Philip J. Ebenezer

NOX Activity Is Increased in Mild Cognitive Impairment

This study was undertaken to investigate the profile of NADPH oxidase (NOX) in the clinical progression of Alzheimers disease (AD). Specifically, NOX activity and expression of the regulatory subunit p47phox and the catalytic subunit gp91phox was evaluated in affected (superior and middle temporal gyri) and unaffected (cerebellum) brain regions from a longitudinally followed group of patients. This group included both control and late-stage AD subjects, and also subjects with preclinical AD and with amnestic mild cognitive impairment (MCI) to evaluate the profile of NOX in the earliest stages of dementia. Data show significant elevations in NOX activity and expression in the temporal gyri of MCI patients as compared with controls, but not in preclinical or late-stage AD samples, and not in the cerebellum. Immunohistochemical evaluations of NOX expression indicate that whereas microglia express high levels of gp91phox, moderate levels of gp91phox also are expressed in neurons. Finally, in vitro experiments showed that NOX inhibition blunted the ability of oligomeric amyloid beta peptides to injure cultured neurons. Collectively, these data show that NOX expression and activity are upregulated specifically in a vulnerable brain region of MCI patients, and suggest that increases in NOX-associated redox pathways in neurons might participate in the early pathogenesis of AD.

Aging and dietary restriction alter proteasome biogenesis and composition in the brain and liver.

Interventions such as dietary restriction (DR) have been reported to ameliorate age-related proteasome inhibition in some tissues. Currently it is not known what effects aging and DR have on proteasome biogenesis in the liver and brain, nor have previous studies identified the links between changes in proteasome composition, biogenesis, and activity in the aging brain and liver. In the present study we demonstrate that the brain and liver exhibit age-dependent decreases in 26S and 20S proteasome activity. Additionally, our studies demonstrate that the brain and liver undergo selective changes in proteasome biology, including increases in proteasome biogenesis in response to aging and DR, with the liver exhibit more robust plasticity as compared to the brain. Lastly, studies demonstrated that aging and DR alter the interaction of Hsp90 with the 20S proteasome complex in the brain and liver. These studies affirm the dynamic nature of the proteasome complexes in both the liver and brain following aging and DR. Additionally, these data indicate that the relationship between proteasome composition/biogenesis and proteasome activity in tissues is extremely complex and tissue specific. These data have implications for understanding the effects of tissue specific effects of aging and DR on protein turnover and proteotoxicity.

Intersection Between Metabolic Dysfunction, High Fat Diet Consumption, And Brain Aging

J. Neurochem. (2010) 114, 344–361.

Neuron specific toxicity of oligomeric amyloid-β: role for JUN-kinase and oxidative stress.

Recent studies have demonstrated a potential role for oligomeric forms of amyloid-β (Aβ) in the pathogenesis of Alzheimers disease (AD), although it remains unclear which aspects of AD may be mediated by oligomeric Aβ. In the present study, we found that primary cultures of rat cortical neurons exhibit a dose-dependent increase in cell death following Aβ oligomer administration, while primary cultures of astrocytes exhibited no overt toxicity with even the highest concentrations of oligomer treatment. Neither cell type exhibited toxicity when treated by equal concentrations of monomeric Aβ. The neuron death induced by oligomer treatment was associated with an increase in reactive oxygen species (ROS), altered expression of mitochondrial fission and fusion proteins, and JUN kinase activation. Pharmacological inhibition of JUN kinase ameliorated oligomeric Aβ toxicity in neurons. These data indicate that oligomeric Aβ is sufficient to selectively induce toxicity in neurons, but not astrocytes, with neuron death occurring in a JUN kinase-dependent manner. Additionally, these observations implicate a role for oligomeric Aβ as a contributor to neuronal oxidative stress and mitochondrial disturbances in AD.

Proteasome alterations during adipose differentiation and aging: links to impaired adipocyte differentiation and development of oxidative stress.

Intracellular proteins are degraded by a number of proteases, including the ubiquitin-proteasome pathway (UPP). Impairments in the UPP occur during the aging of a variety of tissues, although little is known in regards to age-related alterations to the UPP during the aging of adipose tissue. The UPP is known to be involved in regulating the differentiation of a variety of cell types, although the potential changes in the UPP during adipose differentiation have not been fully elucidated. How the UPP is altered in aging adipose tissue and adipocyte differentiation and the effects of proteasome inhibition on adipocyte homeostasis and differentiation are critical issues to elucidate experimentally. Adipogenesis continues throughout the life of adipose tissue, with continual differentiation of preadipocytes essential to maintaining tissue function during aging, and UPP alterations in mature adipocytes are likely to directly modulate adipose function during aging. In this study we demonstrate that aging induces alterations in the activity and expression of principal components of the UPP. Additionally, we show that multiple changes in the UPP occur during the differentiation of 3T3-L1 cells into adipocytes. In vitro data link observed UPP alterations to increased levels of oxidative stress and altered adipose biology relevant to both aging and differentiation. Taken together, these data demonstrate that changes in the UPP occur in response to adipose aging and adipogenesis and strongly suggest that proteasome inhibition is sufficient to decrease adipose differentiation, as well as increasing oxidative stress in mature adipocytes, both of which probably promote deleterious effects on adipose aging.

