Neuronal Preconditioning Requires the Mitophagic Activity of C-terminus of HSC70-Interacting Protein.

The C terminus of HSC70-interacting protein (CHIP, STUB1) is a ubiquitously expressed cytosolic E3-ubiquitin ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction before 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 relocalization 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 tetracopeptide repeat domains of CHIP in primary neurons derived from CHIP-null animals enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests that 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 (four male, four 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.SIGNIFICANCE STATEMENT 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. Although 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 after low-level stress. Together, these data suggest that CHIP is an essential regulator of mitochondrial number, cell signaling, and survival.

Alterations in the E3 ligases Parkin and CHIP result in unique metabolic signaling defects and mitochondrial quality control issues.

E3 ligases are essential scaffold proteins, facilitating the transfer of ubiquitin from E2 enzymes to lysine residues of client proteins via isopeptide bonds. The specificity of substrate binding and the expression and localization of E3 ligases can, however, endow these proteins with unique features with variable effects on mitochondrial, metabolic and CNS function. By comparing and contrasting two E3 ligases, Parkin and C-terminus of HSC70-Interacting protein (CHIP) we seek to highlight the biophysical properties that may promote mitochondrial dysfunction, acute stress signaling and critical developmental periods to cease in response to mutations in these genes. Encoded by over 600 human genes, RING-finger proteins are the largest class of E3 ligases. Parkin contains three RING finger domains, with R1 and R2 separated by an in-between region (IBR) domain. Loss-of-function mutations in Parkin were identified in patients with early onset Parkinson's disease. CHIP is a member of the Ubox family of E3 ligases. It contains an N-terminal TPR domain and forms unique asymmetric homodimers. While CHIP can substitute for mutated Parkin and enhance survival, CHIP also has unique functions. The differences between these proteins are underscored by the observation that unlike Parkin-deficient animals, CHIP-null animals age prematurely and have significantly impaired motor function. These properties make these E3 ligases appealing targets for clinical intervention. In this work, we discuss how biophysical and metabolic properties of these E3 ligases have driven rapid progress in identifying roles for E3 ligases in development, proteostasis, mitochondrial biology, and cell health, as well as new data about how these proteins alter the CNS proteome.

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Dynamic Phosphorylation of Apoptosis Signal Regulating Kinase 1 (ASK1) in Response to Oxidative and Electrophilic Stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a critical cellular stress sensor that senses diverse reactive chemotypes and integrates these chemical signals into a single biological pathway response. It is unknown whether ASK1 senses all stressors in the same way or if unique stress-specific mechanisms detect distinct chemotypes. In order to answer this question, we treated ASK1-expressing cells with two distinct stress activators, H2O2 and 4-hydroxy-2-nonenal (HNE), and monitored the phosphorylation state of ASK1. Phosphorylation is an important regulator for the activity of ASK1, and we hypothesized that these two chemically distinct molecules may produce differences in the phosphorylation state of ASK1. Shotgun mass spectrometry and manual validation identified 12 distinct ASK1 phosphosites. Targeted parallel reaction monitoring assays were used to track the phosphorylation dynamics of each confirmed site in response to treatment. Eleven phosphosites exhibited dynamic response to one or both treatments. Six of these sites were identified in both H2O2- and HNE-treated cells, and four of these exhibited a consistent response between the two molecules. The results confirm that different chemotypes produce distinct phosphorylation patterns in concert with activation of a common MAPK pathway.

Analysis of a Nitroreductase-Based Hypoxia Sensor in Primary Neuronal Cultures.

The ability to assess oxygenation within living cells is much sought after to more deeply understand normal and pathological cell biology. Hypoxia Red manufactured by Enzo Life Sciences is advertised as a novel hypoxia detector dependent on nitroreducatase activity. We sought to use Hypoxia Red in primary neuronal cultures to test cell-to-cell metabolic variability in response to hypoxic stress. Neurons treated with 90 min of hypoxia were labeled with Hypoxia Red. We observed that, even under normoxic conditions neurons expressed fluorescence robustly. Analysis of the chemical reactions and biological underpinnings of this method revealed that the high uptake and reduction of the dye is due to active nitroreductases in normoxic cells that are independent of oxygen availability.

Silent Cerebral Small Vessel Disease in Restless Legs Syndrome.

