No abstract

alpha-amino-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in rat cultured microglia were analyzed precisely using flop- and flip-preferring allosteric modulators of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA) and cyclothiazide (CTZ), respectively. Glutamate (Glu)- or kainite (KA)-induced currents were completely inhibited by a specific blocker of AMPA receptor, LY300164, indicating that functional Glu-receptors in cultured microglia are mostly AMPA receptor but not KA receptor in many cells. Glu- and KA-induced currents were potentiated by PEPA and CTZ in a concentration-dependent manner. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide varied with cells between 0.1 and 0.9, suggesting cell-to-cell heterogeneity of AMPA receptor subunits expressed in microglia. Quantitative RT-PCR revealed that GluR1-3 mainly occurred in the flip forms, which agreed with the stronger potentiation of receptor currents by CTZ vs. PEPA. Finally, the potentiation of microglial AMPA receptors by PEPA and CTZ inhibited the Glu-induced release of tumor necrosis factor-alpha (TNF-alpha) unpredictably. The increase in TNF-alpha release by Glu or KA required extracellular Na+ and Ca2+ ions but not mitogen-activated protein kinase (MAPK), suggesting the effects of PEPA and CTZ were not due to the inhibition of MAPK. These results suggest that potentiation of microglial AMPA receptors serves as a negative feedback mechanism for the regulation of TNF-alpha release and may contribute to the ameliorating effects of allosteric modulators of AMPA receptors.

No abstract

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of alpha-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of alpha-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.

Mammalian neuronal cells abundantly express a de-ubiquitinating isozyme, ubiquitin carboxy-terminal hydrolase L1 (UCH L1). Loss of UCH L1 function causes dying-back type of axonal degeneration. However, the function of UCH L1 in neuronal cells remains elusive. Here we show that overexpression of UCH L1 potentiated ATP-induced currents due to the activation of P2X receptors that are widely distributed in the brain and involved in various biological activities including neurosecretion. ATP-induced inward currents were measured in mock-, wild-type or mutant (C90S)-UCH L1-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in both wild-type and C90S UCH L1-transfected cells, suggesting that hydrolase activity was not involved but increased level of mono-ubiquitin might play an important role. The increased currents were dependent on cAMP-dependent protein kinase (PKA) and Ca 2+ and calmodulin-dependent protein kinase (CaMKII) but not protein kinase C. In addition, ATP-induced currents were likely to be modified via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) that is regulated by PKA and phosphatases. Our finding shows the first evidence that there is a relationship between UCH L1 and neurotransmitter receptor, suggesting that UCH L1 may play an important role in synaptic activity. The ubiquitin-proteasome system is an evolutionarily conserved and energy-dependent proteolytic pathway that functions constitutively to degrade proteins. Recent studies indicate that ubiquitin-mediated proteolysis can also be regulated and is of widespread importance (Wilkinson 1995;Coux et al. 1996). Regulated proteolysis by the ubiquitin-