ASN NEURO Immediate Publications

Role of endolysosomes in HIV-1 Tat-induced neurotoxicity

L Hui, X Chen, N J. Haughey, J D. Geiger — 2012-05-16T13:54:26Z

Combined anti-retroviral therapeutic drugs effectively increase the life span of HIV-1 infected individuals who then have a higher prevalence of HIV-1 associated neurocognitive disorder (HAND). Soluble factors including HIV-1 proteins released from HIV-1 infected cells have been implicated in the pathogenesis of HAND and particular attention has been paid to the HIV-1 transactivator protein (Tat) because of its ability to directly excite neurons and cause neuronal cell death. Because HIV-1 Tat enters cells by receptor-mediated endocytosis and because endolysosomes play an important role in neuronal cell life and death, we tested here the hypothesis that HIV-1 Tat neurotoxicity is associated with changes in endolysosome structure and function as well as autophagy. Following treatment of primary cultured rat hippocampal neurons with HIV-1 Tat, or as controls mutant-Tat or phosphate-buffered saline, neuronal viability was determined using a triple staining method. Preceding observations of HIV-1 Tatinduced neuronal cell death we observed statistically significant changes in the structure and membrane integrity of endolysosomes, endolysosome pH, and autophagy. As early as 24 h after HIV-1 Tat was applied to neurons, HIV-1 Tat accumulated in endolysosomes, endolysosome morphology was affected and their size was increased, endolysosome membrane integrity was disrupted, endolysosome pH was increased, specific activities of endolysosome enzymes were decreased, and autophagy was inhibited as indicated by significant changes in three markers for autophagy. In contrast, statistically significant levels of HIV-1 Tat-induced neuronal cell death was observed only after 48 h of HIV-1 Tat treatment. Our findings suggest that endolysosomes are involved in HIV-1 Tat-induced neurotoxicity and may represent a target for therapeutic intervention against HAND.

Hypoxia Inducible Factor 1 Protects Hypoxic Astrocytes against Glutamate Toxicity

Y Badawi, P Ramamoorthy, H Shi — 2012-04-27T16:21:08Z

Stroke is a major neurological disorder characterized by an increase in the glutamate (Glu) concentration resulting in excitotoxicity and eventually cellular damage and death in the brain. Hypoxia inducible factor-1 (HIF-1), a transcription factor, plays an important protective role in promoting cellular adaptation to hypoxic conditions. It is known that HIF-1α, the regulatable subunit of HIF-1, is expressed by astrocytes under severe ischemia. However, the effect of HIF-1 on astrocytes following Glu toxicity during ischemia has not been well studied. We investigated the role of HIF-1 in protecting ischemic astrocytes against Glu toxicity. Immunostaining with GFAP confirmed the morphological modification of astrocytes in the presence of 1 mM Glu under normoxia. Interestingly, when the astrocytes were exposed to severe hypoxia (0.1% O₂), the altered cell morphology was ameliorated with upregulation of HIF-1α. To ascertain HIF-1’s protective role, effects of two HIF-1α inhibitors, 3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole (YC-1) and 2-methoxyestradiol (2ME2), were tested. Both of the inhibitors decreased the recovery in astrocyte morphology and increased cell death. Given that ischemia increases reactive oxygen species (ROS), we examined the role of glutathione (GSH) in the mechanism for this protection. GSH was increased under hypoxia and this correlated with an increase in HIF-1α stabilization in the astrocytes. Furthermore, inhibition of GSH with L-butathione sulfoximine (BSO) decreased HIF-1α expression, suggesting its role in the stabilization of HIF-1α. Overall, our results indicate that the expression of HIF-1α under hypoxia has a protective effect on astrocytes in maintaining cell morphology and viability in response to Glu toxicity.

Di(2-ethylhexyl)phthalate and Autism Spectrum Disorders

C Testa, F Nuti, J Hayek, C De Felice, M Chelli, P Rovero, G Latini, A Papini — 2012-04-27T11:21:27Z

Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders, still poorly understood, steadily rising in frequency, and treatment-refractory. Extensive research has been so far unable to explain the aetiology of this condition, whereas a growing body of evidence suggests the involvement of environmental factors. Phthalates, given their extensive use and their persistence, are ubiquitous environmental contaminants. They are endocrine-disrupting chemicals suspected to interfere with neurodevelopment. Therefore, they represent interesting candidate risk factors for ASDs pathogenesis. Aim of this study was to evaluate the levels of the primary and secondary metabolites of the di(2-ethylhexyl)phthalate (DEHP) in children with ASDs. A total of 48 children with ASDs [M: 36, F: 12; mean age: 11 years ± 5 years] and 45 age and sex comparable healthy controls (HCs, M: 25, F: 20; mean age: 12 years± 5 years) were enrolled. A diagnostic methodology, based on the determination of urinary concentrations of DEHP metabolites by HPLC-ESI-MS, was applied to urine spot samples. MEHP, 6-OH-MEHP, 5-OH-MEHP, and 5-oxo-MEHP were measured and compared to unequivocally characterised, pure synthetic compounds (<98%) taken as standards. In ASDs patients, significantly increased 5-OH-MEHP (52.1%, median 0.18) and 5-oxo-MEHP (46.0%, median 0.096) urinary concentrations were detected, with a significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP (r_s = 0.668, p < 0.0001). The fully oxidised form 5-oxo-MEHP showed 91.1% specificity in identifying patients with ASDs. Our findings, demonstrate for the first time an association between phthalates exposure and ASDs, thus suggesting a previously unrecognized role for these ubiquitous environmental contaminants in the pathogenesis of autism.