Biomedical subjects
James L Franklin
Publications and source records attributed to James L Franklin.
Bax, reactive oxygen, and cytochrome c release in neuronal apoptosis.
Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.
Prooxidant effects of NGF withdrawal and MEK inhibition in sympathetic neurons.
An increase of mitochondrial-derived reactive oxygen species (ROS) occurs in nerve growth factor (NGF)-deprived sympathetic neurons undergoing apoptotic death. It has been reported that NGF suppresses increased ROS production by the mitochondria in these cells through a mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway because NGF withdrawal inactivates this pathway and the MEK inhibitor, PD98059, increases ROS in the presence of NGF. We show here that treating rat sympathetic neurons in cell culture with PD98059 greatly decreased cellular concentrations of reduced glutathione (GSH), a major cellular antioxidant. Therefore, it is likely that this inhibitor induces a cellular prooxidant state in NGF-maintained sympathetic neurons primarily by decreasing GSH concentration rather than by causing increased mitochondrial ROS production. These data suggest that the MEK/MAP kinase signaling pathway regulates cellular GSH concentration.
A Bax-induced pro-oxidant state is critical for cytochrome c release during programmed neuronal death.
Bax is required for the apoptotic death of sympathetic neurons deprived of nerve growth factor (NGF). After NGF withdrawal, Bax translocates from the cytoplasm to the mitochondria of these cells and induces release of the proapoptotic protein cytochrome c. Here, we report that withdrawing NGF from mouse sympathetic neurons caused an increase of mitochondria-derived reactive oxygen species (ROS). Suppressing these ROS inhibited apoptosis. Bax deletion blocked death and prevented the ROS burst. Inducing a pro-oxidant state similar to that in NGF-deprived, wild-type cells caused cytochrome c release even in neurons lacking Bax. A similar ROS burst in cerebellar granule neurons undergoing apoptosis was also blocked by Bax deletion. These findings indicate that Bax lies upstream from increased ROS in NGF-deprived neurons and suggest that the Bax-induced ROS burst is both necessary and sufficient for cytochrome c redistribution in these cells.
Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling.
It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons.
Gene expression analysis of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia.
Identification of novel modulators of ischemic neuronal death helps in developing new strategies to prevent the stroke-induced neurological dysfunction. Hence, the present study evaluated the gene expression changes in rat cerebral cortex at 6 and 24 h of reperfusion following transient middle cerebral artery occlusion (MCAO) by GeneChip analysis. Transient MCAO resulted in selective increased mRNA levels of genes involved in stress, inflammation, transcription and plasticity, and decreased mRNA levels of genes which control neurotransmitter function and ionic balance. In addition to a number of established ischemia-related genes, many genes not previously implicated in transient focal ischemia-induced brain damage [suppressor of cytokine signaling (SOCS)-3, cAMP responsive element modulator (CREM), cytosolic retinol binding protein (CRBP), silencer factor-B, survival motor neuron (SMN), interferon-gamma regulatory factor-1 (IRF-1), galanin, neurotrimin, proteasome subunit RC8, synaptosomal-associated protein (SNAP)-25 A and B, synapsin 1a, neurexin 1-beta, ras-related rab3, vesicular GABA transporter (VGAT), digoxin carrier protein, neuronal calcium sensor-1 and neurodap] were observed to be altered in the ischemic cortex. Real-time PCR confirmed the GeneChip results for several of these transcripts. SOCS-3 is a gene up-regulated after ischemia which modulates inflammation by controlling cytokine levels. Antisense knockdown of ischemia-induced SOCS-3 protein expression exacerbated transient MCAO-induced infarct volume assigning a neuroprotective role to SOCS-3, a gene not heretofore implicated in ischemic neuronal damage.