Excitotoxic neuronal death is mediated in part by NMDA receptor-induced activation of NOX2, an enzyme that produces superoxide and resultant oxidative stress. It is not known, however, whether the superoxide is generated in the intracellular space, producing oxidative stress in the neurons responding to NMDA receptor activation, or in the extracellular space, producing oxidative stress in neighboring cells. We evaluated these alternatives by preparing cortical neuron cultures from p47(phox-/-) mice, which are unable to form a functional NOX2 complex, and transfecting the cultures at low density with GFP-tagged p47(phox) to reconstitute NOX2 activity in widely scattered neurons. NMDA exposure did not induce oxidative stress or cell death in the nontransfected, p47-phox(-/-) cultures, but did produce oxidative stress and neuronal death in neurons surrounding the transfected, NOX2-competent neurons. This cell-to-cell spread of NMDA-induced oxidative injury was blocked by coincubation with either superoxide dismutase or the anion channel blocker 4'-diisothiocyanostilbene-2,2'-disulphonate, confirming superoxide anion as the mediating oxidant. In neurons plated on a preexisting astrocyte layer, NMDA induced oxidative stress in both the neurons and the astrocytes, and this was also prevented by superoxide dismutase. These findings show that activation of NMDA receptors on one neuron can lead to oxidative stress and cell death in neighboring neurons and astrocytes by a process involving the extracellular release of superoxide by NOX2.

Paroxysmal non-kinesigenic dyskinesia (PNKD) is a rare autosomal dominant movement disorder triggered by stress, fatigue or consumption of either alcohol or caffeine. Attacks last 1-4 h and consist of dramatic dystonia and choreoathetosis in the limbs, trunk and face. The disease is associated with single amino acid changes (A7V or A9V) in PNKD, a protein of unknown function. Here we studied the stability, cellular localization and enzymatic activity of the PNKD protein in cultured cells and transgenic animals. The N-terminus of the wild-type (WT) long PNKD isoform (PNKD-L) undergoes a cleavage event in vitro, resistance to which is conferred by disease-associated mutations. Mutant PNKD-L protein is degraded faster than the WT protein. These results suggest that the disease mutations underlying PNKD may disrupt protein processing in vivo, a hypothesis supported by our observation of decreased cortical Pnkd-L levels in mutant transgenic mice. Pnkd is homologous to a superfamily of enzymes with conserved β-lactamase domains. It shares highest homology with glyoxalase II but does not catalyze the same reaction. Lower glutathione levels were found in cortex lysates from Pnkd knockout mice versus WT littermates. Taken together, our results suggest an important role for the Pnkd protein in maintaining cellular redox status.

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H(+) along with extracellular superoxide, and the intracellular H(+) must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na(+)/H(+) exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na(+)/H(+) exchange and by reduced expression of the Na(+)/H(+) exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na(+)/H(+) exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD⁺ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD⁺ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD⁺ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.Several metabolic factors affect cellular glucose metabolism as well as the innate inflammatory response. Here, the authors show that glucose metabolism regulates pro-inflammatory responses through effects on the cytosolic NADH:NAD+ ratio and the NAD(H)-sensitive transcription co-repressor CtBP.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder. Patients have episodes that last 1 to 4 hours and are precipitated by alcohol, coffee, and stress. Previous research has shown that mutations in an uncharacterized gene on chromosome 2q33-q35 (which is termed PNKD) are responsible for PNKD. Here, we report the generation of antibodies specific for the PNKD protein and show that it is widely expressed in the mouse brain, exclusively in neurons. One PNKD isoform is a membrane-associated protein. Transgenic mice carrying mutations in the mouse Pnkd locus equivalent to those found in patients with PNKD recapitulated the human PNKD phenotype. Staining for c-fos demonstrated that administration of alcohol or caffeine induced neuronal activity in the basal ganglia in these mice. They also showed nigrostriatal neurotransmission deficits that were manifested by reduced extracellular dopamine levels in the striatum and a proportional increase of dopamine release in response to caffeine and ethanol treatment. These findings support the hypothesis that the PNKD protein functions to modulate striatal neuro-transmitter release in response to stress and other precipitating factors.