Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-β (Aβ) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.

Fig. 1. Synaptic depression by Aβ blocked by uncompetitive PKC antagonist

(A and B) Top left: Experimental design (see Materials and Methods). Plots (A) and bar graphs (B) of evoked synaptic response amplitudes recorded in CT100-infected versus noninfected neurons. Shown are individual (black) and average (red) of cell pair responses; sloped line, x = y. Sample traces (B, right) obtained from indicated conditions. Scale bars, 50 ms, 50 pA. Data are means ± SEM from 12 to 13 pairs of cells (one infected, the other not); ^**P < 0.03, by bootstrap test (see Materials and Methods). (C) Normalized average of PKC activity in COS7 cells expressing PKC activity reporter CKAR (C kinase activity reporter) fused to PSD95 (left) or targeted to plasma membrane (right) in response to the competitive (Gö 6983) or uncompetitive (Bis IV) PKC inhibitor, added at the time indicated by the arrow head. Data are means ± SEM from >16 cells; ^****P < 0.0001, by bootstrap test (see Materials and Methods); n.s., not significant.

Fig. 2. PKCα is required for the effects of Aβ on synaptic transmission

(A and B) Plot (A), and example traces (bottom right), of evoked synaptic response amplitudes recorded in infected versus noninfected neurons; genotype and infection indicated. Bar graph (B, left) of same data. ^**P < 0.03, by bootstrap test (see Materials and Methods). wt, wild-type.

Fig. 3. Human genetics of rare PKCα variants

Diagrams indicating number of families, along with phenotype and genotype of individuals, carrying M489V, V636I, or R324W PKCα variants. All PKCα variant carriers (yellow) displayed AD, and both individuals without AD (blue) lacked a PKCα variant.

Fig. 4. AD-associated rare variants in PKCα

(A) PKCα kinase domain structure (53) showing two residues altered in AD:Met⁴⁸⁹ and Val⁶³⁶. Both are near key regulatory phosphorylation sites (stick representation). Enlargement of activation loop segment (right panels) showing that substitution of Met⁴⁸⁹ with Val loosens the structural packing of this segment. (B) Western blot showing phosphorylation of the indicated hemagglutinin (HA)–tagged PKCα proteins. (C) Western blot of COS7 cells expressing wild-type or M489V PKCα and treated with phorbol 12,13-dibutyrate (PDBu) for the indicated times and probed for HA. Quantitative analysis of phosphorylated/total PKC from five independent experiments. ^**P < 0.01, by bootstrap test (see Materials and Methods).

Fig. 5. Live-cell imaging reveals higher signaling output of all three AD-associated rare variants

Left: Normalized FRET ratios (mean ± SEM) representing PKC activity in COS7 cells coexpressing PKC activity reporter, CKAR, (22) and indicated PKCα. Addition of uridine 5′-triphosphate (UTP) (100 μM) where indicated (arrow head). n > 25 cells for each construct. Data are means ± SEM from at least three independent experiments. Right: Area under the curve from 3 to 6 min; ^*P < 0.05, by bootstrap test (see Materials and Methods). r.u., relative units.