Research ArticleNEURODEVELOPMENT

Developmentally regulated KCC2 phosphorylation is essential for dynamic GABA-mediated inhibition and survival

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Science Signaling  15 Oct 2019:
Vol. 12, Issue 603, eaaw9315
DOI: 10.1126/scisignal.aaw9315
  • Fig. 1 Identification of KCC2 phosphorylation sites regulated during CNS development.

    (A) Phosphorylation site mapping. KCC2 was immunopurified from mouse brain, fractionated by SDS-PAGE, and digested with trypsin. Blot is representative of lysates from 19 mice. Schematic lays out how phosphopeptides were subjected to LC-MS/MS. MW, molecular weight. (B) Representative MS/MS spectrum assignment of peptide TLVMEQR (pThr929; presented as human KCC2B pThr906). The phosphorylated precursor ion (478.71 +2) was selected and produced the fragment ion spectrum shown. Specific y and b fragment ions allowed unambiguous identification of the precursor peptide and its phosphorylation at Thr906 (human numbering). Fragment ions with neutral loss of phosphate (−Pb/a1, −Pb/a2, −Pb/a3, etc.) are indicated. (C) Identified KCC2 phosphorylation sites are numbered as in human KCC2B (gene ID 57468). All KCC2 peptides observed at various developmental stages are listed in table S1. (D) Heatmap representation of significant KCC2 phosphorylation sites and their changes during development. Hierarchical clustering showed distinct pattern of KCC2 phosphorylation at these residues. Amino acid residue numbering is referenced to isoform 1 of mouse Slc12a5 (UniProt: Q91V14). (E) Brain lysates were subjected to immunoprecipitation (IP) by pan-KCC2 antibody (KCC2) or by phosphorylation site–specific antibodies recognizing the Thr906- or Thr1007-phosphorylated forms of KCC2, and immunoprecipitated protein was detected with pan-KCC2 antibody [immunoblotting (IB)]. Whole-cell lysates were subjected to immunoblot using antibodies recognizing the indicated proteins or phosphoproteins. D, dimeric KCC2; M, monomeric KCC2. Blot is representative of three experiments. (F) Band intensities represented in (E) were quantitated with ImageJ software. Calculation of intensity ratios was based on the calculation: (phospho-dimeric KCC2 + phospho-monomeric KCC2)/(total dimeric KCC2 + total monomeric KCC2), as described previously (24). ***P < 0.001 and **P < 0.01 by one-way ANOVA with post hoc testing (n = 6; data are means ± SEM).

  • Fig. 2 KCC2 T906E/T1007E (Kcc2E/E) phosphomimetic mice.

    (A) Genomic targeting strategy depicting T906E (exon 22) and T1007E (exon 24). The intron 22 neomycin selection cassette is excised by Cre recombinase. (B) Sanger sequencing trace of KCC2 T906E/T1007E (Kcc2E/E). (C) Genotypes of surviving progeny from Kcc2+/E intercrosses at E18.5, P0, and P10. n is noted in the graph. (D) Consecutive axial brain sections revealed no gross defects in Kcc2E/E mutant mice (Hom, P0). Images are representative of 20 mice. (E) WT brain lysates at indicated ages were immunoprecipitated (IP) with site-specific phospho-antibodies recognizing KCC2 pThr906 or pThr1007. Immunoprecipitates were immunoblotted with pan-KCC2 antibody (IB). Whole-cell lysates were immunoblotted with indicated antibodies. Band intensities were quantitated with ImageJ software (shown in fig. S2C). Blot is representative of three experiments. (F to N) Percentage of WT, heterozygous (Het), and homozygous (Hom) P0 Kcc2E/E mice exhibiting seizures, type of seizure [partial (P), secondary generalized (G), tonic (T), and tonic-clonic types (T-C)], and duration of seizure (with or without opisthotonos: dark and light blue, respectively) provoked by brushing (F to H), tail pinch (I to K), and tail suspension (L to N). **P < 0.01 by χ2 test. Data are from 11 to 13 mice.

  • Fig. 3 Developing Kcc2E/E mouse brains exhibit anomalous distribution of proliferating neurons but normal dendritic spine morphology.

    (A) Neuronal distribution in WT and homozygous Kcc2E/E E14.5 brains. Representative images of EdU-positive neurons in the septum, hypothalamus, hippocampus, and cortex of WT (n = 3) and homozygous Kcc2E/E (n = 4) mouse brains. Proliferating cells were labeled with EdU at E14.5 and then immunostained for EdU at E18.5. EdU-positive cells in each region of interest (ROI) were counted as in Materials and Methods. Images are representative of seven mice. (B) Quantitation of EdU-positive neuron density in WT versus homozygous Kcc2E/E E14.5 brains assessed in the septum, POA, caudate-putamen (CPu), hippocampus, and cortex (ROIs 1 and 2). **P < 0.01 by unpaired t test; n = 4 (Kcc2E/E) and n = 3 (WT). (C) Spine formation in WT and homozygous Kcc2E/E neurons. Representative images of EGFP-transfected DIV 26 primary cultured cortical neurons from WT and homozygous Kcc2E/E mice (each n = 3).

  • Fig. 4 Kcc2E/E neurons exhibit impaired GABA-dependent Cl extrusion and disrupted rhythmogenesis.

