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. In this study we exposed primary rat neurons and astrocyte cultures to established amino acid analogs (Canavanine and Azetidine-2-carboxylic acid), and observed both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity as compared to 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, as compared to astrocytes

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.Fil: Dasuri, Kalavathi. State University of Louisiana; Estados UnidosFil: Ebenezer, Philip J.. State University of Louisiana; Estados UnidosFil: Zhang, Le. State University of Louisiana; Estados UnidosFil: Fernandez Kim, Sun Ok. State University of Louisiana; Estados UnidosFil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Gavilán, Elena. State University of Louisiana; Estados UnidosFil: Di Blasio, Alessia. State University of Louisiana; Estados UnidosFil: Keller, Jeffrey N.. State University of Louisiana; Estados Unido

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. In this study we exposed primary rat neurons and astrocyte cultures to established amino acid analogs (Canavanine and Azetidine-2-carboxylic acid), and observed both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity as compared to 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, as compared to astrocytes.Fil: Dasuri, Kalavathi. State University Of Louisiana; Estados UnidosFil: Ebenezer, Philip J.. State University Of Louisiana; Estados UnidosFil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Universidad Nacional del Sur; ArgentinaFil: Gavilan, Elena. Universidad de Sevilla; EspañaFil: Zhang, Le. State University Of Louisiana; Estados UnidosFil: Fernandez-Kim, Sun O. K.. State University Of Louisiana; Estados UnidosFil: Bruce Keller, Annadora J.. State University Of Louisiana; Estados UnidosFil: Keller, Jeffrey N.. State University Of Louisiana; Estados Unido

Mutant amyloid precursor protein differentially alters adipose biology under obesogenic and non-obesogenic conditions.

Mutations in amyloid precursor protein (APP) have been most intensely studied in brain tissue for their link to Alzheimer's disease (AD) pathology. However, APP is highly expressed in a variety of tissues including adipose tissue, where APP is also known to exhibit increased expression in response to obesity. In our current study, we analyzed the effects of mutant APP (E693Q, D694N, K670N/M671L) expression toward multiple aspects of adipose tissue homeostasis. These data reveal significant hypoleptinemia, decreased adiposity, and reduced adipocyte size in response to mutant APP, and this was fully reversed upon high fat diet administration. Additionally, mutant APP was observed to significantly exacerbate insulin resistance, triglyceride elevations, and macrophage infiltration of adipose tissue in response to a high fat diet. Taken together, these data have significant implications for linking mutant APP expression to adipose tissue dysfunction and global changes in endocrine and metabolic function under both obesogenic and non-obesogenic conditions

Obesity increases cerebrocortical reactive oxygen species and impairs brain function

Nearly two-thirds of the population in the United States is overweight or obese, and this unprecedented level of obesity will undoubtedly have a profound impact on overall health, although little is currently known about the effects of obesity on the brain. The objective of this study was to investigate cerebral oxidative stress and cognitive decline in the context of diet-induced obesity (DIO). We demonstrate for the first time that DIO induces higher levels of reactive oxygen species (ROS) in the brain and promotes cognitive impairment. Importantly, we also demonstrate for the first time in these studies that both body weight and adiposity are tightly correlated with the level of ROS. Interestingly, ROS were not correlated with cognitive decline in this model. Alterations in the antioxidant/detoxification Nrf2 pathway, superoxide dismutase, and catalase activity levels were not significantly altered in response to DIO. However, a significant impairment in glutathione peroxidase was observed in response to DIO. Taken together, these data demonstrate for the first time that DIO increases the levels of total and individual ROS in the brain and highlight a direct relationship between the amount of adiposity and the level of oxidative stress within the brain. These data have important implications for understanding the negative effects of obesity on the brain and are vital to understanding the role of oxidative stress in mediating the effects of obesity on the brain

Autophagic receptor p62 protects against glycation-derived toxicity and enhances viability

Diabetes and metabolic syndrome are associated with the typical American high glycemia diet and result in accumulation of high levels of advanced glycation end products (AGEs), particularly upon aging. AGEs form when sugars or their metabolites react with proteins. Associated with a myriad of age-related diseases, AGEs accumulate in many tissues and are cytotoxic. To date, efforts to limit glycation pharmacologically have failed in human trials. Thus, it is crucial to identify systems that remove AGEs, but such research is scanty. Here, we determined if and how AGEs might be cleared by autophagy. Our in vivo mouse and C. elegans models, in which we altered proteolysis or glycative burden, as well as experiments in five types of cells, revealed more than six criteria indicating that p62-dependent autophagy is a conserved pathway that plays a critical role in the removal of AGEs. Activation of autophagic removal of AGEs requires p62, and blocking this pathway results in accumulation of AGEs and compromised viability. Deficiency of p62 accelerates accumulation of AGEs in soluble and insoluble fractions. p62 itself is subject to glycative inactivation and accumulates as high mass species. Accumulation of p62 in retinal pigment epithelium is reversed by switching to a lower glycemia diet. Since diminution of glycative damage is associated with reduced risk for age-related diseases, including age-related macular degeneration, cardiovascular disease, diabetes, Alzheimer's, and Parkinson's, discovery of methods to limit AGEs or enhance p62-dependent autophagy offers novel potential therapeutic targets to treat AGEs-related pathologies.National Institutes of Health R01AG028664, R21AG058038, R01EY021212, R01EY026979, R01EY028559U.S. Department of Agriculture 8050-51000-089-01SHuman Nutrition Research Center on Aging 2016–08885Ministerio de Economía y Competitividad SAF 2016 78666‐

Serum adipokines.

<p>CAA-CD mice revealed significant hypoleptinemia (C-CD to CAA-CD: p = 0.019; C-HF to CAA-CD: p<0.0001; CAA-HF to CAA-CD: p<0.0001; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043193#pone-0043193-g005" target="_blank">Figure 5A</a>). This was reversed by HF diet treatment as C-HF and CAA-HF had comparable leptin levels, which were also significantly higher than C-CD mice (p<0.0001 and p = 0.015, respectively). No significant differences for the adipokines resistin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043193#pone-0043193-g003" target="_blank">Figure 3B</a>) and adiponectin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043193#pone-0043193-g003" target="_blank">Figure 3C</a>) were observed between groups using ELISA serum analysis.</p

Macrophage infiltration of adipose depots.

<p>In subcutaneous fat, the C-HF and CAA-HF revealed the most “crown-like structures”. These were rarely found in the C-CD or CAA-CD subcutaneous fat samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043193#pone-0043193-g009" target="_blank">Figure 9A</a>). Visceral fat depots revealed a greater number of macrophages and crown –like structures (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043193#pone-0043193-g009" target="_blank">Figure 9B</a>). They were found in CAA-CD, C-HF, and CAA-HF, and rarely found in the C-CD samples. Asterisks mark cells with crown-like structures.</p