MDL 28170

Traumatic brain injury (TBI) is a leading cause of death and morbidity in industrialized countries with considerable associated health care costs. The cost and time associated with pre-clinical development of TBI therapeutics is lengthy and expensive with a poor track record of successful translation to the clinic. The zebrafish is an emerging model organism in research with unique technical and genomic strengths in the study of disease and development. Its high degree of genetic homology and cell signaling pathways relative to mammalian species and amenability to high and medium throughput assays has potential to accelerate the rate of therapeutic drug identification. Accordingly, we developed a novel closed-head model of TBI in adult zebrafish using a targeted, pulsed, high-intensity focused ultrasound (pHIFU) to induce mechanical injury of the brain. Western blot results indicated altered microtubule and neurofilament expression as well as increased expression of cleaved caspase-3 and beta APP (β-APP; p < 0.05). We used automated behavioral tracking software to evaluate locomotor deficits 24 and 48 h post-injury. Significant behavioral impairment included decreased swim distance and velocity (p < 0.

Molecular events under the neuronal degeneration are widely studied but still not defined. Here we compared the effects of both excitotoxic and apoptotic insults on the DNA binding profile of multifunctional transcription factor YY1 protein in cultured cerebellar granule neurons. We report that L-glutamate-induced excitotoxic insult but not ionophore A23187 treatment caused the disappearance of the larger DNA binding complex of YY1 and a simultaneous appearance of the smaller YY1 complex in cerebellar granule neurons. MK-801 (NMDA receptor antagonist) as well as benzamide (PARP inhibitor), MDL 28170 (calpain inhibitor) and roscovitine (cyclin-dependent kinase inhibitor) inhibited the glutamate response to the YY1 complexes. Herbimycin, PD169316, wortmannin, JAK3 inhibitor, KN-93, H-7 and LY294002 were not effective. Apoptosis induced by okadaic acid but not that induced by etoposide or trichostatin A caused a similar excitotoxic reorganization in YY1 complexes. We suggest that despite the different cell death mechanisms, glutamate and okadaic acid activate signalling cascades that affect the formation of YY1 complexes and probably YY1-mediated gene regulation.

BACKGROUND AND PURPOSE: ;Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment.;METHODS: ;Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval.;RESULTS: ;The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170.