    (A) Gramicidin-perforated, voltage-clamped currents (9) recorded at −50-mV holding potential. Two 0.5-s voltage ramps from −100 to 0 mV were applied before and during 30-s puff application of 100 μM GABA; sample I-V curves before (black) and after GABA application (red). EGABA was estimated from the voltage axis intercept (detailed further in Materials and Methods). Insets (top left) are representative GABA-evoked current traces at −50-mV holding potential in ventral spinal cord neurons of acute lumber spinal cord slices from P0 WT (left) and Kcc2E/E mice (right). Data are representative of 12 mice. (B) Neuronal EGABA from WT (−59.6 ± 2.1; n = 5) and homozygous Kcc2E/E mice (−58.7 ± 1.8 mV; n = 7). Data were not significantly different by an unpaired t test. (C) Representative traces of GABA responses in P0 ventral spinal cord neurons of acute lumber spinal cord slices from WT and Kcc2E/E mice. After current-clamp recording of basal GABA responses (3 s, 100 μM GABA puffs every 20 s) in neurons from WT and Kcc2E/E mice, neurons were Cl-loaded by prolonged (20 s) GABA puff during depolarizing voltage clamp (Vh = 0 mV). After Cl loading, responses to brief GABA puffs were again recorded in current-clamp mode, demonstrating 407 ± 78% increased peak neuronal Cl extrusion. Data are representative of 23 mice. (D) Normalized recovery of neuronal GABA responses in WT (black circles; n = 10) and Kcc2E/E mice (red squares; n = 13) after Cl loading. Cl extrusion rate was impaired in Kcc2E/E mice. Each neuronal response was normalized to the GABA pulse peak value (0%) and peak post–Cl loading GABA pulse-induced response (100%) for each neuron. WT peak potentials recovered to initial values (−3.9 ± 3.8%; n = 10), whereas Kcc2E/E peak potentials remained 23.0 ± 4.1% above initial values (n = 13). *P < 0.05 and **P < 0.01 by unpaired t test. Open symbols, single cells; filled symbols, mean values with SE. (E) Respiratory motor neuron recordings from P0 mouse cervical spinal cord ventral rootlets (C4-C5) (42). Spontaneous rhythmic activity was measured in WT (n = 6), T906E/T1007E+/wt (n = 10), and Kcc2E/E mice (n = 11). (F) Respiratory rhythm of WT (10.4 ± 1.1 min−1; n = 6), heterozygous Kcc2E/wt (11 ± 1.1 min−1; n = 10), and Kcc2E/E mice (1.3 ± 0.8 min−1; n = 9). Means ± SEM. **P < 0.01 by Kruskal-Wallis test. (G) P0 L2 ventral root spontaneous activity (upper traces) and locomotor rhythm (lower traces) were induced by perfusion of 20 μM 5-HT (45, 46) in WT (n = 8), heterozygous (n = 8), and Kcc2E/E mice (n = 7). (H) Rate of the locomotor rhythm in WT (7.1 ± 2.2 min−1; n = 8), T906E/T1007E+/wt (8.5 ± 2.7 min−1; n = 8), and Kcc2E/E mice (1.9 ± 0.1 min−1; n = 7). Data are means ± SEM. **P < 0.01 by Kruskal-Wallis test. (I) Coefficient of variation of interburst intervals in WT (0.9 ± 0.04; n = 8), T906E/T1007E+/wt (0.9 ± 0.04; n = 8), and Kcc2E/E mice (0.1 ± 0.001; n = 7). Means ± SEM. **P < 0.01 by Kruskal-Wallis test.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/603/eaaw9315/DC1

    Fig. S1. Sequence alignments.

    Fig. S2. Generation and characterization of KCC2 Thr906/Thr1007 phosphomimetic mice.

    Fig. S3. Hematoxylin and eosin staining of horizontal and sagittal midline sections of P0 mice showing no gross defects of the central nervous or the musculoskeletal system.

    Table S1. In vivo KCC2 phosphorylation sites identified and quantified in all four conditions (E18.5, P0, P20, and adult).

    Table S2. List of candidate protein kinases.

    Movie S1. Seizure triggered by mild brush stroke in a homozygous P0 Kcc2E/E mouse.

    Movie S2. Seizure triggered by tail pinch in a homozygous P0 Kcc2E/E mouse.

    Movie S3. Seizure triggered by tail suspension in a homozygous P0 Kcc2E/E mouse.

    Movie S4. Spontaneous seizure in a homozygous P0 Kcc2E/E mouse.

    Reference (75)

  • The PDF file includes:

    • Fig. S1. Sequence alignments.
    • Fig. S2. Generation and characterization of KCC2 Thr906/Thr1007 phosphomimetic mice.
    • Fig. S3. Hematoxylin and eosin staining of horizontal and sagittal midline sections of P0 mice showing no gross defects of the central nervous or the musculoskeletal system.
    • Table S1. In vivo KCC2 phosphorylation sites identified and quantified in all four conditions (E18.5, P0, P20, and adult).
    • Table S2. List of candidate protein kinases.
    • Legends for movies S1 to S4
    • Reference (75)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.m4v format). Seizure triggered by mild brush stroke in a homozygous P0 Kcc2E/E mouse.
    • Movie S2 (.m4v format). Seizure triggered by tail pinch in a homozygous P0 Kcc2E/E mouse.
    • Movie S3 (.m4v format). Seizure triggered by tail suspension in a homozygous P0 Kcc2E/E mouse.
    • Movie S4 (.m4v format). Spontaneous seizure in a homozygous P0 Kcc2E/E mouse.

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