PKN1 promotes synapse maturation by inhibiting mGluR-dependent silencing through neuronal glutamate transporter activation

Abnormal metabotropic glutamate receptor (mGluR) activity could cause brain disorders; however, its regulation has not yet been fully understood. Here, we report that protein kinase N1 (PKN1), a protein kinase expressed predominantly in neurons in the brain, normalizes group 1 mGluR function by upregulating a neuronal glutamate transporter, excitatory amino acid transporter 3 (EAAT3), and supports silent synapse activation. Knocking out PKN1a, the dominant PKN1 subtype in the brain, unmasked abnormal input-nonspecific mGluR-dependent long-term depression (mGluR-LTD) and AMPA receptor (AMPAR) silencing in the developing hippocampus. mGluR-LTD was mimicked by inhibiting glutamate transporters in wild-type mice. Knocking out PKN1a decreased hippocampal EAAT3 expression and PKN1 inhibition reduced glutamate uptake through EAAT3. Also, synaptic transmission was immature; there were more silent synapses and fewer spines with shorter postsynaptic densities in PKN1a knockout mice than in wild-type mice. Thus, PKN1 plays a critical role in regulation of synaptic maturation by upregulating EAAT3 expression.

2 alternative transcripts that seemed to be driven by separate first translating exons (E1a and E1b) and spliced to the common exon 2 are shown. (b) Scheme of PKN1 genomic DNA, targeting vector, and disrupted gene.
Shown is the targeting vector and a partial map of the PKN1 locus before and after homologous recombination in ES cells. Subsequent breeding of heterozygous mice indicated as PKN1a +/-generates PKN1a KO mice (a) Expression of PKN1 in wild-type and PKN1a KO mice. Whole-cell lysates were prepared from wild-type and PKN1a KO mice, and subjected to immunoblot analysis using the C6 antibody. WT, wild-type mice; KO, PKN1a KO mice. (b) Quantification of PKN1 in various regions of wild-type and PKN1a KO mice. The amount of PKN1 was measured by immunoblot analysis using the C6 antibody. The data are expressed as the mean ± s.e.m from n = 3 mice per genotype. (c) The N1b2 antibody specifically recognizes PKN1b.
Crude lysates were prepared from COS7 cells transfected with PKN1a or PKN1b expression vector. Equal amounts of protein were loaded on all lanes and detected using the N1b2 antibody or C6 antibody. (d) PKN1b is expressed in PKN1a KO mice. The brain and spleen supernatant from mice (left) and immunoprecipitates from the supernatants prepared using N2, which is a variant nonselective anti-PKN1 antibody (right) were subjected to immunoblot analysis using a monoclonal antibody against PKN1 (BD Transduction Laboratories) or the N1b2 antibody. The sensitivity of the N1b2 antibody is not sufficient to detect endogenous PKN1b in whole-cell lysates. However, PKN1b was detected using the N1b2 antibody more clearly in N2 immunoprecipitates from PKN1a KO mice than in those from wild-type mice. (e) PKN2 expression in various regions of the mouse brain in wild-type and PKN1a KO mice. Crude homogenates were prepared from various brain regions of mice and subjected to immunoblot analysis using the  ParN2 antibody or C6 antibody. No compensatory increase in PKN2 expression level was detected in the cortex, the CA1 region of the hippocampus, and the amygdala in PKN1a KO mice. (f, g) Immunohistochemical staining of PKN1 in CA1 (f) and the dentate gyrus (g) of the hippocampus in wild-type mice using the C6 antibody. PKN1 signals were detected in cell layers in the CA1 region and dentate gyrus, and dendrite-like processes in the stratum radiatum of the CA1 region. Scale, 50 m. 69.0 ± 2.7%, n = 12 from 11 mice; D-APV, 80.5 ± 3.1%, n = 12 from 7 mice, F(2, 31) = 6.55, p = 0.018; 9 LY341495, 82.7 ± 2.7%, n = 10 from 4 mice; p = 0.0069; one-way ANOVA with Tukey-Kramer test). D-APV showed no effects; however, LY341495 suppressed heterosynaptic LTD in KO mice (no drug, 76.1 ± 4.5%; APV, 80.2 ± 3.5%; LY, 95.3 ± 3.1%, F(2, 31) = 6.39, p = 0.0047; one-way ANOVA with Tukey-Kramer test).
(b) Examples (1, 2) and summary (3)  Original images shown in Fig. 5e and Coomassie staining of the same membranes. A P11-16 wild-type or KO mouse was taken from the home cage at the Animal Facility and immediately anesthetized with isoflurane.
Then, the brain was removed and placed in ice-cold oxygenated (95% O2/5% CO2) ACSF. The two hippocampi were dissected out, placed in a sample tube, and quick-frozen in liquid nitrogen. Hippocampal lysates were prepared and subjected to immunoblot analysis using an anti-EAAT3 antibody (#12179, Cell Signaling Technology). Summary of the effects of preceding homo-and heterosynaptic LTD on hetero-(A) and homosynaptic LTDs in P12-14 KO mice. (B). Heterosynaptic LTD was completely occluded by two LFS (116.0 ± 7.1% of baseline 50 min after third LFS, n = 9 from 5 mice). However, homosynaptic LTD was not completely occluded by heterosynaptic LTD (89.5 ± 4.8%), presumably because NMDAR-dependent LTD was not induced in heterosynaptic pathways by two LFS applied to homosynaptic pathways and there was still room for synaptic depression by NMDA receptor activation in the heterosynaptic pathways when the third LFS was applied. Data are shown as mean ± s.e.m.