Conditional deletion of KCC2 impairs synaptic plasticity and both spatial and nonspatial memory.

The postsynaptic inhibition through GABAA receptors (GABAAR) relies on two mechanisms, a shunting effect due to an increase in the postsynaptic membrane conductance and, in mature neurons, a hyperpolarization effect due to an entry of chloride into postsynaptic neurons. The second effect requires the action of the K+-Cl- cotransporter KCC2 which extrudes Cl- from the cell and maintains its cytosolic concentration very low. Neuronal chloride equilibrium seems to be dysregulated in several neurological and psychiatric conditions such as epilepsy, anxiety, schizophrenia, Down syndrome, or Alzheimer's disease. In the present study, we used the KCC2 Cre-lox knockdown system to investigate the role of KCC2 in synaptic plasticity and memory formation in adult mice. Tamoxifen-induced conditional deletion of KCC2 in glutamatergic neurons of the forebrain was performed at 3 months of age and resulted in spatial and nonspatial learning impairment. On brain slices, the stimulation of Schaffer collaterals by a theta burst induced long-term potentiation (LTP). The lack of KCC2 did not affect potentiation of field excitatory postsynaptic potentials (fEPSP) measured in the stratum radiatum (dendrites) but increased population spike (PS) amplitudes measured in the CA1 somatic layer, suggesting a reinforcement of the EPSP-PS potentiation, i.e., an increased ability of EPSPs to generate action potentials. At the cellular level, KCC2 deletion induced a positive shift in the reversal potential of GABAAR-driven Cl- currents (EGABA), suggesting an intracellular accumulation of chloride subsequent to the downregulation of KCC2. After treatment with bumetanide, an antagonist of the Na+-K+-Cl- cotransporter NKCC1, spatial memory impairment, chloride accumulation, and EPSP-PS potentiation were rescued in mice lacking KCC2. The presented results emphasize the importance of chloride equilibrium and GABA-inhibiting ability in synaptic plasticity and memory formation.

Overexpression of wild-type human amyloid precursor protein alters GABAergic transmission.

The function of the amyloid precursor protein (APP) is not fully understood, but its cleavage product amyloid beta (Aß) together with neurofibrillary tangles constitute the hallmarks of Alzheimer's disease (AD). Yet, imbalance of excitatory and inhibitory neurotransmission accompanied by loss of synaptic functions, has been reported much earlier and independent of any detectable pathological markers. Recently, soluble APP fragments have been shown to bind to presynaptic GABAB receptors (GABABRs), subsequently decreasing the probability of neurotransmitter release. In this body of work, we were able to show that overexpression of wild-type human APP in mice (hAPPwt) causes early cognitive impairment, neuronal loss, and electrophysiological abnormalities in the absence of amyloid plaques and at very low levels of Aß. hAPPwt mice exhibited neuronal overexcitation that was evident in EEG and increased long-term potentiation (LTP). Overexpression of hAPPwt did not alter GABAergic/glutamatergic receptor components or GABA production ability. Nonetheless, we detected a decrease of GABA but not glutamate that could be linked to soluble APP fragments, acting on presynaptic GABABRs and subsequently reducing GABA release. By using a specific presynaptic GABABR antagonist, we were able to rescue hyperexcitation in hAPPwt animals. Our results provide evidence that APP plays a crucial role in regulating inhibitory neurotransmission.

Correction: Gualdani, R.; et al. Store-Operated Calcium Entry Contributes to Cisplatin-Induced Cell Death in Non-Small Cell Lung Carcinoma. Cancers 2019, 11, 430.

The authors wish to make the following corrections to this paper [...].

Role of the TRPC1 Channel in Hippocampal Long-Term Depression and in Spatial Memory Extinction.

Group I metabotropic glutamate receptors (mGluR) are involved in various forms of synaptic plasticity that are believed to underlie declarative memory. We previously showed that mGluR5 specifically activates channels containing TRPC1, an isoform of the canonical family of Transient Receptor Potential channels highly expressed in the CA1-3 regions of the hippocampus. Using a tamoxifen-inducible conditional knockout model, we show here that the acute deletion of the Trpc1 gene alters the extinction of spatial reference memory. mGluR-induced long-term depression, which is partially responsible for memory extinction, was impaired in these mice. Similar results were obtained in vitro and in vivo by inhibiting the channel by its most specific inhibitor, Pico145. Among the numerous known postsynaptic pathways activated by type I mGluR, we observed that the deletion of Trpc1 impaired the activation of ERK1/2 and the subsequent expression of Arc, an immediate early gene that plays a key role in AMPA receptors endocytosis and subsequent long-term depression.

Store-Operated Calcium Entry Contributes to Cisplatin-Induced Cell Death in Non-Small Cell Lung Carcinoma.

Cisplatin (CDDP) is one of the principal chemotherapeutic agents used for the first-line treatment of many malignancies, including non-small cell lung carcinoma (NSCLC). Despite its use for over 40 years, its mechanism of action is not yet fully understood. Store-operated calcium entry (SOCE), the main pathway allowing Ca2+ entry in non-excitable cells, is involved in tumorogenesis, cancer progression and chemoresistance. It has become an attractive target in cancer treatment. In this study, we showed that siRNA-mediated depletion of stromal interaction molecule 1 (STIM1) and transient receptor potential channel 1 (TRPC1), two players of the store-operated calcium entry, dramatically reduced CDDP cytotoxicity in NSCLC cells. This was associated with an inhibition of the DNA damage response (DDR) triggered by CDDP. Moreover, STIM1 depletion also reduced CDDP-dependent oxidative stress. In parallel, SOCE activation induced Ca2+ entry into the mitochondria, a major source of reactive oxygen species (ROS) within the cell. This effect was highly decreased in STIM1-depleted cells. We then conclude that mitochondrial Ca2+ peak associated to the SOCE contributes to CDDP-induced ROS production, DDR and subsequent apoptosis. To the best of our knowledge, this is the first time that it is shown that Ca2+ signalling constitutes an initial step in CDDP-induced apoptosis.