STUDY OBJECTIVES: Growing literature suggests that patients with restless legs syndrome (RLS) may be at increased risk for hypertension, heart disease, and stroke. Cerebral small vessel disease (SVD) is a known risk factor for clinical stroke. This study evaluated silent cerebral SVD by MRI in patients with RLS, in the absence of a history of previous clinical stroke or known stroke risk factors and taking into account disease duration. METHODS: Fifty-three patients with RLS < 10 y were prospectively recruited along with 44 with RLS > 10 y and 74 normal controls. A magnetic resonance imaging study was obtained from all subjects and scans were analyzed for area and volume of SVD. RESULTS: There was a significant increase in SVD area in the entire group of RLS patients compared to controls (P = 0.036); this was almost entirely driven by the group with RLS > 10 y. SVD area and volume were significantly increased in patients with RLS > 10 y with respect to both controls (P < 0.0001 and P < 0.0014, respectively) and RLS < 10 y (P < 0.00022 and P < 0.003, respectively). Age, duration of RLS, and the interaction of age and duration of RLS were independent predictors of SVD disease. Duration of RLS was an independent predictor of the burden of cerebral SVD (area P < 0.00012 and volume P < 0.0025), whereas sex and insomnia were not. CONCLUSION: RLS duration should be taken into account when analyzing the association between RLS and cerebrovascular disease; our data support the hypothesis that a long-lasting RLS and its accompanying periodic limb movements in sleep are a risk factor for silent SVD and perhaps for the development of clinical stroke.

Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.

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 protein-protein 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 0.1:1 or less. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14-3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.

Intraarterial administration of norcantharidin attenuates ischemic stroke damage in rodents when given at the time of reperfusion: novel uses of endovascular capabilities.

OBJECT Matrix metalloprotease-9 (MMP-9) plays a critical role in infarct progression, blood-brain barrier (BBB) disruption, and vasogenic edema. While systemic administration of MMP-9 inhibitors has shown neuroprotective promise in ischemic stroke, there has been little effort to incorporate these drugs into endovascular modalities. By modifying the rodent middle cerebral artery occlusion (MCAO) model to allow local intraarterial delivery of drugs, one has the ability to mimic endovascular delivery of therapeutics. Using this model, the authors sought to maximize the protective potential of MMP-9 inhibition by intraarterial administration of an MMP-9 inhibitor, norcantharidin (NCTD). METHODS Spontaneously hypertensive rats were subjected to 90-minute MCAO followed immediately by local intraarterial administration of NCTD. The rats' neurobehavioral performances were scored according to the ladder rung walking test results and the Garcia neurological test for as long as 7 days after stroke. MRI was also conducted 24 hours after the stroke to assess infarct volume and BBB disruption. At the end of the experimental protocol, rat brains were used for active MMP-9 immunohistochemical analysis to assess the degree of MMP-9 inhibition. RESULTS NCTD-treated rats showed significantly better neurobehavioral scores for all days tested. MR images also depicted significantly decreased infarct volumes and BBB disruption 24 hours after stroke. Inhibition of MMP-9 expression in the ischemic region was depicted on immunohistochemical analysis, wherein treated rats showed decreased active MMP-9 staining compared with controls. CONCLUSIONS Intraarterial NCTD significantly improved outcome when administered at the time of reperfusion in a spontaneously hypertensive rat stroke model. This study suggests that supplementing endovascular revascularization with local neuroprotective drug therapy may be a viable therapeutic strategy.

CHIP Is an Essential Determinant of Neuronal Mitochondrial Stress Signaling.

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, PTEN-inducible 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 p66(shc). 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.

Poised for success: implementation of sound conditioning strategies to promote endogenous protective responses to stroke in patients.

The following perspective represents our summary of questions, ideas, concerns, and recommendations expressed by speakers and discussants at the second Biennial Translational Preconditioning Workshop held in Miami in December 2011.

Enrichment of elevated plasma F2t-isoprostane levels in individuals with autism who are stratified by presence of gastrointestinal dysfunction.

Etiology is unknown in the majority of individuals with autism spectrum disorder (ASD). One strategy to investigate pathogenesis is to stratify this heterogeneous disorder based on a prominent phenotypic feature that enriches for homogeneity within population strata. Co-occurring gastrointestinal dysfunction (GID) characterizes a subset of children with ASD. Our current objective was to investigate a potential pathophysiological measure to test the hypothesis that children with both ASD and GID have a more severe metabolic dysfunction than children with ASD-only, given that the highly metabolically active brain and gastrointestinal system may additively contribute measurable impairment. Plasma levels of F2t-Isoprostanes (F2-IsoPs), a gold standard biomarker of oxidative stress, were measured in 87 children in four groups: ASD-GID, ASD-only, GID-only and Unaffected. F2-IsoP levels were elevated in all 3 clinical groups compared to the Unaffected group, with the ASD-GID group significantly elevated above the ASD-only group (mean, SD in pg/mg: ASD-GID 53.6, 24.4; ASD-only 36.5, 13.3; p = 0.007). Adjusting for age, sex, and triglyceride levels, F2-IsoP levels remained significantly different between study groups, with a moderate effect size of η(p)(2) = 0.187 (p = 0.001). Elevation in peripheral oxidative stress is consistent with, and may contribute to, the more severe functional impairments in the ASD-GID group. With unique medical, metabolic, and behavioral features in children with ASD-GID, the present findings serve as a compelling rationale for both individualized approaches to clinical care and integrated studies of biomarker enrichment in ASD subgroups that may better address the complex etiology of ASD.

