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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 Aβ42/40 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.

INTRODUCTION Blood tests have the potential to improve the accuracy of Alzheimer's 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 Alzheimer's 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. Highlights Plasma p‐tau217 measures most accurately classified amyloid and tau status. Plasma Aβ42/Aβ40 had relatively low accuracy in classification of amyloid status. Plasma p‐tau217 measures had higher correlations with cortical thickness than NfL. Correlations of plasma biomarkers with dementia symptoms were relatively low.

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. This is an factual error as written; however, total oxygen consumption was decreased. 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.

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.

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.

Background TANGO was a Phase 2 clinical study designed to assess the safety and efficacy of gosuranemab, an anti‐tau monoclonal antibody, in participants with mild cognitive impairment due to Alzheimer’s disease (AD) or with mild AD dementia. Despite robust target engagement of unbound N‐terminal tau, the clinical efficacy endpoint was not met. In this exploratory analysis of TANGO participants, we examine plasma p‐tau217 levels to assess the feasibility of using this biomarker to identify patients with AD pathology and predict disease progression. Methods Plasma was collected from 554 randomized TANGO participants at baseline and up to Week 78. Plasma p‐tau217 was measured using the ALZpath Quanterix Simoa assay. Plasma p‐tau181 was measured using the Quanterix Simoa p‐tau181 V2 Advantage assay. Tau PET was measured in a sub‐study of 357 participants at baseline and Weeks 52 and 78 using 18F‐MK‐6240. Amyloid PET was measured at screening using 18F‐florbetapir in n = 322 participants Clinical decline was measured using the CDR‐SB, MMSE, ADAS‐Cog13 and ADCS‐ADL cognitive assessment scales. Statistical analysis was performed using Spearman correlation coefficient. Results Plasma p‐tau217 and plasma p‐tau181 were correlated at baseline (Spearman = 0.7617, p‐value <0.0001). Baseline plasma p‐tau217 levels were correlated with both baseline amyloid PET using SUVR in a composite region of interest (Spearman = 0.4294, p‐value <0.0001) and baseline Tau PET using SUVR in composite regions of interest corresponding to Braak stages I‐II, III‐IV, and V‐VI (Spearman range = 0.4075‐0.6163, p = value <0.0001). Participants with higher concentrations of plasma ptau217 at baseline showed a greater rate of clinical decline at Week 78. Conclusions The correlations of plasma p‐tau217 with amyloid and tau PET at baseline suggest a relationship with both underlying pathological hallmarks of AD. Similar to results from observational cohorts, correlation of baseline plasma p‐tau217 and clinical decline observed during the TANGO trial supports the utility of plasma p‐tau217 as a potential prognostic biomarker of disease. Data from additional studies are needed to define appropriate use cases and relevant thresholds for plasma p‐tau217 as a biomarker of disease pathology that can be used to prescreen patients in clinical trials.

All cells must respond to changes in their environment including a plethora of physiologic and pathologic stresses in order to maintain homeostasis and survive. Protein homeostasis is particularly critical to cell survival and cells utilize multiple highly specialized and integrated methods of protein quality control (PQC) to ensure that proteins are appropriately folded and terminally misfolded proteins are eliminated to prevent proteotoxicity. PQC depends on an elegant collaboration between molecular chaperones and the ubiquitin-proteasome system (UPS). Disruption of PQC and subsequent proteotoxicity is an underlying molecular phenotype in disease pathologies in the brain and heart. Understanding the molecular mechanisms underlying diseases where disruption of PQC is central to disease pathology is key to our ability to intervene therapeutically. To this end, this thesis focuses on understanding the function of E3 ubiquitin ligases and how mutations in these key players in the UPS can drive disease pathology in the heart and brain. First, I describe and validate a novel method for the identification of E3 ubiquitin ligase substrates addressing a significant technological limitation in the field. Next, I describe the first discovery of human mutation in the E3 ubiquitin ligase CHIP in a form of spinocerebellar ataxia, Gordon Holmes Syndrome that has led to the establishment of a new disease designation, autosomal recessive spinocerebellar ataxia-16 (SCAR16) to describe spinocerebellar ataxia caused by homozygous or compound heterozygous mutation in CHIP. Finally, I expanded upon this discovery to define the structural and functional consequences of CHIP mutation in SCAR16 and explore the deficits associated with this mutation in a genomic context utilizing a mouse model system providing the first in vivo, disease-relevant model of partial CHIP dysfunction. Together these studies provide novel tools to further our understanding of the UPS and reveal fascinating insight into the molecular mechanisms underlying CHIP mutation in SCAR16 disease that not only may facilitate the development of therapies for this devastating disease, but also contribute to our basic understanding of the UPS and its role in disease pathogenesis to drive successful investment, innovation, preclinical investigation and clinical study design in other disease areas.