title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration.
abstract: Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.
authors.affiliation: Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: White
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Cagla
authors.initials: C
authors.lastname: Akay
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Lindl
authors.forename: Dennis L
authors.initials: DL
authors.lastname: Kolson
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Jordan-Sciutto
mesh_terms: D015526:AIDS Dementia Complex / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D008264:Macrophages / Q000378:metabolism / Q000473:pathology / Q000821:virology; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000172:deficiency; D028361:Mitochondrial Diseases / Q000473:pathology* / Q000821:virology*; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology* / Q000821:virology; D009474:Neurons / Q000378:metabolism / Q000473:pathology* / Q000821:virology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines
chemical_list: D008869:Microtubule-Associated Proteins; D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester; D002154:Calpain