Minocycline mediates neuroprotection in experimental models of neurodegeneration. It inhibits the activity of caspase-1, caspase-3, inducible form of nitric oxide synthetase (iNOS) and p38 mitogen-activated protein kinase (MAPK). Although minocycline does not directly inhibit these enzymes, the effects may result from interference with upstream mechanisms resulting in their secondary activation. Because the above-mentioned factors are important in amyotrophic lateral sclerosis (ALS), we tested minocycline in mice with ALS. Here we report that minocycline delays disease onset and extends survival in ALS mice. Given the broad efficacy of minocycline, understanding its mechanisms of action is of great importance. We find that minocycline inhibits mitochondrial permeability-transition-mediated cytochrome c release. Minocycline-mediated inhibition of cytochrome c release is demonstrated in vivo, in cells, and in isolated mitochondria. Understanding the mechanism of action of minocycline will assist in the development and testing of more powerful and effective analogues. Because of the safety record of minocycline, and its ability to penetrate the blood-brain barrier, this drug may be a novel therapy for ALS.

Enoxaparin ͉ neurodegeneration ͉ transgenic mouse ͉ mitochondria ͉ Lovenox H untington's disease (HD) has onset usually between 35 and 50 years with chorea and psychiatric disturbances and gradual but inexorable intellectual decline to death after 15-20 years (1). Neuropathological analysis reveals selective and progressive neuronal loss in the striatum (1), particularly affecting the GABAergic medium spiny neurons (MSN). At the molecular level, the cause of HD is a polyglutamine expansion (exp) in the amino terminus of huntingtin (Htt), a 350-kDa ubiquitously expressed cytoplasmic protein (2). Despite significant progress, cellular mechanisms that link the Htt exp mutation with the disease are poorly understood (3).A number of transgenic HD mouse models have been generated that reproduce many HD-like features (4). In the yeast artificial chromosome (YAC128) mouse model, the full-length human Htt protein with polyglutamine exp (128Q) is expressed under the control of its endogenous promoter and regulatory elements (5). The onset of a motor deficit before striatal neuronal loss in the YAC128 mouse model accurately recapitulates the progression of HD (5). Thus, the YAC128 mouse model is ideal for understanding the cellular mechanisms that lead to neurodegeneration in HD, as well as for validating potential therapeutic agents.Previous studies demonstrated that Htt exp facilitates activity of the NR2B subtype of NMDA receptors (NMDARs) (6-8) and the type 1 inositol 1,4,5-trisphosphate receptors (InsP 3 R1) (9). A connection between disturbed Ca 2ϩ signaling and neuronal apoptosis is well established (10, 11), and we therefore proposed that Htt exp -induced Ca 2ϩ overload results in degeneration of MSN in HD (12). To test this hypothesis, we analyzed Ca 2ϩ signals and apoptotic cell death in primary cultures of MSN from the YAC128 mice. Our results provide further support to the hypothesis that disturbed Ca 2ϩ underlies neuronal cell death in HD (12) and allowed us to identify a number of potential therapeutic targets for HD treatment. Materials and MethodsPrimary Neuronal Cultures. Generation and breeding of YAC18 and YAC128 transgenic mice (FVBN͞NJ background strain) are described in refs. 5 and 13. Heterozygous male YAC128 or YAC18 mice were crossed with the wild-type (WT) female mice and resulting litters were collected at postnatal days 1-2. The pups were genotyped by PCR with primers specific for exons 44 and 45 of human Htt gene and the medium spiny neuronal (MSN) or hippocampal neuronal (HN) cultures of WT, YAC18, and YAC128 mice were established and maintained as described in ref. 9.Ca 2؉ Imaging Experiments. Fura-2 Ca 2ϩ imaging experiments with 14-to 16-DIV (days in vitro) MSN cultures were performed as described in ref. 9, using a DeltaRAM illuminator, an IC-300 camera, and IMAGEMASTER PRO software (all from PTI, South Brunswick, NJ). The cells were maintained in artificial cerebrospinal fluid (aCSF) (140 mM NaCl͞5 mM KCl͞1 mM MgCl 2 ͞2 mM CaCl 2 ͞10 mM Hepes, pH 7.3) at 37°C during measurements (PH1 heat...

Minocycline is broadly protective in neurologic disease models featuring cell death and is being evaluated in clinical trials. We previously demonstrated that minocycline-mediated protection against caspase-dependent cell death related to its ability to prevent mitochondrial cytochrome c release. These results do not explain whether or how minocycline protects against caspaseindependent cell death. Furthermore, there is no information on whether Smac͞Diablo or apoptosis-inducing factor might play a role in chronic neurodegeneration. In a striatal cell model of Huntington's disease and in R6͞2 mice, we demonstrate the association of cell death͞disease progression with the recruitment of mitochondrial caspase-independent (apoptosis-inducing factor) and caspase-dependent (Smac͞Diablo and cytochrome c) triggers. We show that minocycline is a drug that directly inhibits both caspase-independent and -dependent mitochondrial cell death pathways. Furthermore, this report demonstrates recruitment of Smac͞Diablo and apoptosis-inducing factor in chronic neurodegeneration. Our results further delineate the mechanism by which minocycline mediates its remarkably broad neuroprotective effects.

