Aims: Determine the mechanism by which C-terminus of HSC70-interacting protein (CHIP) induction alters neuronal survival under conditions of mitochondrial stress induced by oxygen glucose deprivation. Results: We report that animals deficient in the E3 ubiquitin ligase, CHIP, have high baseline levels of central nervous system protein oxidation and lipid peroxidation, reduced antioxidant defenses, and decreased energetic status. Stress-associated molecules typically linked to Parkinson's disease such as the mitochondrial kinase, PTENinducible putative kinase 1 (PINK1), and another E3 ligase, Parkin, are upregulated in brains from CHIP knockout (KO) animals. Utilizing a novel biotin-avidin capture technique, we found that the oxidation status of Parkin and the mitochondrial fission protein, dynamin-related protein 1 (Drp1), are altered in a CHIP-dependent manner. We also found that following oxygen-glucose deprivation (OGD), the expression of CHIP, PINK1, and the autophagic marker, LC3, increase and there is activation of the redox-sensitive kinase p66shc . Under conditions of OGD, CHIP relocalizes from the cytosol to mitochondria. Mitochondria from CHIP KO mice have profound impairments in stress response induced by calcium overload, resulting in accelerated permeability transition activity. While CHIP-deficient neurons are morphologically intact, they are more susceptible to OGD consistent with a previously unknown neuroprotective role for CHIP in maintaining mitochondrial homeostasis. Innovation: CHIP relocalization to the mitochondria is essential for the regulation of mitochondrial integrity and neuronal survival following OGD. Conclusions: CHIP is an essential regulator of neuronal bioenergetics and redox tone. Altering the expression of this protein has profound effects on neuronal survival when cells are exposed to OGD. Antioxid. Redox Signal. 23, 535-549.

Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles. ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 proteinprotein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14 -3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0

C-terminus of HSC70-interacting protein (CHIP, ) is a ubiquitously expressed cytosolic E3-ubiquitin ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction prior to premature death. This phenotype is more consistent with animal models in which master regulators of autophagy are affected rather than with the mild phenotype of classic E3-ubiquitin ligase mutants. The cellular and biochemical events that contribute to neurodegeneration and premature aging in CHIP KO models remain poorly understood. Electron and fluorescent microscopy demonstrates that CHIP deficiency is associated with greater numbers of mitochondria, but these organelles are swollen and misshapen. Acute bioenergetic stress triggers CHIP induction and re-localization to mitochondria where it plays a role in the removal of damaged organelles. This mitochondrial clearance is required for protection following low-level bioenergetic stress in neurons. CHIP expression overlaps with stabilization of the redox stress sensor PTEN-inducible kinase 1 (PINK1) and is associated with increased LC3-mediated mitophagy. Introducing human promoter-driven vectors with mutations in either the E3 ligase or TPR domains of CHIP in primary neurons derived from CHIP-null animals enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests the increase in mitochondrial CHIP is likely due to diminished clearance of these CHIP-tagged organelles. Proteomic analysis of WT and CHIP KO mouse brains (4 male, 4 female per genotype) reveals proteins essential for maintaining energetic, redox and mitochondrial homeostasis undergo significant genotype-dependent expression changes. Together these data support the use of CHIP deficient animals as a predictive model of age-related degeneration with selective neuronal proteotoxicity and mitochondrial failure. Mitochondria are recognized as central determinants of neuronal function and survival. We demonstrate that C-terminus of HSC70-Interacting Protein (CHIP) is critical for neuronal responses to stress. CHIP upregulation and localization to mitochondria is required for mitochondrial autophagy (mitophagy). Unlike other disease-associated E3 ligases such as Parkin and Mahogunin, CHIP controls homeostatic and stress-induced removal of mitochondria. While CHIP deletion results in greater numbers of mitochondria, these organelles have distorted inner membranes without clear cristae. Neuronal cultures derived from animals lacking CHIP are more vulnerable to acute injuries, and transient loss of CHIP renders neurons incapable of mounting a protective response following low-level stress. Together these data suggest that CHIP is an essential regulator of mitochondrial number, cell signaling and survival.