Activation of TRPC1 Channel by Metabotropic Glutamate Receptor mGluR5 Modulates Synaptic Plasticity and Spatial Working Memory.

Group I metabotropic glutamate receptors, in particular mGluR5, have been implicated in various forms of synaptic plasticity that are believed to underlie declarative memory. We observed that mGluR5 specifically activated a channel containing TRPC1, an isoform of the canonical family of transient receptor potential (TRPC) channels highly expressed in CA1-3 regions of the hippocampus. TRPC1 is able to form tetrameric complexes with TRPC4 and/or TRPC5 isoforms. TRPC1/4/5 complexes have recently been involved in the efficiency of synaptic transmission in the hippocampus. We therefore used a mouse model devoid of TRPC1 expression to investigate the involvement of mGluR5-TRPC1 pathway in synaptic plasticity and memory formation. Trpc1-/- mice showed alterations in spatial working memory and fear conditioning. Activation of mGluR increased synaptic excitability in neurons from WT but not from Trpc1-/- mice. LTP triggered by a theta burst could not maintain over time in brain slices from Trpc1-/- mice. mGluR-induced LTD was also impaired in these mice. Finally, acute inhibition of TRPC1 by Pico145 on isolated neurons or on brain slices mimicked the genetic depletion of Trpc1 and inhibited mGluR-induced entry of cations and subsequent effects on synaptic plasticity, excluding developmental or compensatory mechanisms in Trpc1-/- mice. In summary, our results indicate that TRPC1 plays a role in synaptic plasticity and spatial working memory processes.

Store operated calcium entry is altered by the inhibition of receptors tyrosine kinase.

SOCE (Store-Operated Calcium Entry) is the main mechanism by which external Ca2+ enters into non-excitable cells after endoplasmic reticulum emptying. It is implicated in several processes such as proliferation and migration. Alterations in SOCE could initiate or support the development of hallmarks of cancer. In this project, we showed that disruption of the EGFR/ErbB2-dependent signalling by lapatinib and CP-724714, two inhibitors of the receptor tyrosine kinase (RTK), dramatically reduced the amplitude of the SOCE in breast cancer cells. LY294002 and MK2206, two inhibitors of the PI3K/Akt pathway, mimicked the effect of the inhibition of EGFR/ErbB2. In contrast, inhibitors of the MAPK pathway had no effect on SOCE. The involvement of EGFR/ErbB2 receptors and the PI3K/Akt pathway in the regulation of SOCE was confirmed in other cell lines derived from various cancer types. All these results showed that SOCE is positively regulated by the PI3K/Akt pathway and that this effect may be suppressed by the inhibition of the upstream RTKs. Inhibition of SOCE might therefore contribute to the anticancer effects of RTK inhibitors.

Osmosensation in TRPV2 dominant negative expressing skeletal muscle fibres.

Increased plasma osmolarity induces intracellular water depletion and cell shrinkage (CS) followed by activation of a regulatory volume increase (RVI). In skeletal muscle, the hyperosmotic shock-induced CS is accompanied by a small membrane depolarization responsible for a release of Ca(2+) from intracellular pools. Hyperosmotic shock also induces phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK). TRPV2 dominant negative expressing fibres challenged with hyperosmotic shock present a slower membrane depolarization, a diminished Ca(2+) response, a smaller RVI response, a decrease in SPAK phosphorylation and defective muscle function. We suggest that hyperosmotic shock induces TRPV2 activation, which accelerates muscle cell depolarization and allows the subsequent Ca(2+) release from the sarcoplasmic reticulum, activation of the Na(+) -K(+) -Cl(-) cotransporter by SPAK, and the RVI response. Increased plasma osmolarity induces intracellular water depletion and cell shrinkage followed by activation of a regulatory volume increase (RVI). In skeletal muscle, this is accompanied by transverse tubule (TT) dilatation and by a membrane depolarization responsible for a release of Ca(2+) from intracellular pools. We observed that both hyperosmotic shock-induced Ca(2+) transients and RVI were inhibited by Gd(3+) , ruthenium red and GsMTx4 toxin, three inhibitors of mechanosensitive ion channels. The response was also completely absent in muscle fibres overexpressing a non-permeant, dominant negative (DN) mutant of the transient receptor potential, V2 isoform (TRPV2) ion channel, suggesting the involvement of TRPV2 or of a TRP isoform susceptible to heterotetramerization with TRPV2. The release of Ca(2+) induced by hyperosmotic shock was increased by cannabidiol, an activator of TRPV2, and decreased by tranilast, an inhibitor of TRPV2, suggesting a role for the TRPV2 channel itself. Hyperosmotic shock-induced membrane depolarization was impaired in TRPV2-DN fibres, suggesting that TRPV2 activation triggers the release of Ca(2+) from the sarcoplasmic reticulum by depolarizing TTs. RVI requires the sequential activation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NKCC1, a Na(+) -K(+) -Cl(-) cotransporter, allowing ion entry and driving osmotic water flow. In fibres overexpressing TRPV2-DN as well as in fibres in which Ca(2+) transients were abolished by the Ca(2+) chelator BAPTA, the level of P-SPAK(Ser373) in response to hyperosmotic shock was reduced, suggesting a modulation of SPAK phosphorylation by intracellular Ca(2+) . We conclude that TRPV2 is involved in osmosensation in skeletal muscle fibres, acting in concert with P-SPAK-activated NKCC1.