intensive, and is also associated with high interassay variability. In addition to these limitations, a growing body of research suggests that unique subtypes of CJD are often undetected or misdiagnosed using standard diagnostic western blotting protocols. Consequently, we successfully optimized and developed a capillary-based western assay using the JESS Simple Western (ProteinSimple) to detect and characterize prion proteins from patients with CJD. We found that this novel assay consistently differentiated CJD type 1 and type 2 cases with a limit of detection 10 to 100× higher than traditional western blotting. Cases with CJD in which type 1 and type 2 coexist within the same brain region can be detected using type 1-specific and type 2-specific antibodies, and we found that there was remarkable specificity for the detection of cases with variably protease-sensitive prionopathy. The assay presented displays outstanding sensitivity, allowing for the preservation of valuable samples and enhancing current detection methods.

Proteomic Screen of Brain Glycoproteome Reveals Prion Specific Marker of Pathogenesis.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE-infected and aged-matched control mice using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Since the brain proteome is too complex to resolve all proteins using 2D-DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat-germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin-3 (CNN3), peroxiredoxin-6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE-infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.

MicroRNA-16 targets mRNA involved in neurite extension and branching in hippocampal neurons during presymptomatic prion disease.

The mechanisms that lead to neuronal death in neurodegenerative diseases are poorly understood. Prion diseases, like many more common disorders such as Alzheimer's and Parkinson's diseases, are characterized by the progressive accumulation of misfolded disease-specific proteins. The earliest changes observed in brain tissue include a reduction in synaptic number and retraction of dendritic spines, followed by reduced length and branching of neurites. These pathologies are observable during presymptomatic stages of disease and are accompanied by altered expression of transcripts that include miRNAs. Here we report that miR-16 localized within hippocampal CA1 neurons is increased during early prion disease. Modulating miR-16 expression in mature murine hippocampal neurons by expression from a lentivirus, thus mimicking the modest increase seen in vivo, was found to induce neurodegeneration. This was characterized by retraction of neurites and reduced branching. We performed immunoprecipitation of the miR-16 enriched RISC complex, and identified associated transcripts from the co-immunoprecipitated RNA (Ago2 RIP-Chip). These transcripts were enriched with predicted binding sites for miR-16, including the validated miR-16 targets APP and BCL2, as well as numerous novel targets. In particular, genes within the neurotrophin receptor mediated MAPK/ERK pathway were potentially regulated by miR-16; including TrkB (NTRK2), MEK1 (MAP2K1) and c-Raf (RAF). Increased miR-16 expression in neurons during presymptomatic prion disease and reduction in proteins involved in MAPK/ERK signaling represents a possible mechanism by which neurite length and branching are decreased during early stages of disease.

Non-Productive Infection of Glial Cells with SARS-CoV-2 in Hamster Organotypic Cerebellar Slice Cultures.

The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement.

Regulation of cardiomyocyte Glut4 expression by ZAC1.

The transcription factor ZAC1 is expressed in a variety of tissues including the developing heart, but its physiological role is unclear. We examined the role of ZAC1 in regulating expression of the insulin-responsive glucose transporter GLUT4 and whether ZAC1 expression is altered in cardiomyocyte hypertrophy. We demonstrated expression of Zac1 mRNA and protein in rat cardiomyocytes by PCR and Western blotting, respectively. Using a combination of chromatin immunoprecipitation and luciferase assays, we showed that ZAC1 regulates Glut4 expression via a specific binding site in the Glut4 promoter. Overexpression of ZAC1 increased Glut4 mRNA and protein expression and resulted in increased glucose uptake in cardiomyocytes as determined by a fluorescent analog uptake assay. Induction of hypertrophy by phenylephrine or isoproterenol resulted in increased Zac1 expression. We identified a novel putative promoter in the Zac1 gene and demonstrated increased binding of MEF2 to this promoter in response to hypertrophic stimulation. MEF2 regulated transactivation of the Zac1 promoter and ZAC1 protein expression. This work identifies ZAC1 as a novel and previously unknown regulator of cardiomyocyte Glut4 expression and glucose uptake. Our results also implicate MEF2 as a regulator of ZAC1 expression in response to induction of hypertrophy.

Factors affecting the accuracy of urine-based biomarkers of BSE.

