Amy M. Palubinsky

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 Parkinsons 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.

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.

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.

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 Parkinsons 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.

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.

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.

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

ABSTRACT 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 Parkinsons 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. HIGHLIGHTSParkin is a RING E3 ligase mutated in forms of early‐onset Parkinsons disease.Structure‐function studies identify Parkin as a regulator of mitochondria.Parkin shares client proteins with Ubox E3 ligase CHIP.Recent studies reveal that CHIP is a key regulator of mitochondrial energetics.Uncovering the functions of such E3 ligases can advance therapy for CNS diseases.

Genetic Models of Parkinson's Disease

Parkinsons disease (PD) is the most common neurodegenerative movement disorder in the United States. Although the disease can either be inherited or arise spontaneously, only 10–20% of all PD cases arise from inherited mutations. Despite this discrepancy, modeling PD in animals by means of genetic manipulations has significantly advanced our understanding of the pathogenesis of this disease. In this chapter, we provide a comprehensive review of the pathological, neurochemical, and behavioral phenotypes in mouse genetic models of PD. We discuss the incidence of given polymorphisms in PD patients and the nature of the mutations associated with inherited forms of the disease. Special attention is paid to the advantages and limitations of each model. Lastly, we identify groups of animals with the greatest utility in identifying environmental agents that may alter the course of PD-associated symptoms.

Genetic Models of Parkinson’s Disease: Behavior, Signaling, and Pathological Features

Parkinsons disease (PD) is the most common neurodegenerative movement disorder in the United States. Although the disease can either be inherited or arise spontaneously, only 10–20% of all PD cases arise from inherited mutations. Despite this discrepancy, modeling PD in animals by means of genetic manipulations has significantly advanced our understanding of the pathogenesis of this disease. In this chapter, we provide a comprehensive review of the pathological, neurochemical, and behavioral phenotypes in mouse genetic models of PD. We discuss the incidence of given polymorphisms in PD patients and the nature of the mutations associated with inherited forms of the disease. Special attention is paid to the advantages and limitations of each model. Lastly, we identify groups of animals with the greatest utility in identifying environmental agents that may alter the course of PD-associated symptoms.

Chapter 14 – Genetic Models of Parkinson’s Disease: Behavior, Signaling, and Pathological Features

Parkinsons disease (PD) is the most common neurodegenerative movement disorder in the United States. Although the disease can either be inherited or arise spontaneously, only 10–20% of all PD cases arise from inherited mutations. Despite this discrepancy, modeling PD in animals by means of genetic manipulations has significantly advanced our understanding of the pathogenesis of this disease. In this chapter, we provide a comprehensive review of the pathological, neurochemical, and behavioral phenotypes in mouse genetic models of PD. We discuss the incidence of given polymorphisms in PD patients and the nature of the mutations associated with inherited forms of the disease. Special attention is paid to the advantages and limitations of each model. Lastly, we identify groups of animals with the greatest utility in identifying environmental agents that may alter the course of PD-associated symptoms.