Redox modification of proteins as essential mediators of CNS autophagy and mitophagy.

Production of cellular reactive oxygen species (ROS) is typically associated with protein and DNA damage, toxicity, and death. However, ROS are also essential regulators of signaling and work in concert with redox-sensitive proteins to regulate cell homeostasis during stress. In this review, we focus on the redox regulation of mitophagy, a process that contributes to energetic tone as well as mitochondrial form and function. Mitophagy has been increasingly implicated in diseases including Parkinson's, Amyotrophic Lateral Sclerosis, and cancer. Although these disease states employ different genetic mutations, they share the common factors of redox dysregulation and autophagic signaling. This review highlights key redox sensitive signaling molecules which can enhance neuronal survival by promoting temporally and spatially controlled autophagic signaling and mitophagy.

Alteration of isocitrate dehydrogenase following acute ischemic injury as a means to improve cellular energetic status in neuroadaptation.

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.

Neurovascular unit on a chip: implications for translational applications.

The blood-brain barrier (BBB) dynamically controls exchange between the brain and the body, but this interaction cannot be studied directly in the intact human brain or sufficiently represented by animal models. Most existing in vitro BBB models do not include neurons and glia with other BBB elements and do not adequately predict drug efficacy and toxicity. Under the National Institutes of Health Microtissue Initiative, we are developing a three-dimensional, multicompartment, organotypic microphysiological system representative of a neurovascular unit of the brain. The neurovascular unit system will serve as a model to study interactions between the central nervous system neurons and the cerebral spinal fluid (CSF) compartment, all coupled to a realistic blood-surrogate supply and venous return system that also incorporates circulating immune cells and the choroid plexus. Hence all three critical brain barriers will be recapitulated: blood-brain, brain-CSF, and blood-CSF. Primary and stem cell-derived human cells will interact with a variety of agents to produce critical chemical communications across the BBB and between brain regions. Cytomegalovirus, a common herpesvirus, will be used as an initial model of infections regulated by the BBB. This novel technological platform, which combines innovative microfluidics, cell culture, analytical instruments, bioinformatics, control theory, neuroscience, and drug discovery, will replicate chemical communication, molecular trafficking, and inflammation in the brain. The platform will enable targeted and clinically relevant nutritional and pharmacologic interventions for or prevention of such chronic diseases as obesity and acute injury such as stroke, and will uncover potential adverse effects of drugs. If successful, this project will produce clinically useful technologies and reveal new insights into how the brain receives, modifies, and is affected by drugs, other neurotropic agents, and diseases.

Metabolic multianalyte microphysiometry reveals extracellular acidosis is an essential mediator of neuronal preconditioning.

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.

Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP) produces specific behavioral impairments.

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.

The role of central nervous system development in late-onset neurodegenerative disorders.

The human brain is dependent upon successfully maintaining ionic, energetic and redox homeostasis within exceptionally narrow margins for proper function. The ability of neurons to adapt to genetic and environmental perturbations and evoke a 'new normal' can be most fully appreciated in the context of neurological disorders in which clinical impairments do not manifest until late in life, although dysfunctional proteins are expressed early in development. We now know that proteins controlling ATP generation, mitochondrial stability, and the redox environment are associated with neurological disorders such as Parkinson's disease and amyotrophic lateral sclerosis. Generally, focus is placed on the role that early or long-term environmental stress has in altering the survival of cells targeted by genetic dysfunctions; however, the central nervous system undergoes several periods of intense stress during normal maturation. One of the most profound periods of stress occurs when 50% of neurons are removed via programmed cell death. Unfortunately, we have virtually no understanding of how these events proceed in individuals who harbor mutations that are lethal later in life. Moreover, there is a profound lack of information on circuit formation, cell fate during development and neurochemical compensation in either humans or the animals used to model neurodegenerative diseases. In this review, we consider the current knowledge of how energetic and oxidative stress signaling differs between neurons in early versus late stages of life, the influence of a new group of proteins that can integrate cell stress signals at the mitochondrial level, and the growing body of evidence that suggests early development should be considered a critical period for the genesis of chronic neurodegenerative diseases.