A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain.

Maladaptive plasticity involving increased expression of AMPA-type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell-permeable, high-affinity (~2 nM) peptide inhibitor, Tat-P4 -(C5)2 , of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat-P4 -(C5)2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA-receptor surface expression in vivo. Moreover, Tat-P4 -(C5)2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat-P4 -(C5)2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non-tandem protein-protein interaction domains.

Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes.

Glycogen phosphorylase (GP) is activated to degrade glycogen in response to different stimuli, to support both the astrocyte's own metabolic demand and the metabolic needs of neurons. The regulatory mechanism allowing such a glycogenolytic response to distinct triggers remains incompletely understood. In the present study, we used siRNA-mediated differential knockdown of the two isoforms of GP expressed in astrocytes, muscle isoform (GPMM), and brain isoform (GPBB), to analyze isoform-specific regulatory characteristics in a cellular setting. Subsequently, we tested the response of each isoform to phosphorylation, triggered by incubation with norepinephrine (NE), and to AMP, increased by glucose deprivation in cells in which expression of one GP isoform had been silenced. Successful knockdown was demonstrated on the protein level by Western blot, and on a functional level by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE, with glycogen levels significantly reduced only after 60 min. In contrast, allosteric activation of GP by AMP, induced by glucose deprivation, seemed to mainly affect GPBB, as only knockdown of GPBB, but not of GPMM, delayed the glycogenolytic response to glucose deprivation. Our results indicate that the two GP isoforms expressed in astrocytes respond to different physiological triggers, therefore conferring distinct metabolic functions of brain glycogen.