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...