Alzheimer's disease biomarker utilization at first referral enhances differential diagnostic precision with simultaneous exclusion of Creutzfeldt-Jakob disease.

Most suspected Creutzfeldt-Jakob disease (CJD) cases are eventually diagnosed with other disorders. We assessed the utility of investigating Alzheimer's disease (AD) biomarkers and neurofilament light (NfL) in patients when CJD is suspected. The study cohort consisted of cerebrospinal fluid (CSF) samples referred for CJD biomarker screening wherein amyloid beta 1-42 (Aß1-42), phosphorylated tau 181 (p-tau181), and total tau (t-tau) could be assessed via Elecsys immunoassays (n = 419) and NfL via enzyme-linked immunosorbent assay (ELISA; n = 161). In the non-CJD sub cohort (n = 371), 59% (219/371) had A+T- (abnormal Aß1-42 only) and 21% (79/371) returned A+T+ (abnormal Aß1-42 and p-tau181). In the 48 CJD subjects, a similar AD biomarker profile distribution was observed. To partially address the prevalence of likely pre-symptomatic AD, NfL was utilized to assess for neuronal damage. NfL was abnormal in 76% (25/33) of A+T- subjects 40 to 69 years of age, 80% (20/25) of whom had normal t-tau. This study reinforces AD as an important differential diagnosis of suspected CJD, highlighting that incorporating AD biomarkers and NfL at initial testing is worthwhile.

Distribution of microRNA profiles in pre-clinical and clinical forms of murine and human prion disease.

Prion diseases are distinguished by long pre-clinical incubation periods during which prions actively propagate in the brain and cause neurodegeneration. In the pre-clinical stage, we hypothesize that upon prion infection, transcriptional changes occur that can lead to early neurodegeneration. A longitudinal analysis of miRNAs in pre-clinical and clinical forms of murine prion disease demonstrated dynamic expression changes during disease progression in the affected thalamus region and serum. Serum samples at each timepoint were collected whereby extracellular vesicles (EVs) were isolated and used to identify blood-based biomarkers reflective of pathology in the brain. Differentially expressed EV miRNAs were validated in human clinical samples from patients with human sporadic Creutzfeldt-Jakob disease (sCJD), with the molecular subtype at codon 129 either methionine-methionine (MM, n = 14) or valine-valine (VV, n = 12) compared to controls (n = 20). EV miRNA biomarkers associated with prion infection predicted sCJD with an AUC of 0.800 (85% sensitivity and 66.7% specificity) in a second independent validation cohort (n = 26) of sCJD and control patients with MM or VV subtype. This study discovered clinically relevant miRNAs that benefit diagnostic development to detect prion-related diseases and therapeutic development to inhibit prion infectivity.

Strain variation in treatment and prevention of human prion diseases.

Transmissible spongiform encephalopathies or prion diseases describe a number of different human disorders that differ in their clinical phenotypes, which are nonetheless united by their transmissible nature and common pathology. Clinical variation in the absence of a conventional infectious agent is believed to be encoded by different conformations of the misfolded prion protein. This misfolded protein is the target of methods designed to prevent disease transmission in a surgical setting and reduction of the misfolded seed or preventing its continued propagation have been the focus of therapeutic strategies. It is therefore possible that strain variation may influence the efficacy of prevention and treatment approaches. Historically, an understanding of prion disease transmission and pathogenesis has been focused on research tools developed using agriculturally relevant strains of prion disease. However, an increased understanding of the molecular biology of human prion disorders has highlighted differences not only between different forms of the disease affecting humans and animals but also within diseases such as Creutzfeldt-Jakob Disease (CJD), which is represented by several sporadic CJD specific conformations and an additional conformation associated with variant CJD. In this chapter we will discuss whether prion strain variation can affect the efficacy of methods used to decontaminate prions and whether strain variation in pre-clinical models of prion disease can be used to identify therapeutic strategies that have the best possible chance of success in the clinic.

PrPSc Oligomerization Appears Dynamic, Quickly Engendering Inherent M1000 Acute Synaptotoxicity.

