No abstract

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Impaired axonal transport may play a key role in Parkinson’s disease. To test this notion, a microchamber system was adapted to segregate axons from cell bodies using green fluorescent protein-labeled mouse dopamine (DA) neurons. Transport was examined in axons challenged with the DA neurotoxin MPP+. MPP+ rapidly reduced overall mitochondrial motility in DA axons; among motile mitochondria, anterograde transport was slower yet retrograde transport was increased. Transport effects were specific for DA mitochondria, which were smaller and transported more slowly than their non-DA counterparts. MPP+ did not affect synaptophysin-tagged vesicles or any other measureable moving particle. Toxin effects on DA mitochondria were not dependent upon ATP, calcium, free radical species, JNK, or caspase3/PKC pathways but were completely blocked by the thiol-anti-oxidant N-acetyl-cysteine or membrane-permeable glutathione. Since these drugs also rescued processes from degeneration, these findings emphasize the need to develop therapeutics aimed at axons as well as cell bodies to preserve “normal” circuitry and function as long as possible.

Recent studies suggest that the degree of mitochondrial dysfunction in cerebral ischemia may be an important determinant of the final extent of tissue injury. Although loss of mitochondrial membrane potential ( m ), one index of mitochondrial dysfunction, has been documented in neurons exposed to ischemic conditions, it is not yet known whether astrocytes, which are relatively resistant to ischemic injury, experience changes in m under similar conditions. To address this, we exposed cortical astrocytes cultured alone or with neurons to oxygen-glucose deprivation (OGD) and monitored m using tetramethylrhodamine ethyl ester. Both neurons and astrocytes exhibited profound loss of m after 45-60 min of OGD. However, although this exposure is lethal to nearly all neurons, it is hours less than that needed to kill astrocytes. Astrocyte m was rescued during OGD by cyclosporin A, a permeability transition pore blocker, and G N-nitro-arginine, a nitric oxide synthase inhibitor. Loss of mitochondrial membrane potential in astrocytes was not accompanied by depolarization of the plasma membrane. Recovery of astrocyte m after reintroduction of O 2 and glucose occurred over a surprisingly long period (Ͼ1 hr), suggesting that OGD caused specific, reversible changes in astrocyte mitochondrial physiology beyond the simple lack of O 2 and glucose. Decreased m was associated with a cyclosporin A-sensitive loss of cytochrome c but not with activation of caspase-3 or caspase-9. Our data suggest that astrocyte mitochondrial depolarization could be a previously unrecognized event early in ischemia and that strategies that target the mitochondrial component of ischemic injury may benefit astrocytes as well as neurons. Key words: tetramethylrhodamine ethyl ester; mitochondrial permeability transition pore; nitric oxide synthase; cyclosporin A; confocal microscopy; cortical cell culturesMitochondrial dysfunction is an early feature in nervous system ischemia. Functional studies on mitochondria isolated from ischemic brain (Sims et al., 1986;Sims, 1991) and metabolic imaging studies of brain during ischemia (Watanabe et al., 1994;Shiino et al., 1998;McCleary et al., 1999;Shadid et al., 1999) indicate that brief periods of ischemia result in transient mitochondrial respiratory defects that normalize rapidly after reperfusion (Schutz et al., 1973;Hillered et al., 1984;Sims et al., 1986). Longer periods of ischemia, however, result in a secondary, irreversible decline in mitochondrial function that occurs minutes to hours later (for review, see Siesjo et al., 1999). Although mitochondrial failure is associated with loss of mitochondrial membrane potential ( m ) in many injury conditions (Green and Reed, 1998), it is not yet known whether impaired mitochondrial respiration during cerebral ischemia is accompanied by loss of m , because of the lack of sensitive and specific probes for m in the intact brain. Suggestive evidence comes from reports by Fujimura et al. (1998), Andreyev et al. (1998), and Perez-Pinzon et al. (1999, who observed releas...

Background and Purpose-Recent measurements in intracerebral hemorrhage (ICH) patients suggest a primary reduction in brain metabolism is responsible for reduced cerebral blood flow and low oxygen extraction surrounding the hematoma. We sought to determine whether reduced mitochondrial respiratory function could account for reduced metabolic demand in ICH patients. Methods-Brain-tissue samples from 6 patients with acute spontaneous ICH and 6 control patients undergoing brain resection for management of seizure were evaluated. Only tissue removed from the brain adjacent to the hematoma was studied. Specimens were collected in the operating room; mitochondrial studies were begun within 1-hour. Mitochondrial oxygen consumption was measured after the addition of pyruvate, malate, and ADP, followed by oligomycin and carbonylcyanide. Results-The ICH patients ranged in age from 40 to 54 years; 2 were female and half black. Hemorrhages were located in the temporal lobe (3), cerebellum (2) and parietal lobe (1).