A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo

Abstract

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically-interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific vs. shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified PKC phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via MARCKS is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (Pcdhg^S/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (Pcdhg^CTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remains unchanged in both Pcdhg^S/A and Pcdhg^CTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the Pcdhg^CTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point, and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

Figure 1.. Generation of the Pcdhg^S/A and Pcdhg^CTD mice.

A, Schematic representation of (top to bottom): the mouse clustered protocadherin loci, comprising Pcdha, Pcdhb, and Pcdhg clusters; the Pcdhg locus, comprising 22 variable exons (γA-subtype: light blue, γB-subtype: dark blue, and γC-subtype: purple) which are spliced to three constant exons (black); Pcdhg transcripts comprising one large variable exon and three constant exons; and the resultant γ-Pcdh protein domain structure, with six extracellular cadherin (EC) repeats, a transmembrane (TM) domain, a short variable cytoplasmic domain (VCD; all encoded by the variable exon) followed by a shared cytoplasmic domain (encoded by the three constant exons). B, Schematic of the FAK/PKC/MARCKS signaling pathway identified in Garrett et al. (2012) and Keeler et al. (2015) in the presence (B) and absence (C) of γ-Pcdh constant domain phosphorylation. Pink lines represent actin filaments. D, Schematic representation of (top to bottom): the wildtype γ-Pcdh protein with an intact constant domain; the serine-to-alanine mutation in Pcdhg^S/A, and the C-terminal deletion in Pcdhg^CTD. E, sgRNAs targeting the third constant exon to generate Pcdhg^S/A (blue) and Pcdhg^CTD (red), resulting in a thymine-to-guanine point mutation and insertion of an early stop codon, respectively. F, Chromatograms of Pcdhg^S/A and Pcdhg^CTD genomic DNA sequencing compared to wildtype.

Figure 2.. Validation of the Pcdhg^S/A and Pcdhg^CTD mice.

A, Cryosections of adult control, Pcdhg^S/A, and Pcdhg^CTD homozygous mutant cortices stained with the indicated layer- and cell type-specific markers (NeuN, neurons; GFAP, astrocytes; Cux1, upper layer neurons; FoxP2, deep layer neurons; cleaved caspase-3 [CC3], apoptotic cells; Parvalbumin, interneuron subset), revealing grossly normal morphology, neuronal survival, and cell types in Pcdhg^S/A and Pcdhg^CTD mutants. Scale bar: 100 μm. B, Immunoprecipitation of γ-Pcdhs using a pan-γ-Pcdh antibody from control and Pcdhg^S/A homozygous mutant brain lysates followed by blotting with an antibody specific to phosphoserine PKC substrates, revealing loss of PKC-phosphorylated γ-Pcdhs in Pcdhg^S/A. C, Western blot of control, Pcdhg^S/A homozygous mutants, and Pcdhg^CTD homozygous mutants using a peptide antibody raised against the final 15 amino acids of the constant domain and a pan-γ-Pcdh antibody (epitope in constant exon 1/2) reveals that Pcdhg^CTD mutants specifically lack the final 15 amino acids of the third constant exon as expected, but still express truncated γ-Pcdh proteins, apparently at a lower level (see Figure 6). GAPDH antibody was used as a loading control for western blots.

Figure 3.. Increased exuberance of cortical dendritic arborization due to nonphosphorylatable γ-Pcdhs in Pcdhg^S/A and Pcdhg^CTD mice in vivo.

A, Sholl analysis graph showing dendritic crossings of concentric circles drawn at increasing 10 μm intervals from the cell body of layer V neurons from control and Pcdhg^S/A and Pcdhg^CTD homozygous mutant mice at 6 weeks of age. B, Representative tracings of layer V pyramidal neurons from the three genotypes, color coded as in the graphs. C, Graph showing area under the curve of the Sholl graph in panel A. Other graphs indicate total dendritic length (D), average branch length (E), and number of branch points (F). Significant increases in dendritic complexity are seen in Pcdhg^S/A and Pcdhg^CTD homozygous mutant neurons compared to control. N ≤ 80 neurons from 3-4 mice per genotype. Scale bar in B: 100 μm. Western blots of P21 brain lysate using pan-FAK and Y397 phosphorylated FAK (P-FAK) antibodies (G), or pan-MARCKS and S152/156 phosphorylated MARCKS antibodies (P-MARCKS) (H) reveal a non-significant downward trend in relative P-FAK levels in Pcdhg^S/A and Pcdhg^CTD homozygous mutants compared to controls (G), and a significant reduction in P-MARCKS levels in Pcdhg^S/A and Pcdhg^CTD homozygous mutants compared to controls (H). Duplicate blots were probed with pan- and phospho-specific antibodies, and each were normalized to GAPDH (loading control). Normalized Phos/Pan ratios are plotted as arbitrary units, setting control values at 1. Data are averaged from three replicate experiments. N = 6-9 brains per genotype. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM; *p<.05, **p<.01, ***p<.001, ****p<.0001; ns = not significant.