Prion diseases are neurodegenerative disorders pathogenically linked to cellular prion protein (PrPC) misfolding into abnormal conformers (PrPSc), with PrPSc underpinning both transmission and synaptotoxicity. Although the biophysical features of PrPSc required to induce acute synaptic dysfunction remain incompletely defined, we recently reported that acutely synaptotoxic PrPSc appeared to be oligomeric. We herein provide further insights into the kinetic and requisite biophysical characteristics of acutely synaptotoxic ex vivo PrPSc derived from the brains of mice dying from M1000 prion disease. Pooled fractions of M1000 PrPSc located within the molecular weight range approximating monomeric PrP (mM1000) generated through size exclusion chromatography were found to harbor acute synaptotoxicity equivalent to preformed oligomeric fractions (oM1000). Subsequent investigation showed mM1000 corresponded to PrPSc rapidly concatenating in physiological buffer to exist as predominantly, closely associated, small oligomers. The oligomerization of PrP in mM1000 could be substantially mitigated by treatment with the antiaggregation compound epigallocatechin gallate, thereby maintaining the PrPSc as primarily nonoligomeric with completely abrogated acute synaptotoxicity; moreover, despite epigallocatechin gallate treatment, pooled oM1000 remained oligomeric and acutely synaptotoxic. A similar tendency to rapid formation of oligomers was observed for PrPC when monomeric fractions derived from size exclusion chromatography of normal brain homogenates (mNBH) were pooled, but neither mNBH nor preformed higher-order NBH complexes (oNBH) were acutely synaptotoxic. Oligomers formed from mNBH could be reduced to mainly monomers (<100 kDa) after enzymatic digestion of nucleic acids, whereas higher-order PrP assemblies derived from pooled mM1000, oM1000, and oNBH resisted such treatment. Collectively, these findings support that oligomerization of PrPSc into small multimeric assemblies appears to be a critical biophysical feature for engendering inherent acute synaptotoxicity, with preformed oligomers found in oM1000 appearing to be stable, tightly self-associated ensembles that coexist in dynamic equilibrium with mM1000, with the latter appearing capable of rapid aggregation, albeit initially forming smaller, weakly self-associated, acutely synaptotoxic oligomers.

Chronic isolation stress is associated with increased colonic and motor symptoms in the A53T mouse model of Parkinson's disease.

BACKGROUND: Chronic stress exacerbates motor deficits and increases dopaminergic cell loss in several rodent models of Parkinson's disease (PD). However, little is known about effects of stress on gastrointestinal (GI) dysfunction, a common non-motor symptom of PD. We aimed to determine whether chronic stress exacerbates GI dysfunction in the A53T mouse model of PD and whether this relates to changes in α-synuclein distribution. METHODS: Chronic isolation stress was induced by single-housing WT and homozygote A53T mice between 5 and 15 months of age. GI and motor function were compared with mice that had been group-housed. KEY RESULTS: Chronic isolation stress increased plasma corticosterone and exacerbated deficits in colonic propulsion and whole-gut transit in A53T mice and also increased motor deficits. However, our results indicated that the novel environment-induced defecation response, a common method used to evaluate colorectal function, was not a useful test to measure exacerbation of GI dysfunction, most likely because of the reported reduced level of anxiety in A53T mice. A53T mice had lower corticosterone levels than WT mice under both housing conditions, but single-housing increased levels for both genotypes. Enteric neuropathy was observed in aging A53T mice and A53T mice had a greater accumulation of alpha-synuclein (αsyn) in myenteric ganglia under both housing conditions. CONCLUSIONS & INFERENCES: Chronic isolation stress exacerbates PD-associated GI dysfunction, in addition to increasing motor deficits. However, these changes in GI symptoms are not directly related to corticosterone levels, worsened enteric neuropathy, or enteric αsyn accumulation.

Method for Folding of Recombinant Prion Protein to Soluble ß-Sheet Secondary Structure.

A key event in the pathogenesis of prion diseases is the change in structure of the normal cellular form of the prion protein from a predominantly α-helix form to the ß-sheet-rich prion protein found in disease-associated tissue. To allow more detailed structural research into PrP misfolding, it is necessary to have techniques which enable enrichment of the ß-sheet content in recombinant PrP.This method describes the procedure for inducing ß-folding of recombinant PrP to resemble a disease-associated structure and ultimately produce soluble ß-folded recombinant PrP.

Analysis of Prion Protein Conformation Using Circular Dichroism Spectroscopy.

According to the protein-only hypothesis of prion propagation, the pathogenesis of prion disease is due to the misfolding of cellular PrP (PrPC) which gives rise to disease-associated PrPSc. This misfolding results in the predominantly α-helix secondary structure of PrP becoming increasingly ß-sheet. Prion protein researchers often employ circular dichroism (CD) spectroscopy to rapidly analyze and identify the degree of α-helix and ß-sheet content in their recombinant protein and peptide samples. CD is a nondestructive method of determining protein secondary structure and can be used to monitor the protein structural changes in various environments, e.g., pH and temperature. CD can also be used to investigate kinetic and thermodynamic characteristics of proteins and peptides.

Preparation and Immunostaining of the Myenteric Plexus of Prion-Infected Mice.

Intracerebral inoculation of mice with the M1000 strain of mouse-adapted human prions results in the consistent accumulation of PrPSc in the ileum of the gastrointestinal tract (GIT) of mice with clinical signs of prion disease. The accumulation of PrPSc in the ileum is accompanied by caspase activation and loss of immunoreactivity in subpopulations of neurons in the enteric nervous system. This suggests that like neurons in the central nervous system, cells in the enteric nervous system are also susceptible to prion-induced toxicity. In this chapter we describe the immunostaining of cells in myenteric plexus preparations of whole mounts prepared from the gastrointestinal tract of prion-infected mice.

Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.

Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP(C), to a misfolded isoform called PrP(Sc). Although PrP(Sc) is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP(Sc). Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP(Sc) deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP(C) and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a ß-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.