Background: Mitochondrial dysfunction and aggregation of ␣-synuclein both contribute to Parkinson disease. Results: Prefibrillar ␣-synuclein oligomers reduce the Ca 2ϩ retention time of isolated mitochondria respiring with complex I but not II substrates. Conclusion: Oligomeric ␣-synuclein promotes mitochondrial dysfunction in a Ca

Background and Purpose-The identification of a neuroprotective drug for stroke remains elusive. Given that mitochondria play a key role both in maintaining cellular energetic homeostasis and in triggering the activation of cell death pathways, we evaluated the efficacy of newly identified inhibitors of cytochrome c release in hypoxia/ischemia induced cell death. We demonstrate that methazolamide and melatonin are protective in cellular and in vivo models of neuronal hypoxia. Methods-The effects of methazolamide and melatonin were tested in oxygen/glucose deprivation-induced death of primary cerebrocortical neurons. Mitochondrial membrane potential, release of apoptogenic mitochondrial factors, pro-IL-1␤ processing, and activation of caspase -1 and -3 were evaluated. Methazolamide and melatonin were also studied in a middle cerebral artery occlusion mouse model. Infarct volume, neurological function, and biochemical events were examined in the absence or presence of the 2 drugs. Results-Methazolamide and melatonin inhibit oxygen/glucose deprivation-induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1␤ processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, they decrease infarct size and improve neurological scores after middle cerebral artery occlusion in mice. Conclusions-We demonstrate that methazolamide and melatonin are neuroprotective against cerebral ischemia and provide evidence of the effectiveness of a mitochondrial-based drug screen in identifying neuroprotective drugs. Given the proven human safety of melatonin and methazolamide, and their ability to cross the blood-brain-barrier, these drugs are attractive as potential novel therapies for ischemic injury.

Huntington's disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosinecytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

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Brown adipose tissue (BAT) mitochondria exhibit high oxidative capacity and abundant expression of both electron transport chain components and uncoupling protein 1 (UCP1). UCP1 dissipates the mitochondrial proton motive force (Δp) generated by the respiratory chain and increases thermogenesis. Here we find that in mice genetically lacking UCP1, cold-induced activation of metabolism triggers innate immune signaling and markers of cell death in BAT. Moreover, global proteomic analysis reveals that this cascade induced by UCP1 deletion is associated with a dramatic reduction in electron transport chain abundance. UCP1-deficient BAT mitochondria exhibit reduced mitochondrial calcium buffering capacity and are highly sensitive to mitochondrial permeability transition induced by reactive oxygen species (ROS) and calcium overload. This dysfunction depends on ROS production by reverse electron transport through mitochondrial complex I, and can be rescued by inhibition of electron transfer through complex I or pharmacologic depletion of ROS levels. Our findings indicate that the interscapular BAT of Ucp1 knockout mice exhibits mitochondrial disruptions that extend well beyond the deletion of UCP1 itself. This finding should be carefully considered when using this mouse model to examine the role of UCP1 in physiology.brown fat | mitochondria | ROS | UCP1 | electron transport chain U ncoupling protein 1 (UCP1) plays a role in acute adaptive thermogenesis in interscapular brown adipose tissue (BAT). UCP1 dissipates the mitochondrial protonmotive force (Δp) generated by the electron transport chain (ETC) and is important for thermal homeostasis in rodents and human infants (1, 2). Ucp1 orthologs are not limited to mammals, but are also expressed in ectothermic vertebrates (3) and protoendothermic mammals (4), suggesting that UCP1 may have an important role in biology beyond thermal control. For example, it is becoming increasingly evident that in specific respiratory states, UCP1 can reduce reactive oxygen species (ROS) levels in vitro (4-9). The mitochondrial ETC is a major source of ROS production in the cell, and ROS play important roles in physiology and pathophysiology (10-12). Reverse electron transport (RET) through mitochondrial complex I is a key mechanism by which ROS are generated in vivo (11, 13). Interestingly, RET relies critically on high Δp, whereas dissipation of Δp by UCP1 can lower ROS levels in isolated mitochondria (5-7).Thermogenic respiration in BAT is triggered by external stimuli that activate adrenergic signaling (14). Most notably, environmental cold induces the capacity for adrenergic-mediated BAT respiration in wild type (WT) animals, but only minimally in UCP1-KO animals (15, 16). It is understood that the respiratory response of BAT under these conditions is indicative of UCP1-mediated respiration; however, the rate of maximal chemically uncoupled oxygen consumption, an UCP1-independent parameter, is also lower in UCP1-KO adipocytes compared with WT (15, 16).Moreover, the basal respiratory rat...