Figure 4.. Increased exuberance of cortical dendritic arborization in heterozygous Pcdhg^CTD mice in vivo.

A, Sholl analysis graph showing dendritic crossings of concentric circles drawn at increasing 10 μm intervals from the cell body of layer V neurons from control and heterozygous (HET) Pcdhg^S/A and Pcdhg^CTD mice at 6 weeks of age. B, Representative tracings of layer V pyramidal neurons from the three genotypes, color coded as in the graphs. C, Graph showing area under the curve of the Sholl graph in A. Other graphs indicate total dendritic length (D), average branch length (E), and number of branch points (F). Significant increases in dendritic complexity are seen in heterozygous Pcdhg^CTD compared to controls and heterozygous Pcdhg^S/A mice. Scale bar in B: 100 μm. N ≤ 60 neurons from 3-5 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM; *p<.05, **p<.01, ***p<.001; ns = not significant.

Figure 5.. Normal density and maturation of dendritic spines in γ-Pcdh C-terminal mutant mice.

A, Representative images showing dendritic spines of Thy1-YFPH-labeled layer V pyramidal neurons from the cortex of five-week-old controls and Pcdhg^S/A and Pcdhg^CTD homozygous mutant mice. Scale bar: 5 μm. Quantification of total dendritic spine density (B) and of the density of thin, stubby, and mushroom dendritic spines (C). No significant differences were observed between controls and either Pcdhg^S/A or Pcdhg^CTD homozygous neurons. N ≤50 dendritic segments from 7-8 mice per genotype. D, Western blots of P21 brain lysates reveal no molecular alterations in mutants compared to wildtype (WT) controls, assessed by antibodies against markers of post-synaptic compartments (PSD-95, GluR2, Nlg1, and SAP102). Blots were normalized to GAPDH and values plotted as arbitrary units compared to control values set at 1. Western blotting data were averaged from 3 replicate experiments. N = 6-9 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM; ns = not significant.

Figure 6.. Significantly decreased γ-Pcdh protein levels in Pcdhg^CTD mutant brain.

A, B, Western blots using pan-γ-Pcdh antibody along with a panel of isoform-specific γ-Pcdh antibodies reveal a significant reduction of all γ-Pcdhs, γ-Pcdh subgroup A isoforms, γB2, and γC5 in Pcdhg^CTD homozygous brain lysates compared to controls and Pcdhg^S/A homozygous mutants (a non-significant downward trend in γC3 protein levels was also observed in Pcdhg^CTD mutants). Data were averaged from three replicate experiments. N = 9 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM; *p<.05, **p<.01, ***p<.001; ns = not significant.

Figure 7.. Decreased γ-Pcdh protein levels in Pcdhg^CTD neurons are associated with reduced transcript levels and are not attributable to increased protein degradation.

A, Western blots of lysates from cultured control, Pcdhg^S/A homozygous mutant, and Pcdhg^CTD homozygous mutant cortical neurons (day in vitro (DIV)8) treated with cycloheximide (left) or vehicle (right) for the indicated times and probed with pan-γ-Pcdh antibody. B, Ratio of γ-Pcdh protein levels from blots in (A) at each time point post cycloheximide addition (or just media change) normalized to baseline levels (0h). Comparison of area under the curves in (B) reveals no significant difference in protein degradation rate among genotypes. Data were collected from three separate cultures and experiments. C, Quantitative RT-PCR analysis using primers situated in Pcdhg constant exon 1 and 2 as shown reveals a significant decrease in pan-Pcdhg RNA levels in Pcdhg^CTD homozygous mutant brains compared to controls and Pcdhg^S/A homozygous mutants. Relative Pcdhg abundance was calculated using the ΔΔCt method, normalized to GAPDH and control mice. Data were averaged from three replicate experiments. N = 7-8 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SEM; ****p<.0001; ns = not significant.