Reduction of increased calcineurin activity rescues impaired homeostatic synaptic plasticity in presenilin 1 M146V mutant

Abstract

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases characterized by memory loss and cognitive impairment. Whereas most AD cases are sporadic, some are caused by mutations in early-onset familial AD (FAD) genes. One FAD gene encodes presenilin 1 (PS1), and a PS1 mutation in methionine 146 impairs homeostatic synaptic plasticity (HSP). We have previously shown that Ca(2+) and calcineurin activity are critical regulators of HSP. Here, we confirm that endoplasmic reticulum-mediated Ca(2+) signals are increased in mutant PS1 neurons. We further show that calcineurin activity is abnormally elevated in the mutant and that inhibition of increased calcineurin activity stabilizes GluA1 phosphorylation, promoting synaptic trafficking of Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, contributing to the recovery of impaired HSP found in the mutant. Because HSP is suggested to have roles during learning and memory formation, increased calcineurin activity-induced impairment of HSP can cause cognitive decline in FAD. Thus, reducing abnormally increased calcineurin activity in AD brain may be beneficial for improving AD-related cognitive decline.

Keywords: AMPA receptor; Alzheimer’s disease; Calcineurin; Homeostatic synaptic plasticity; Presenilin 1.

Figure 1. Enhanced ER-mediated Ca²⁺ signaling in PS1 M146V hippocampal neurons

a) Example bar graphs of Ca²⁺ signals in each condition. Each bar represents the GCaMP5 fluorescence intensity detected in a single exposure frame. b) Normalized average of total Ca²⁺ activity in each condition showing that PS1 mutant neurons have higher ER-mediated Ca²⁺ signals (n=number of neurons, **p<0.01, ***p<0.001, and ****p<0.0001, one-way ANOVA, uncorrected Fisher's LSD).

Figure 2. Elevated calcineurin activity in PS1 M146V can be reduced by FK506 treatment

Representative images of CFP channel, FRET channel, and pseudocolored emission ratio (FRET/CFP) in each condition (Blue (L): low emission ratio and red (H): high emission ratio). A scale bar indicates 10μm. A summary graph showing an increase in calcineurin activity in PS1 M146V, which can be reduced by FK506 treatment (n=number of cells, *p<0.05, **p<0.01, and ****p<0.0001, one-way ANOVA, uncorrected Fisher's LSD).

Figure 3. Elevated calcineurin activity in PS1 M146V can decrease GluA1 S845 phosphorylation

a) Representative immunoblots and quantitative analysis of synaptosomes from cultured hippocampal neurons of WT and PS1 M146V showing that selective reduction of GluA1 S845 phosphorylation [pGluA1(S845)] (n=6 experiments, ***p<0.001, unpaired two-tailed student's t-test). b) Representative immunoblots and quantitative analysis of PSD from the hippocampus of 18-month old heterozygotes (het) and homozygotes (PS1) of PS1 M146V showing GluA1 and GluA1 S845 phosphorylation are decreased (n=5 WT and 5 KO animals, *p<0.05 and ****p<0.0001, unpaired two-tailed student's t-test).

Figure 4. FK506 treatment is sufficient for inducing synaptic scaling in PS1 M146V

a) Representative traces of mEPSC recordings in each condition (n=number of cells). b) An average mEPSC amplitude graph showing FK506 treatment increases the CPAR-dependent amplitude (****p<0.0001, one-way ANOVA, uncorrected Fisher's LSD). c) An average mEPSC frequency graph showing FK506 treatment increases the CPAR-mediated frequency (***p<0.001, one-way ANOVA, uncorrected Fisher's LSD). d) An average decay time (peak to 10%) graph showing that FK506 treatment induces synaptic expression of CPARs (*p<0.05, one-way ANOVA, uncorrected Fisher's LSD). e) Average cumulative probability of mEPSC amplitude. FK506-induced distribution is significantly different from the DMSO-treated control (p<0.0001, K-S test). Distribution of FK506 scaled down by a factor of 1.27 does not fitted to the DMSO-treated control (p<0.05, K-S test).

Figure 5. FK506 treatment-induced selective synaptic trafficking of GluA1

a) Representative immunoblots and quantitative analysis of synaptosomes from cultured mutant hippocampal neurons in the presence and absence of FK506 treatment showing a selective increase in total GluA1 and GluA1 S845 phosphorylation [pGluA1(S845)] (n=10 experiments, *p<0.05 and ***p<0.001, unpaired two-tailed student's t-test). b) Representative immunoblots of surface biotinylation and a summary graph in DMSO (D) or FK506 (F)-treated neurons showing that FK506 treatment selectively increases surface GluA1 levels (n=10 experiments, *p<0.05, unpaired two-tailed student's t-test).