Comparative analytical performance of multiple plasma Aß42 and Aß40 assays and their ability to predict positron emission tomography amyloid positivity.

INTRODUCTION: This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aß) peptides in plasma and the extent to which they improve the prediction of amyloid positivity. METHODS: Alzheimer's Disease Neuroimaging Initiative plasma samples with corresponding amyloid positron emission tomography (PET) data were run on six plasma Aß assays. Statistical tests were performed to determine whether the plasma Aß measures significantly improved the area under the receiver operating characteristic curve for predicting amyloid PET status compared to age and apolipoprotein E (APOE) genotype. RESULTS: The age and APOE genotype model predicted amyloid status with an area under the curve (AUC) of 0.75. Three assays improved AUCs to 0.81, 0.81, and 0.84 (P < .05, uncorrected for multiple comparisons). DISCUSSION: Measurement of Aß in plasma contributes to addressing the amyloid component of the ATN (amyloid/tau/neurodegeneration) framework and could be a first step before or in place of a PET or cerebrospinal fluid screening study. HIGHLIGHTS: The Foundation of the National Institutes of Health Biomarkers Consortium evaluated six plasma amyloid beta (Aß) assays using Alzheimer's Disease Neuroimaging Initiative samples. Three assays improved prediction of amyloid status over age and apolipoprotein E (APOE) genotype. Plasma Aß42/40 predicted amyloid positron emission tomography status better than Aß42 or Aß40 alone.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP.

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo.

MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1's role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1-/- mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. However, total oxygen consumption was decreased [corrected]. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1-/- hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2's regulation of mitochondrial function.

Head-to-head comparison of leading blood tests for Alzheimer's disease pathology.

INTRODUCTION: Blood tests have the potential to improve the accuracy of Alzheimer disease (AD) clinical diagnosis, which will enable greater access to AD-specific treatments. This study compared leading commercial blood tests for amyloid pathology and other AD-related outcomes. METHODS: Plasma samples from the Alzheimers Disease Neuroimaging Initiative were assayed with AD blood tests from C2N Diagnostics, Fujirebio Diagnostics, ALZPath, Janssen, Roche Diagnostics, and Quanterix. Outcomes measures were amyloid positron emission tomography (PET), tau PET, cortical thickness, and dementia severity. Logistic regression models assessed the classification accuracies of individual or combined plasma biomarkers for binarized outcomes, and Spearman correlations evaluated continuous relationships between individual plasma biomarkers and continuous outcomes. RESULTS: Measures of plasma p-tau217, either individually or in combination with other plasma biomarkers, had the strongest relationships with all AD outcomes. DISCUSSION: This study identified the plasma biomarker analytes and assays that most accurately classified amyloid pathology and other AD-related outcomes.

Independent study demonstrates amyloid probability score accurately indicates amyloid pathology.

BACKGROUND: The amyloid probability score (APS) is the model read-out of the analytically validated mass spectrometry-based PrecivityAD® blood test that incorporates the plasma Aß42/40 ratio, ApoE proteotype, and age to identify the likelihood of brain amyloid plaques among cognitively impaired individuals being evaluated for Alzheimer's disease. PURPOSE: This study aimed to provide additional independent evidence that the pre-established APS algorithm, along with its cutoff values, discriminates between amyloid positive and negative individuals. METHODS: The diagnostic performance of the PrecivityAD test was analyzed in a cohort of 200 nonrandomly selected Australian Imaging, Biomarker & Lifestyle Flagship Study of Aging (AIBL) study participants, who were either cognitively impaired or healthy controls, and for whom a blood sample and amyloid PET imaging were available. RESULTS: In a subset of the dataset aligned with the Intended Use population (patients aged 60 and older with CDR ≥0.5), the pre-established APS algorithm predicted amyloid PET with a sensitivity of 84.9% (CI: 72.9-92.1%) and specificity of 96% (CI: 80.5-99.3%), exclusive of 13 individuals for whom the test was inconclusive. INTERPRETATION: The study shows individuals with a high APS are more likely than those with a low APS to have abnormal amounts of amyloid plaques and be on an amyloid accumulation trajectory, a dynamic and evolving process characteristic of progressive AD pathology. Exploratory data suggest APS retains its diagnostic performance in healthy individuals, supporting further screening studies in the cognitively unimpaired.

Non-radiometric Cell-free Assay to Measure the Effect of Molecular Chaperones on AMP-activated Kinase Activity.

AMP-activated kinase (AMPK) is a trimeric protein holoenzyme with kinase activity. AMPK plays an important role in cellular metabolism and is thought to function as a fuel sensor within the cell, exerting kinase activity to activate energy-conserving pathways and simultaneously inhibit energy-consuming pathways. Traditional in vitro methods to measure AMPK activity to test potential agonists or antagonists utilize radiolabeled ATP with a peptide substrate. Although radiolabeling provides a high level of sensitivity, this approach is not ideal for medium to high-throughput screening, dose-response curves, or kinetic analyses. Our protocol utilizes Invitrogen's Z'-LYTE™ Kinase Assay Kit (Ser/Thr 23 Peptide) to measure changes in the enzymatic activity of AMPKɑ2ß1γ1 in the presence of a molecular chaperone. The Z'-LYTE™ platform is based on Fluorescence Resonance Energy Transfer (FRET). The AMPK peptide substrate (S/T 23 peptide: MRPRKRQGSVRRRV) is a self-contained FRET system, using coumarin as the donor and fluorescein as the acceptor. When the peptide is phosphorylated, it is sensitive to cleavage by a site-specific protease. The cleavage of the phospho-peptide eliminates the FRET pair, and the ratiometric analysis of FRET is used as an indirect measure of AMPK kinase activity. This method does not require the use of radiolabeling or antibodies and is used in a multi-well format, with high reproducibility and throughput capabilities.

CHIP protects against cardiac pressure overload through regulation of AMPK.

Protein quality control and metabolic homeostasis are integral to maintaining cardiac function during stress; however, little is known about if or how these systems interact. Here we demonstrate that C terminus of HSC70-interacting protein (CHIP), a regulator of protein quality control, influences the metabolic response to pressure overload by direct regulation of the catalytic α subunit of AMPK. Induction of cardiac pressure overload in Chip-/- mice resulted in robust hypertrophy and decreased cardiac function and energy generation stemming from a failure to activate AMPK. Mechanistically, CHIP promoted LKB1-mediated phosphorylation of AMPK, increased the specific activity of AMPK, and was necessary and sufficient for stress-dependent activation of AMPK. CHIP-dependent effects on AMPK activity were accompanied by conformational changes specific to the α subunit, both in vitro and in vivo, identifying AMPK as the first physiological substrate for CHIP chaperone activity and establishing a link between cardiac proteolytic and metabolic pathways.

Diggin' on u(biquitin): a novel method for the identification of physiological E3 ubiquitin ligase substrates.

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing tandem ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.