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

The C terminus of HSC70-interacting protein (CHIP, ) 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.

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.

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.

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, HO and 4-hydroxy-2-nonenal (HNE), and monitored the phosphorylation state of ASK1.

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.

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.

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).

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.

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.

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.

Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding.

Perioperative plasma F(2)-Isoprostane levels correlate with markers of impaired ventilation in infants with single-ventricle physiology undergoing stage 2 surgical palliation on the cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) produces inflammation and oxidative stress, which contribute to postoperative complications after cardiac surgery. F(2)-Isoprostanes (F(2)-IsoPs) are products of lipid oxidative injury and represent the most accurate markers of oxidative stress. In adults undergoing cardiac surgery, CPB is associated with elevated IsoPs.

The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation.

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation.

Assessing neuronal bioenergetic status.

Drug discovery and therapeutic development for disorders of the central nervous system (CNS) represents one of the largest unmet markets in modern medicine. We have increasingly recognized that the lack of stringent assessment of mitochondrial function during the discovery process has resulted in drug recalls, black box warnings, and an urgent need to understand the metabolic liability of small molecules in neural systems. Given that the brain is the most energetically demanding organ, even modest perturbations in neuronal energetic pathways have been shown to impact growth, signaling, connectivity, and the restorative capacity of the CNS.

p66(shc)'s role as an essential mitophagic molecule in controlling neuronal redox and energetic tone.

Stroke is the leading cause of adult disability in the U.S. and is now recognized as a global epidemic.

C-terminus of heat shock cognate 70 interacting protein increases following stroke and impairs survival against acute oxidative stress.

The decision to remove or refold oxidized, denatured, or misfolded proteins by heat shock protein 70 and its binding partners is critical to determine cell fate under pathophysiological conditions. Overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. The ability of CHIP to alter cell fate after acute neurological injury has not been assessed.

Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation.

Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level.

Neurotoxic lipid peroxidation species formed by ischemic stroke increase injury.

Stroke is the third leading cause of death in the United States, yet no neuroprotective agents for treatment are clinically available. There is a pressing need to understand the signaling molecules that mediate ischemic cell death and identify novel neuroprotective targets. Cyclopentenone isoprostanes (IsoPs), formed after free radical-mediated peroxidation of arachidonic acid, are used as markers of stress, but their bioactivity is poorly understood.

Manganese exposure is cytotoxic and alters dopaminergic and GABAergic neurons within the basal ganglia.

Manganese is an essential nutrient, integral to proper metabolism of amino acids, proteins and lipids. Excessive environmental exposure to manganese can produce extrapyramidal symptoms similar to those observed in Parkinson's disease (PD). We used in vivo and in vitro models to examine cellular and circuitry alterations induced by manganese exposure.

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species.

Neurotoxic potential of depleted uranium effects in primary cortical neuron cultures and in Caenorhabditis elegans.

Depleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate.