Cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are required for neurite outgrowth and cortical lamination. Proteolytic cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimer's disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons. Here we describe the mechanism of conversion of p35 to p25. In cultured primary cortical neurons, excitotoxins, hypoxic stress and calcium influx induce the production of p25. In fresh brain lysates, addition of calcium can stimulate cleavage of p35 to p25. Specific inhibitors of calpain, a calcium-dependent cysteine protease, effectively inhibit the calcium-induced cleavage of p35. In vitro, calpain directly cleaves p35 to release a fragment with relative molecular mass 25,000. The sequence of the calpain cleavage product corresponds precisely to that of p25. Application of the amyloid beta-peptide A beta(1-42) induces the conversion of p35 to p25 in primary cortical neurons. Furthermore, inhibition of cdk5 or calpain activity reduces cell death in A beta-treated cortical neurons. These observations indicate that cleavage of p35 to p25 by calpain may be involved in the pathogenesis of Alzheimer's disease.

Huntingtin is a 350-kilodalton protein of unknown function that is mutated in Huntington's disease (HD), a neurodegenerative disorder. The mutant protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Here we demonstrate that wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. We show that this beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This leads to insufficient neurotrophic support for striatal neurons, which then die. Restoring wild-type huntingtin activity and increasing BDNF production may be therapeutic approaches for treating HD.

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.

No abstract

Huntington disease is an autosomal dominant neurodegenerative disease with no effective treatment. Minocycline is a tetracycline derivative with proven safety. After ischemia, minocycline inhibits caspase-1 and inducible nitric oxide synthetase upregulation, and reduces infarction. As caspase-1 and nitric oxide seem to play a role in Huntington disease, we evaluated the therapeutic efficacy of minocycline in the R6/2 mouse model of Huntington disease. We report that minocycline delays disease progression, inhibits caspase-1 and caspase-3 mRNA upregulation, and decreases inducible nitric oxide synthetase activity. In addition, effective pharmacotherapy in R6/2 mice requires caspase-1 and caspase-3 inhibition. This is the first demonstration of caspase-1 and caspase-3 transcriptional regulation in a Huntington disease model.

The interleukin 1␤ converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1␤ (IL-1␤) levels increased in ischemic mouse brain.

Mutations in the copper/zinc superoxide dismutase (SOD1) gene produce an animal model of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. To test a new therapeutic strategy for ALS, we examined the effect of caspase inhibition in transgenic mice expressing mutant human SOD1 with a substitution of glycine to alanine in position 93 (mSOD1(G93A)). Intracerebroventricular administration of zVAD-fmk, a broad caspase inhibitor, delays disease onset and mortality. Moreover, zVAD-fmk inhibits caspase-1 activity as well as caspase-1 and caspase-3 mRNA up-regulation, providing evidence for a non-cell-autonomous pathway regulating caspase expression. Caspases play an instrumental role in neurodegeneration in transgenic mSOD1(G93A) mice, which suggests that caspase inhibition may have a protective role in ALS.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT), its associated G protein, and β-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT signal-transduction pathway inhibiting stress-mediated cytochrome release and caspase activation. These findings coupled with our observation that mitochondrial MT overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.