Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2

Abstract

Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. Here we report a unique feature of hippocampal slices from GCN2(-/-) mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2(-/-) slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2(-/-) mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2(-/-) mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway.

Figure 1

Unusual properties of LTP induced in slices from GCN2 -/- mice. A) One train (100 Hz for 1s) of high-frequency stimulation (HFS) elicited E-LTP in WT slices but produced a robust and L-LTP in GCN2 -/- slices. B) Sustained LTP in GCN2 -/- slices is reduced by anisomycin (ANISO, 40μM), actinomycin-D (ACTD, 40μM) or the PKA inhibitor KT5720 (1μM). C) The enhanced LTP in GCN2 -/- slices is reduced by actinomycin-D, at later time points (>90 min) when applied 15 min prior to and 45 min post-tetanus. D) L-LTP induced by four 100 Hz trains at 5 min intervals is stable in WT slices but not in GCN2 -/- slices.

Figure 2

ATF4 mRNA translation is downregulated in GCN2 -/- mice. A) Western blots performed on hippocampal extracts show that eIF2α phosphorylation is reduced in GCN2 -/- (n=3) as compared to WT mice (n=3). B) In polysome profiles from hippocampal extracts, ATF4 mRNA is in lighter fractions in GCN2 -/- (left panel) than in WT (right panel) controls as determined by RT-PCR analysis. C) Quantification of the band intensities in each fraction from ATF4 mRNA, panel B. D) for data in B, band intensities are quantified for each fraction of β-actin mRNA. E) In pooled hippocampal extracts, expression of ATF4 is reduced in GCN2 -/- mice. F) Real-time RT-PCR analysis reveals increased expression of CREB-dependent genes in hippocampal extracts from GCN2 -/- vs. WT mice (for both, n=5); mRNA expression is given as % of controls; *p < 0.05, **p < 0.01. G) Forskolin decreases GCN2 and eIF2α phosphorylation. In immunoblots of homogenates of CA1 region (from slices frozen immediately after tetanic stimulation), phosphorylated GCN2 (top) and eIF2α (middle panel) are decreased five minutes after forskolin application.

Figure 3

GCN2 -/- mice are impaired in contextual but not auditory fear conditioning. A) Though acquisition of contextual freezing (with two pairings) is similar in GCN2 -/- (filled squares, n=10) and WT (open squares, n=12) mice, contextual freezing, measured during 2 min period before first shock (pre-shocks) and 1 min period after last shock (post-shocks), is impaired in GCN2 -/- mice 1 and 10 days after acquisition. B) Auditory fear conditioning is intact in freezing to tone over two pairings and in long term memory tests at 1 and 10 days later in GCN2 -/- mice. Labels indicate genotype and whether freezing was to tone (CS) or during 2 min before tone (pre-CS) (Means ± SEM).

Figure 4

Long-term spatial memory of GCN2 -/- mice is enhanced after weak training but impaired after more intense training (in the Morris water maze) A) Escape latencies in hidden platform tests (3 trial per day), plotted as function of training days (WT, open squares, n = 16 and GCN2 -/-, filled squares, n = 15), are shorter for WT than GCN2 -/- mice. B) After completion of training, WT mice show preferential quadrant occupancy. C) WT mice crossed the previous site where the platform was located more times than GCN2 -/- mice (p < 0.001). D) When locating hidden platform (1 trial per day), escape latencies were consistently shorter for GCN2 -/- mice than for WT (for both, n=15). E) In occupancy test, GCN2 -/- mice spent more time in the trained quadrant (Means ± SEM).