Albert W. Probert

Evaluation of HIV-1 Tat induced neurotoxicity in rat cortical cell culture

In a substantial number of cases, Human Immunodeficiency Virus type 1 (HIV-1) infection causes neuronal cell loss and leads to the development of AIDS associated dementia. Several studies have suggested that both host and viral factors contribute to neuronal loss. Here we studied the effect of HIV-1 Tat in primary rat neuronal cells as a model to understand mechanism of neuronal cell death. At nano molar concentration, recombinant Tat induced cell death in primary rat mixed cortical neurons. Tat could also induce uptake of calcium in primary rat cultures. When cells were incubated with NMDA receptor antagonists, MK-801 and D-CPP, cell death and 45Ca uptake were inhibited. Under similar conditions non-NMDA antagonists, NBQX, DNQX and CNQX, and sodium channel antagonist, TTX, did not inhibit Tat induced neuronal cell death. In a similar way HIV associated products from in vitro HIV-1 infected cells induced neuronal cell death which was inhibited by NMDA receptor antagonist. Results presented in this paper suggest that activation of NMDA receptors by HIV-1 Tat is responsible for neuronal cell death in primary rat cortical neurons.

Selective release of calpain produced αII-spectrin (α-fodrin) breakdown products by acute neuronal cell death

Abstract Activation of calpain results in the breakdown of α II spectrin (αfodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cellconditioned media from SHSY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150- specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a timedependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTXinduced SBDP150 release. The cellconditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R()-3-(2-carbopiperazine-4-yl)propyl-1- phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody based detection of SBDP150 in the cellconditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Treatment with conotoxin, an ‘N-type’ calcium channel blocker, in neuronal hypoxic-ischemic injury

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit. In cell cultures, CgTx inhibited hypoxia-induced 45Ca accumulation and neuronal injury minimally, compared to the NMDA antagonist ketamine. In vivo, the duration of spinal cord ischemia which produced permanent paraplegia in 50% of control animals (ET50) was 24.0 +/- 2.6 min. Animals treated 2 h prior to ischemia with 0.5 nmol CgTx in the subarachnoid space had an ET50 of 26.9 +/- 1.8 min (P = 0.36). Animals treated 24 h prior to ischemia (all had persistent systemic tremor) had a ET50 of 28.9 +/- 1.8 min (P = 0.13). We conclude that pharmacologic modulation of the N-type calcium channel does not provide a significant protective effect against neuronal hypoxic-ischemic injury.

Alpha-Mercaptoacrylic Acid Derivatives as Novel Selective Calpain Inhibitors

Calpain is a family of cytosolic cysteine proteases activated by calcium (1–5). There are two major isoforms, µ- and m-calpain, requiring low and high micromolar calcium for in vitro activity, respectively. Both µ- and m-calpain have a large catalytic subunit (80 kDa) and a small regulatory subunit (29 kDa). Both subunits contain EF-hand calcium-binding structures which control the proteolytic activity of the enzyme. Calpains are implicated in a number of pathological conditions, including stroke, myocardial ischemia and cataract, where intracellular calcium levels are elevated markedly (6). In cerebral ischemia, synaptic glutamate (excitotoxin) buildup leads to excessive activation of ionotropic N-methyl-D-aspartate NMDA- and AMPA/kainate-receptors (7–8). The resultant calcium influx triggers calpain activation. Calpain then degrades cytoskeletal proteins which leads to the loss of cell integrity and cell death (9). Several peptide calpain inhibitors (e.g., calpain inhibitor I (acetyl-Leu-Leu-Nle-H) and MDL28170 (carbobenzoxy-Val-Phe-H)) were found to have neuroprotective effects in various models of excitotoxicity and/or ischemia (10-12). However, these agents are nonselective and cross-inhibit other cysteine proteases. Thus, it is highly desirable to develop selective and potent calpain inhibitors.