Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Sex-Dependent Effects of Chronic Microdrive Implantation on Acquisition of Trace Eyeblink Conditioning.

Neuroscience techniques, including in vivo recording, have allowed for a great expansion in knowledge; however, this technology may also affect the very phenomena researchers set out to investigate. Including both female and male mice in our associative learning experiments shed light on sex differences on the impact of chronic implantation of tetrodes on learning. While previous research showed intact female mice acquired trace eyeblink conditioning faster than male and ovariectomized females, implantation of chronic microdrive arrays showed sexually dimorphic effects on learning. Microdrive implanted male mice acquired the associative learning paradigm faster than both intact and ovariectomized females. These effects were not due to the weight of the drive alone, as there were no significant sex-differences in learning of animals that received "dummy drive" implants without tetrodes lowered into the brain. Tandem mass tag mass spectrometry and western blot analysis suggest that significant alterations in the MAPK pathway, acute inflammation, and brain derived neurotrophic factor may underlie these observed sex- and surgery-dependent effects on learning.

Long-lived mitochondrial cristae proteins in mouse heart and brain.

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)-based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.

Using stable isotope labeling to advance our understanding of Alzheimer's disease etiology and pathology.

Stable isotope labeling with mass spectrometry (MS)-based proteomic analysis has become a powerful strategy to assess protein steady-state levels, protein turnover, and protein localization. Applying these analyses platforms to neurodegenerative disorders may uncover new aspects of the etiology of these devastating diseases. Recently, stable isotopes-MS has been used to investigate early pathological mechanisms of Alzheimer's disease (AD) with mouse models of AD-like pathology. In this review, we summarize these stable isotope-MS experimental designs and the recent application in the context of AD pathology. We also describe our current efforts aimed at using nuclear magnetic resonance (NMR) analysis of stable isotope-labeled amyloid fibrils from AD mouse model brains. Collectively, these methodologies offer new opportunities to study proteome changes in AD and other neurodegenerative diseases by elucidating mechanisms to target for treatment and prevention.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aß) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aß accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aß. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper's transparent peer review process is included in the Supplemental Information.

Amyloid Accumulation Drives Proteome-wide Alterations in Mouse Models of Alzheimer's Disease-like Pathology.

Amyloid beta (Aß) peptides impair multiple cellular pathways and play a causative role in Alzheimer's disease (AD) pathology, but how the brain proteome is remodeled by this process is unknown. To identify protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at young and old ages. Our analysis revealed a robust increase in Apolipoprotein E (ApoE) levels in nearly all brain regions with increased Aß levels. Taken together with prior findings on ApoE driving Aß accumulation, this analysis points to a pathological dysregulation of the ApoE-Aß axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission. Analysis of the AMPA receptor (AMPAR) complex revealed specific loss of TARPγ-2, a key AMPAR-trafficking protein. Expression of TARPγ-2 in hAPP transgenic mice restored AMPA currents. This proteomic database represents a resource for the identification of protein alterations responsible for AD.

NAAG Peptidase Inhibitors Act via mGluR3: Animal Models of Memory, Alzheimer's, and Ethanol Intoxication.

Glutamate carboxypeptidase II (GCPII) inactivates the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Inhibitors of GCPII increase extracellular NAAG levels and are efficacious in animal models of clinical disorders via NAAG activation of a group II metabotropic glutamate receptor. mGluR2 and mGluR3 knock-out (ko) mice were used to test the hypothesis that mGluR3 mediates the activity of GCPII inhibitors ZJ43 and 2-PMPA in animal models of memory and memory loss. Short- (1.5 h) and long- (24 h) term novel object recognition tests were used to assess memory. Treatment with ZJ43 or 2-PMPA prior to acquisition trials increased long-term memory in mGluR2, but not mGluR3, ko mice. Nine month-old triple transgenic Alzheimer's disease model mice exhibited impaired short-term novel object recognition memory that was rescued by treatment with a NAAG peptidase inhibitor. NAAG peptidase inhibitors and the group II mGluR agonist, LY354740, reversed the short-term memory deficit induced by acute ethanol administration in wild type mice. 2-PMPA also moderated the effect of ethanol on short-term memory in mGluR2 ko mice but failed to do so in mGluR3 ko mice. LY354740 and ZJ43 blocked ethanol-induced motor activation. Both GCPII inhibitors and LY354740 also significantly moderated the loss of motor coordination induced by 2.1 g/kg ethanol treatment. These data support the conclusion that inhibitors of glutamate carboxypeptidase II are efficacious in object recognition models of normal memory and memory deficits via an mGluR3 mediated process, actions that could have widespread clinical applications.