BACKGROUND: Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD. OBJECTIVE: Urine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test. RESULTS: Two-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender. CONCLUSION: Despite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.

Analysis of clusterin glycoforms in the urine of BSE-infected Fleckvieh-Simmental cows.

Currently approved tests for bovine spongiform encephalopathy (BSE) monitoring in cattle are based on the detection of the disease-related isoform of the prion protein in brain tissue and consequently are only suitable for postmortem diagnosis. Previously, to meet the demand for an antemortem test based on a matrix that would permit easy access and repeated sampling, two-dimensional differential gel electrophoresis (2D-DIGE) was used to perform an unbiased screen of bovine urine. Data demonstrated the altered abundance of particular isoforms of the multifunctional glycoprotein clusterin in urine samples obtained from BSE-infected and age-matched Fleckvieh-Simmental cattle. Unfortunately, the use of particular isoforms of a relatively abundant urine protein such as clusterin for diagnosis faces many of the same challenges encountered in tests based on PrP(d) detection. In both instances the specific detection of the marker protein is complicated by the high background levels of proteins with identical amino acid sequences, but lacking the disease-specific posttranslational modifications or configuration. The goal of the current study was to define the distinguishing characteristics of the clusterin isoforms observed. Biochemical and mass spectrometry analyses in combination with the generation of bovine clusterin subunit-specific antibodies enabled us to demonstrate that it was ß-subunits of clusterin possessing N-linked glycans of complex structure that exhibited differential abundance in response to BSE infection. The charateristic highly glycosylated clusterin ß-subunit was detectable as early as 16 mo post infection (mpi) by one-dimensional (1D) Western blot analysis of urine obtained from BSE-infected cattle.

Evaluation of a commercially-available surrogate virus neutralization test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.

Exposure Risk of Chronic Wasting Disease in Humans.

The majority of human prion diseases are sporadic, but acquired disease can occur, as seen with variant Creutzfeldt-Jakob disease (vCJD) following consumption of bovine spongiform encephalopathy (BSE). With increasing rates of cervid chronic wasting disease (CWD), there is concern that a new form of human prion disease may arise. Currently, there is no evidence of transmission of CWD to humans, suggesting the presence of a strong species barrier; however, in vitro and in vivo studies on the zoonotic potential of CWD have yielded mixed results. The emergence of different CWD strains is also concerning, as different strains can have different abilities to cross species barriers. Given that venison consumption is common in areas where CWD rates are on the rise, increased rates of human exposure are inevitable. If CWD was to infect humans, it is unclear how it would present clinically; in vCJD, it was strain-typing of vCJD prions that proved the causal link to BSE. Therefore, the best way to screen for CWD in humans is to have thorough strain-typing of harvested cervids and human CJD cases so that we will be in a position to detect atypical strains or strain shifts within the human CJD population.

The basic helix-loop-helix transcription factor scleraxis regulates fibroblast collagen synthesis.

The transcription factor scleraxis has been implicated in regulating the development of collagen-rich tissues such as tendons and cardiac valves, but its role in general collagen synthesis in the heart is unknown. Scleraxis expression in cardiac fibroblasts was examined, and its ability to regulate gene expression of collagen I alpha 2, the predominant cardiac collagen isoform, was assayed. Using real-time PCR, we demonstrate here that scleraxis mRNA is up-regulated by the profibrotic agonist TGF-beta(1) in rat cardiac myofibroblasts, and that phenoconversion of fibroblasts to myofibroblasts similarly increases scleraxis expression. Over-expression of scleraxis in NIH-3T3 or primary rat cardiac fibroblasts by adenoviral gene delivery is sufficient to significantly increase collagen I alpha 2 gene expression. Using luciferase reporter assays, we demonstrate that scleraxis transactivates the human collagen I alpha 2 promoter in a DNA- and protein-binding dependent manner. Intriguingly, examination of infarcted rat hearts reveals a nearly four-fold increase in scleraxis expression in the infarct scar, but not in non-infarcted tissue. These data support a novel and previously unknown role for scleraxis in the regulation of collagen gene expression in the heart, including in post-infarct scar formation.

Efficient knockdown of human prnp mRNA expression levels using hybrid hammerhead ribozymes.

