Because the release of cytochrome c
from mitochondria triggers caspase activation, blocking this critical step should interfere with the cell death program thereby rescue dying neurons. Release of cytochrome c
and activation of downstream cell death pathways have been clearly identified in vivo
in a broad number of acute (i.e., ischemia, spinal cord injury, and traumatic brain injury) and chronic (i.e., ALS and Huntington’s disease) neurodegenerative diseases. However, evidence clearly demonstrating the functional role of cytochrome c
release in neurologic disease models is limited. Although minocycline inhibits cytochrome c
release and it is neuroprotective, it may be other functions that may explain its protective function. What is needed to provide further support for the functional role of cytochrome c
release in HD and other neurodegenerative diseases is to identify new inhibitors of cytochrome c
release. With this goal in mind, we developed a cell-free screening assay to identify inhibitors of mitochondrial cytochrome c
release. Purified mitochondria exposed to Ca2+
ions provide the requisite in vitro
system. Released cytochrome c
(i.e., that which remains in the supernatant once the organelles have been removed by centrifugation) can easily be detected by ELISA. Those compounds that reduce the resulting signal are promising candidates for neuroprotective drugs. Compounds from the resulting “short list” were then assayed for their ability to rescue cultured cells challenged with a cell death stimuli, incubation at the nonpermissive temperature. Drugs/compounds that are protective in these cellular systems are further evaluated in trials on animal models. Ultimately, biologically safe and potentially curative agents can be tested on humans afflicted by chronic neurodegenerative diseases such as HD. Minocycline and doxycycline, two compounds known a priori to slow neurodegeneration (Yrjänheikki et al., 1998
; Chen et al., 2000
; Brundula et al., 2002
; Wu et al., 2002
; Zhu et al., 2002
; Friedlander, 2003
; Wang et al., 2003
), are particularly effective at inhibiting the release of cytochrome c
from purified mitochondria. These observations are reassuring, as they suggest that the in vitro
assay has predictive value. However, a positive result in the isolated mitochondrial screen does not, itself, give definitive indication of eventual efficacy or safety of an agent. Gossypol could be an example of a “false” positive agent. Gossypol is a well known respiratory poison (uncoupler), considering a strong mitochondrial respiratory toxin will score positive as an inhibitor of any event which requires an active proton gradient, such as calcium transport, it is reasonable to account for our observation that gossypol is effective in the isolated mitochondrial screen (rank in first) but toxic in intact cells (cellular screen) (Floridi et al., 1984
Because different screening campaigns and different model systems may yield different hit compounds, until now, no single powerful and validated model exists for Huntington’s or other neurodegenerative diseases. Using our three-tiered selection process (an assay for activity in a cell-free system, observation of effects on cultured cells, and finally trials in animal models) we successively narrowed the list of potentially therapeutic compounds. Of the 21 compounds selected in the initial screen, 16 proved protective of cells in culture. Our study thus provides proof of principle that the screen for inhibitors of cytochrome c release from purified mitochondria is a useful technique for identifying drugs with the potential to slow neurodegeneration.
Furthermore, our observations constitute the first report of the ability of methazolamide to benefit R6/2 mice. CA is a zinc-containing enzyme that catalyzes the reversible hydration of carbon dioxide: CO2+ H2O<–>HCO3(−)+H+. There are seven mammalian CA isozymes, CA I-VII. Thus far, the mechanism of action of methazolamide consists in inhibition of target CA isozymes, with the reduction of bicarbonate, and profound effect on pH. Furthermore, methazolamide has been reported to be an effective inhibitor of human CA II (hCA II) and mitochondrial hCA V. Considering methazolamide inhibits cytochrome c/Smac/apoptosis-inducing factor release and slows the dissipation of the mitochondrial potential gradient in this study, we hypothesize that mitochondrial hCA V may involve in the neuroprotection by methazolamide in mutant-htt ST14A living cells and in R6/2 transgenic mice. However, methazolamide may act independent of CA isozymes, it may act on other targets in cellular and/or animal model of HD, such as the NADH oxidase, or inhibit neuronal cell death with involvement of reactive oxygen species. In light of the fact that methazolamide in current clinical usage as chronic therapy for patients with glaucoma, although it is a weak diuretic, should not have prohibitive side effects if used in trials on humans. It is our hope that methazolamide will also be of clinical value for the treatment of persons afflicted by neurodegenerative diseases.
Together, these experiments have demonstrated that methazolamide is protective in an animal model of chronic neurodegeneration. The conclusions reached above pertain to this drug in particular. The study has broader significance, however. It demonstrates that the in vitro assay for cytochrome c release from Ca2+-stimulated mitochondria can be used to screen for potentially therapeutic compounds and provides further in vivo evidence for the importance of cytochrome c release in the process of neurodegeneration. At the physiological level, it validates targeting that molecular process as a rational approach to designing therapies for chronic neurological diseases such as HD.