Robert M. Friedlander

Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice

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.

Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease

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.

Neurotoxicity induces cleavage of p35 to p25 by calpain

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 Alzheimers 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 β-peptide Aβ(1–42) induces the conversion of p35 to p25 in primary cortical neurons. Furthermore, inhibition of cdk5 or calpain activity reduces cell death in Aβ-treated cortical neurons. These observations indicate that cleavage of p35 to p25 by calpain may be involved in the pathogenesis of Alzheimers disease.

Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease.

Huntingtons disease is an autosomal-dominant progressive neurodegenerative disorder resulting in specific neuronal loss and dysfunction in the striatum and cortex. The disease is universally fatal, with a mean survival following onset of 15–20 years and, at present, there is no effective treatment. The mutation in patients with Huntingtons disease is an expanded CAG/polyglutamine repeat in huntingtin, a protein of unknown function with a relative molecular mass of 350,000 (M r 350K). The length of the CAG/polyglutamine repeat is inversely correlated with the age of disease onset. The molecular pathways mediating the neuropathology of Huntingtons disease are poorly understood. Transgenic mice expressing exon 1 of the human huntingtin gene with an expanded CAG/polyglutamine repeat develop a progressive syndrome with many of the characteristics of human Huntingtons disease. Here we demonstrate evidence of caspase-1 activation in the brains of mice and humans with the disease. In this transgenic mouse model of Huntingtons disease, expression of a dominant-negative caspase-1 mutant extends survival and delays the appearance of neuronal inclusions, neurotransmitter receptor alterations and onset of symptoms, indicating that caspase-1 is important in the pathogenesis of the disease. In addition, we demonstrate that intracerebroventricular administration of a caspase inhibitor delays disease progression and mortality in the mouse model of Huntingtons disease.

Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease

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 caspase-independent cell death. Furthermore, there is no information on whether Smac/Diablo or apoptosis-inducing factor might play a role in chronic neurodegeneration. In a striatal cell model of Huntingtons disease and in R6/2 mice, we demonstrate the association of cell death/disease progression with the recruitment of mitochondrial caspase-independent (apoptosis-inducing factor) and caspase-dependent (Smac/Diablo 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 Smac/Diablo and apoptosis-inducing factor in chronic neurodegeneration. Our results further delineate the mechanism by which minocycline mediates its remarkably broad neuroprotective effects.

Inhibition of ICE slows ALS in mice

Amyotrophic lateral sclerosis (ALS) is a progressive age-dependent disease involving degeneration of motor neurons in the brain, brainstem and spinal cord. ALS is universally fatal, with the median survival of patients being five years from diagnosis. In a transgenic mouse model of ALS, we now show that a dominant negative inhibitor of a cell-death gene, the interleukin-1β-converting enzyme (ICE), significantly slows the symptomatic progression of ALS.

Minocycline Reduces Traumatic Brain Injury-mediated Caspase-1 Activation, Tissue Damage, and Neurological Dysfunction

OBJECTIVECaspase-1 plays an important functional role mediating neuronal cell death and dysfunction after experimental traumatic brain injury (TBI) in mice. Minocycline, a derivative of the antibiotic tetracycline, inhibits caspase-1 expression. This study investigates whether minocycline can ameliorate TBI-mediated injury in mice. METHODSBrains from mice subjected to traumatic brain injury underwent immunohistochemical analyses for caspase-1, caspase-3, and a neuronal specific marker (NeuN). Minocycline- and saline-treated mice subjected to traumatic brain injury were compared with respect to neurological function, lesion volume, and interleukin-1&bgr; production. RESULTSImmunohistochemical analysis revealed that activated caspase-1 and caspase-3 are present in neurons 24 hours after TBI. Intraperitoneal administration of minocycline 12 hours before or 30 minutes after TBI in mice resulted in improved neurological function when compared with mice given saline control, as assessed by Rotarod performance 1 to 4 days after TBI. The lesion volume, assessed 4 days after trauma, was significantly decreased in mice treated with minocycline before or after trauma when compared with saline-treated mice. Caspase-1 activity, quantified by measuring mature interleukin-1&bgr; production by enzyme-linked immunosorbent assay, was considerably increased in mice that underwent TBI, and this increase was significantly diminished in minocycline-treated mice. CONCLUSIONWe show for the first time that caspase-1 and caspase-3 activities localize specifically within neurons after experimental brain trauma. Further, these results indicate that minocycline is an effective pharmacological agent for reducing tissue injury and neurological deficits that result from experimental TBI, likely through a caspase-1-dependent mechanism. These results provide an experimental rationale for the evaluation of minocycline in human trauma patients.