Prion diseases are invariably fatal infectious diseases of the central nervous system. The prion protein has been identified as the underlying causative agent as PrP knockout mice (prnp(0/0)) are resistant to infection. This suggests that a significant reduction in the expression levels of PrP(c) should interrupt disease progression. Accomplishing this in vivo, upon presentation of symptoms, requires a mechanism that significantly reduces prnp mRNA levels while lacking potential side effects that may be cytotoxic or lethal to the host. Hybrid hammerhead ribozymes (HyHamRzs) include both a helicase recruitment signal and a tRNA(Val) promoter. HyHamRzs offer a means of highly specific and significant mRNA cleavage. In this study, data demonstrate increased activity granted to HamRzs by the addition of the helicase recruitment signal. Results show that three different HyHamRzs, targeting different locations along the full length prnp mRNA, reduced expression levels by greater than 95% relative to the control. It is postulated that HyHamRzs, modified to enhance serum stability and delivered intravenously to neurons by forming a complex with the modified rabies virus G protein (RVG), may offer a potential gene therapy strategy.

The identification of disease-induced biomarkers in the urine of BSE infected cattle.

BACKGROUND: The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob Disease (vCJD) have led to profound changes in the production and trade of agricultural goods. The rapid tests currently approved for BSE monitoring in slaughtered cattle are all based on the detection of the disease related isoform of the prion protein, PrPd, in brain tissue and consequently are only suitable for post-mortem diagnosis. OBJECTIVES: In instances such as assessing the health of breeding stock for export purposes where post-mortem testing is not an option, there is a demand for an ante-mortem test based on a matrix or body fluid that would permit easy access and repeated sampling. Urine and urine based analyses would meet these requirements. RESULTS: Two dimensional differential gel eletrophoresis (2D-DIGE) and mass spectrometry analyses were used to identify proteins exhibiting differential abundance in the urine of BSE infected cattle and age matched controls over the course of the disease. Multivariate analyses of protein expression data identified a single protein able to discriminate, with 100% accuracy, control from infected samples. In addition, a subset of proteins were able to predict with 85% +/- 13.2 accuracy the time post infection that the samples were collected. CONCLUSION: These results suggest that in principle it is possible to identify biomarkers in urine useful in the diagnosis, prognosis and monitoring of disease progression of transmissible spongiform encephalopathy diseases (TSEs).

Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.

A radiation-induced adaptive response prolongs the survival of prion-infected mice.

Previously, it has been demonstrated that an "adaptive response" that includes the prevention, repair, and removal of oxidative damage can be evoked by radiation at dose rates substantially lower than those at which risks have been observed. The exact pathogenic mechanism of prion diseases is unknown, but circumstantial evidence suggests that oxidative stress plays a central role. Exposure of prion-infected mice to four 500 mGy/fraction doses of (60)Co γ-radiation administered every other day at a low dose rate (0.5 mGy/min) starting at 2 days before infection, 7 days postinfection (dpi), or 50 dpi significantly prolonged the survival of infected mice. The 500-mGy radiation treatments started at 50 dpi also significantly prolonged the symptom-free period of the disease and caused a significant delay in the rise of the 8-hydroxydeoxyguanosine concentration observed in the urine of nonirradiated infected mice at 98 dpi. The duration of the reduction in oxidative stress achieved by the radiation treatments was similar in length to the prolonged survival of the irradiated mice. This suggests that the adaptive response induced by low-dose whole-body radiation treatments prolongs the survival of prion-infected mice by reducing oxidative stress.

The role of sex in cardiac function and disease.

In the past decade, increasing attention has been paid to the importance of sex in the etiology of cardiac dysfunction. While focus has been primarily on how sex modulates atherogenesis, it is becoming clear that sex is both a predictor of outcome and an independent risk factor for a number of other cardiac diseases. Animal models and human studies have begun to shed light on the mechanisms by which sex influences the function of cardiomyocytes in health and disease. This review will survey the current literature on cardiac diseases that are influenced by sex and discuss the intracellular mechanisms by which steroid sex hormones affect heart function. A theory on how sex may regulate myocardial energy metabolism to affect disease susceptibility and progression will be presented, as well as a discussion of how sex may influence outcomes of experiments on isolated cardiomyocytes by epigenetic marking.