The human leukocyte antigen (HLA) system is the most polymorphic in the human genome that has been associated with protection and predisposition to a broad array of infectious, autoimmune, and malignant diseases. More recently over the last two decades, HLA class I alleles have been strongly associated with T‐cell‐mediated drug hypersensitivity reactions. In the case of abacavir hypersensitivity and HLA‐B*57:01, the 100% negative predictive value and low number needed to test to prevent a single case has led to a durable and effective global preprescription screening strategy. However, HLA associations are still undefined for most drugs clinically associated with different delayed drug hypersensitivity phenotypes, and an HLA association relevant to one population is not generalizable across ethnicities. Furthermore, while a specific risk HLA allele is necessary for drug‐induced T‐cell activation, it is not sufficient. The low and incomplete positive predictive value has hindered efforts at clinical implementation for many drugs but has provided the impetus to understand the mechanisms of HLA class I restricted T‐cell‐mediated drug hypersensitivity reactions. Current research has focused on defining the contribution of additional elements of the adaptive immune response and other genetic and ecologic risk factors that contribute to drug hypersensitivity risk. In this review we focus on new insights into immunological, pharmacological, and genetic mechanisms underpinning HLA‐associated drug reactions and the implications for future translation into clinical care.

The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress.

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Metabolic adaptation to stress is a crucial yet poorly understood phenomenon, particularly in the central nervous system (CNS). The ability to identify essential metabolic events which predict neuronal fate in response to injury is critical to developing predictive markers of outcome, for interpreting CNS spectroscopic imaging, and for providing a richer understanding of the relevance of clinical indices of stress which are routinely collected. In this work, real-time multianalyte microphysiometry was used to dynamically assess multiple markers of aerobic and anaerobic respiration through simultaneous electrochemical measurement of extracellular glucose, lactate, oxygen, and acid. Pure neuronal cultures and mixed cultures of neurons and glia were compared following a 90 min exposure to aglycemia. This stress was cytotoxic to neurons yet resulted in no appreciable increase in cell death in age-matched mixed cultures. The metabolic profile of the cultures was similar in that aglycemia resulted in decreases in extracellular acidification and lactate release in both pure neurons and mixed cultures. However, oxygen consumption was only diminished in the neuron enriched cultures. The differences became more pronounced when cells were returned to glucose-containing media upon which extracellular acidification and oxygen consumption never returned to baseline in cells fated to die. Taken together, these data suggest that lactate release is not predictive of neuronal survival. Moreover, they reveal a previously unappreciated relationship of astrocytes in maintaining oxygen uptake and a correlation between metabolic recovery of neurons and extracellular acidification.

The isocitrate dehydrogenase (IDH) enzymes were initially identified as essential components of the Krebs cycle. IDH mutations were thought to be incompatible with cell survival. However, 90% of glioblastomas were recently shown to be associated with somatic mutations in these enzymes, indicating a possible role for IDH in promoting cellular survival in hypoxic environments. Our proteomic analysis of rats given 10 minutes of middle cerebral artery occlusion to induce transient ischemia demonstrates a significant decrease in IDH expression. We have recapitulated this decrease in an in vitro model using primary cortical neurons exposed to acute oxygen and glucose deprivation. Given the role of IDHs in energy metabolism and antioxidant production, we hypothesize that the IDHs may serve as first-line, rapid-response enzymes that regulate survival in environments of energetic or oxidative stress. In order to identify the specific events that regulate IDH enzymes, HT-22 neural cells were subjected to either a selective energetic challenge or a pure oxidative stress. In response to the non-lethal energetic challenge induced by substituting galactose for glucose, we observed increased IDH1, 2, and 3 expression and cessation of cellular proliferation. No change in expression of any IDH isoform was observed when neural cells were subjected to subtoxic oxidative stress via glutathione depletion. Taken together, these data imply that IDH expression rapidly responds to changes in energetic status, but not to oxidative stress. These data also suggest that IDH enzymes respond not only to allosteric modulation, but can also change patterns of expression in response to moderate stress in an effort to maximize ATP production and survival.