Attenuation of Transient Focal Cerebral Ischemic Injury in Transgenic Mice Expressing a Mutant ICE Inhibitory Protein

We used transgenic mice expressing a dominant negative mutation of interleukin-1β converting enzyme (ICE) (C285G) in a model of transient focal ischemia in order to investigate the role of ICE in ischemic brain damage. Transgenic mutant ICE mice (n = 11) and wild-type littermates (n = 9) were subjected to 3 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Cerebral infarcts and brain swelling were reduced by 44% and 46%, respectively. Neurological deficits were also significantly reduced. Regional CBF, blood pressure, core temperature, and heart rate did not differ between groups when measured for up to 1 h after reperfusion. Increases in immunoreactive IL-1β levels, observed in ischemic wild-type brain at 30 min after reperfusion, were 77% lower in the mutant strain, indicating that proIL-1β cleavage is inhibited in the mutants. DNA fragmentation was reduced in the mutants 6 and 24 h after reperfusion. Hence, endogenous expression of an ICE inhibitor confers resistance to cerebral ischemia and brain swelling. Our results indicate that down-regulation of ICE expression might provide a useful therapeutic target in cerebral ischemia.

Caspase Cascades in Human Immunodeficiency Virus-Associated Neurodegeneration

Many patients infected with human immunodeficiency virus-1 (HIV-1) develop a syndrome of neurologic deterioration known as HIV-associated dementia (HAD). Neurons are not productively infected by HIV-1; thus, the mechanism of HIV-induced neuronal injury remains incompletely understood. Several investigators have observed evidence of neuronal injury, including dendritic degeneration, and apoptosis in CNS tissue from patients with HAD. Caspase enzymes, proteases associated with the process of apoptosis, are synthesized as inactive proenzymes and are activated in a proteolytic cascade after exposure to apoptotic signals. Here we demonstrate that HAD is associated with active caspase-3-like immunoreactivity that is localized to the soma and dendrites of neurons in affected regions of the human brain. Additionally, the cascade of caspase activation was studied using anin vitro model of HIV-induced neuronal apoptosis. Increased caspase-3 proteolytic activity and mitochondrial release of cytochrome c were observed in cerebrocortical cultures exposed to the HIV coat protein gp120. Specific inhibitors of both the Fas/tumor necrosis factor-α/death receptor pathway and the mitochondrial caspase pathway prevented gp120-induced neuronal apoptosis. Caspase inhibition also prevented the dendrite degeneration observed in vivo in transgenic mice with CNS expression of HIV/gp120. These findings suggest that pharmacologic interventions aimed at the caspase enzyme pathways may be beneficial for the prevention or treatment of HAD.

Additive neuroprotective effects of minocycline with creatine in a mouse model of ALS

The known neuroprotective effects of minocycline and creatine in animal models of amyotrophic lateral sclerosis (ALS) led us to examine whether the combination of these agents would result in increased neuroprotection. As previously reported, we confirmed in ALS mice that either minocycline or creatine treatment results in improvement in motor performance and extended survival. We report that combination of minocycline and creatine resulted in additive neuroprotection, suggesting this to be a novel potential strategy for the treatment of ALS. To our knowledge, this is the first report demonstrating additive neuroprotection of a combinatorial approach in a mouse model of ALS. Adding relevancy to our findings, minocycline and creatine, are relatively safe, cross the blood–brain barrier, and are currently available for human evaluation.