Abstract Cortical spreading depression (CSD) is characterized by slowly propagating waves of neuronal/astrocytic depolarization and metabolic changes, followed by a period of quiescent neuronal and electroencephalographic activity. CSD acts as a preconditioning stimulus in brain, reducing cell death when elicited up to several days prior to an ischemic insult. Precise mechanisms associated with this neuroprotection are not known, although CSD increases the expression of a number of potentially neuroprotective genes/proteins. The nitric oxide (NO) system may be of particular importance, as it is acutely activated and chronically up‐regulated in cerebral cortex by CSD, and NO can ameliorate and exacerbate cell death under different conditions. Several molecules have recently been identified that modulate the production and/or cellular actions of NO, but it is not known whether their expression is altered by CSD. Therefore, the present study examined the effect of CSD on the spatiotemporal expression of PIN, CAPON, PSD‐95, Mn‐SOD and Cu/Zn‐SOD mRNA in the rat brain. In situ hybridization using specific [ 35 S]‐labelled oligonucleotides revealed that levels of PIN mRNA were significantly increased in the cortex and claustrum (∼30–180%; p ≤ 0.01) after 6 h and 1 and 2 days, but were again equivalent to contralateral (control) cortical values at 7, 14 and 28 days. CAPON mRNA levels were increased (∼30–180%; p ≤ 0.05) in the ipsilateral cortical hemisphere at 6 h and 2 days post treatment, but not at the other times examined. In contrast, levels of PSD‐95, Mn‐ and Cu/Zn‐SOD mRNA were not altered at any time after CSD. These results suggest that following CSD, nNOS activity and NO levels may be tightly regulated by both transcriptional and translational alterations in a range of nNOS adaptor proteins, which may contribute to CSD‐induced neuroprotection against subsequent ischemia.
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.
Mouse Numb homologs antagonize Notch1 signaling pathways through largely unknown mechanisms. Here we demonstrate that conditional mouse mutants with deletion of numb and numblike in developing sensory ganglia show a severe reduction in axonal arborization in afferent fibers, but no deficit in neurogenesis. Consistent with these results, expression of Cre recombinase in sensory neurons from numb conditional mutants results in reduced endocytosis, a significant increase in nuclear Notch1, and severe reductions in axon branch points and total axon length. Conversely, overexpression of Numb, but not mutant Numb lacking α-adaptin-interacting domain, leads to accumulation of Notch1 in markedly enlarged endocytic–lysosomal vesicles, reduced nuclear Notch1, and dramatic increases in axonal length and branch points. Taken together, our data provide evidence for previously unidentified functions of Numb and Numblike in sensory axon arborization by regulating Notch1 via the endocytic–lysosomal pathways.
Sustained activation of poly(ADP-ribose) polymerase-1 (PARP-1) and extracellular signal-regulated kinases 1/2 (ERK1/2) both promote neuronal death. Here we identify a direct link between these two cell death pathways. In a rat model of hypoglycemic brain injury, neuronal PARP-1 activation and subsequent neuronal death were blocked by the ERK1/2 inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059). In neuron cultures, PARP-1-mediated neuronal death induced by N -methyl- d -aspartate, peroxynitrite, or DNA alkylation was similarly blocked by ERK1/2 pathway inhibitors. These inhibitors also blocked PARP-1 activation and PARP-1-mediated death in astrocytes. siRNA down-regulation of ERK2 expression in astrocytes also blocked PARP-1 activation and cell death. Direct effects of ERK1/2 on PARP-1 were evaluated by using isolated recombinant enzymes. The activity of recombinant human PARP-1 was reduced by incubation with alkaline phosphatase and restored by incubation with active ERK1 or ERK2. Putative ERK1/2 phosphorylation sites on PARP-1 were identified by mass spectrometry. Using site-directed mutagenesis, these sites were replaced with alanine (S372A and T373A) to block phosphorylation, or with glutamate (S372E and T373E) to mimic constitutive phosphorylation. Transfection of PARP-1 deficient mouse embryonic fibroblasts with the mutant PARP-1 species showed that the S372A and T373A mutations impaired PARP-1 activation, whereas the S372E and T373E mutations increased PARP-1 activity and eliminated the effect of ERK1/2 inhibitors on PARP-1 activation. These results suggest that PARP1 phosphorylation by ERK1/2 is required for maximal PARP-1 activation after DNA damage.
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