Ten nights of moderate hypoxia improves insulin sensitivity in obese humans.

Hypoxia in obese adipose tissue (AT) plays an important role in the development of whole-body insulin resistance by inducing local inflammation and the release of proinflammatory cytokines (1). Yet, living at high altitude is associated with a lower prevalence of impaired fasting glucose and type 2 diabetes compared with living at low altitude (2). Furthermore, exposure to hypoxic environments increases whole-body glucose fluxes in healthy males and glucose uptake in human and murine skeletal muscle (3). In addition, exercising under hypoxic conditions improves glucose tolerance more than exercising under normoxia (4), strongly suggesting an insulin-sensitizing effect of hypoxia. Therefore, we hypothesized that exposing obese men to 10 consecutive nights of moderate hypoxia (15 ± 0.5% O2, ∼2,400 m elevation) would improve insulin sensitivity.nnEight healthy obese men (4 Caucasians, 3 African Americans, and 1 Hispanic of mean ± SEM age 28 ± 1 years, weight 96.5 ± 5.3 kg, and BMI 32.7 ± 1.3 kg/m2) without evidence of chronic disease or sleep apnea and taking no medication participated in this study. The protocol was approved by the institutional review board at Pennington Biomedical Research Center (Baton Rouge, LA). Subjects slept for 10 consecutive nights (∼10 h/night, ≥100 h in total) in a hypoxic tent (Hypoxico Inc., New York, NY) maintained at ∼15% O2 (range 14.5–15.5% O2, ∼2,400 m above sea level) using nitrogen dilution. Biopsies of abdominal subcutaneous AT and skeletal muscle were obtained at baseline and on day 11 under …

Increased protein hydrophobicity in response to aging and Alzheimer disease.

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.

Selective vulnerability of neurons to acute toxicity after proteasome inhibitor treatment: Implications for oxidative stress and insolubility of newly synthesized proteins

Maintaining protein homeostasis is vital to cell viability, with numerous studies demonstrating a role for proteasome inhibition occurring during the aging of a variety of tissues and, presumably, contributing to the disruption of cellular homeostasis during aging. In this study we sought to elucidate the differences between neurons and astrocytes in regard to basal levels of protein synthesis, proteasome-mediated protein degradation, and sensitivity to cytotoxicity after proteasome inhibitor treatment. In these studies we demonstrate that neurons have an increased vulnerability, compared to astrocyte cultures, to proteasome-inhibitor-induced cytotoxicity. No significant difference was observed between these two cell types in regard to the basal rates of protein synthesis, or basal rates of protein degradation, in the pool of short-lived proteins. After proteasome inhibitor treatment neuronal crude lysates were observed to undergo greater increases in the levels of ubiquitinated and oxidized proteins and selectively exhibited increased levels of newly synthesized proteins accumulating within the insoluble protein pool, compared to astrocytes. Together, these data suggest a role for increased oxidized proteins and sequestration of newly synthesized proteins in the insoluble protein pool, as potential mediators of the selective neurotoxicity after proteasome inhibitor treatment. The implications for neurons exhibiting increased sensitivity to acute proteasome inhibitor exposure, and the corresponding changes in protein homeostasis observed after proteasome inhibition, are discussed in the context of both aging and age-related disorders of the nervous system.

Role of physiological levels of 4-hydroxynonenal on adipocyte biology: implications for obesity and metabolic syndrome

Abstract Lipid peroxidation products such as 4-hydroxynonenal (HNE) are known to be increased in response to oxidative stress, and are known to cause dysfunction and pathology in a variety of tissues during periods of oxidative stress. The aim of the current study was to determine the chronic (repeated HNE exposure) and acute effects of physiological concentrations of HNE toward multiple aspects of adipocyte biology using differentiated 3T3-L1 adipocytes. Our studies demonstrate that acute and repeated exposure of adipocytes to physiological concentrations of HNE is sufficient to promote subsequent oxidative stress, impaired adipogenesis, alter the expression of adipokines, and increase lipolytic gene expression and subsequent increase in free fatty acid (FFA) release. These results provide an insight in to the role of HNE-induced oxidative stress in regulation of adipocyte differentiation and adipose dysfunction. Taken together, these data indicate a potential role for HNE promoting diverse effects toward adipocyte homeostasis and adipocyte differentiation, which may be important to the pathogenesis observed in obesity and metabolic syndrome.

Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: Implications for protein misfolding and TDP‐43 regulation

Amino acid analogs promote translational errors that result in aberrant protein synthesis and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP‐43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. This study exposed primary rat neurons and astrocyte cultures to established amino acid analogs (canavanine and azetidine‐2‐carboxylic acid) and showed that both cell types undergo a dose‐dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity compared with astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP‐43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP‐43 in response to increased protein misfolding compared with astrocytes.