Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons.

Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression.

ATP P2X receptors downregulate AMPA receptor trafficking and postsynaptic efficacy in hippocampal neurons.

P2X receptors (P2XRs) are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons or glia. Although purinergic signaling has multiple effects on synaptic transmission and plasticity, P2XR function at brain synapses remains to be established. Here, we show that activation of postsynaptic P2XRs by exogenous ATP or noradrenaline-dependent glial release of endogenous ATP decreases the amplitude of miniature excitatory postsynaptic currents and AMPA-evoked currents in cultured hippocampal neurons. We also observed a P2X-mediated depression of field potentials recorded in CA1 region from brain slices. P2X2Rs trigger dynamin-dependent internalization of AMPA receptors (AMPARs), leading to reduced surface AMPARs in dendrites and at synapses. AMPAR alteration required calcium influx through opened ATP-gated channels and phosphatase or CamKII activities. These findings indicate that postsynaptic P2XRs play a critical role in regulating the surface expression of AMPARs and thereby regulate the synaptic strength.