benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and malonic-acid

Transgenic Huntington's disease (HD) mice, expressing exon 1 of the HD gene with an expanded CAG repeat, are totally resistant to striatal lesion induced by excessive NMDA receptor activation. We now show that striatal lesions induced by the mitochondrial toxin malonate are reduced by 70-80% in transgenic HD mice compared with wild-type littermate controls. This occurred in 6- and 12-week-old HD mice with 150 CAG repeats (line R6/2) and in 18-week-old, but not 6-week-old, HD mice with 115 CAG repeats (line R6/1). Therefore, we show for the first time that the resistance to neurotoxin in transgenic HD mice is dependent on both the CAG repeat length and the age of the mice. Importantly, most HD patients develop symptoms in adulthood and exhibit an inverse relationship between CAG repeat length and age of onset. Transgenic mice expressing a normal CAG repeat (18 CAG) were not resistant to malonate. Although endogenous glutamate release has been implicated in malonate-induced cell death, glutamate release from striatal synaptosomes was not decreased in HD mice. Malonate-induced striatal cell death was reduced by 50-60% in wild-type mice when they were treated with either the NMDA receptor antagonist MK-801 or the caspase inhibitor zVAD-fmk. These two compounds did not reduce lesion size in transgenic R6/1 mice. This might suggest that NMDA receptor- and caspase-mediated cell death pathways are inhibited and that the limited malonate-induced cell death still occurring in HD mice is independent of these pathways. There were no changes in striatal levels of the two anti cell death proteins Bcl-X(L) and X-linked inhibitor of apoptosis protein (XIAP), before or after the lesion in transgenic HD mice. We propose that mutant huntingtin causes a sublethal grade of metabolic stress which is CAG repeat length-dependent and results in up-regulation over time of cellular defense mechanisms against impaired energy metabolism and excitotoxicity.

Topics: Aging; Amino Acid Chloromethyl Ketones; Animals; bcl-X Protein; Blood Glucose; Cell Death; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Humans; Huntingtin Protein; Huntington Disease; Immunoblotting; Immunohistochemistry; Male; Malonates; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Nuclear Proteins; Proteins; Proto-Oncogene Proteins c-bcl-2; Succinate Dehydrogenase; Synaptosomes; Trinucleotide Repeats; X-Linked Inhibitor of Apoptosis Protein

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain; Caspase 2; Caspase 3; Caspase Inhibitors; Caspases; Corpus Striatum; Cycloheximide; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Drug Synergism; Genes, bcl-2; Humans; Hypoxia, Brain; In Situ Nick-End Labeling; Male; Malonates; Mice; Mice, Transgenic; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley