BDNF facilitates L-LTP maintenance in the absence of protein synthesis through PKMζ

Abstract

Late-phase long term potentiation (L-LTP) is thought to be the cellular basis for long-term memory (LTM). While LTM as well as L-LTP is known to depend on transcription and translation, it is unclear why brain-derived neurotrophic factor (BDNF) could sustain L-LTP when protein synthesis is inhibited. The persistently active protein kinase ζ (PKMζ) is the only molecule implicated in perpetuating L-LTP maintenance. Here, in mouse acute brain slices, we show that inhibition of PKMζ reversed BDNF-dependent form of L-LTP. While BDNF did not alter the steady-state level of PKMζ, BDNF together with the L-LTP inducing theta-burst stimulation (TBS) increased PKMζ level even without protein synthesis. Finally, in the absence of de novo protein synthesis, BDNF maintained TBS-induced PKMζ at a sufficient level. These results suggest that BDNF sustains L-LTP through PKMζ in a protein synthesis-independent manner, revealing an unexpected link between BDNF and PKMζ.

Figure 1. BDNF-dependent late phase LTP is mediated by PKMζ.

(A, B) 12 TBS-induced L-LTP was reversed by PKMζ inhibitor ZIP. Field EPSP (fEPSP) was evoked in CA1 stratum radiatum by stimulating Schaffer Collateral in adult C57BL/6 mice. (A) After a stable baseline was obtained, 12 TBS was conducted. LTP was sustained at least for 3 hours. ZIP (5 µM) or scrambled ZIP peptide (5 µM) was applied at 1 hour after stimulation. (B) Quantification of the initial slope value from the last 5 minutes recording. (C, D) Forskolin-induced L-LTP was abolished by PKMζ inhibitor ZIP. The experiments were done identically as in (A), except that L-LTP was induced by a transient perfusion of forskolin (50 µM) and IBMX (30 µM) for 15 minutes. ZIP or scrambled ZIP was applied at 80 minutes after chemical induction when stable L-LTP was fully established. Numbers of slices and mice used in each condition are indicated at the top of each plot. In this and all other figures, data are presented as mean ± s.e.m. * p<0.05, ** p<0.01,*** p<0.001, Student's t-Test.

Figure 2. Steady-State PKMζ protein level is not regulated by BDNF.

(A) PKMζ protein level in cortex and hippocampus of BDNF KO and WT littermates. At postnatal day 18, cortex or hippocampus from BDNF KO and WT littermates were dissected and subjected to Western blot. Representative blots and quantification of data were shown. GAPDH was used as loading control. (n = 5–8 independent experiments). (B) PKMζ expression in primary neuron cultures derived from different genotypes after BDNF treatment. DIV 7 cortical primary cultures of WT, Het or KO genotype were separately treated with BDNF (100 ng/ml) or vehicle for 24 hours. For each experiment, BDNF treatment group was normalized against vehicle treatment group. Representative blots are shown on top of the quantification of data. (n = 5 independent experiments).

Figure 3. BDNF modulates activity-dependent PKMζ level to sustain L-LTP in the absence of protein synthesis.

(A, B) Rescuing L-LTP impairment by BDNF in the presence of anisomycin is PKMζ-dependent. (A) Applications of various drugs were indicated by horizontal bars. Anisomycin (40 µM) was used throughout the entire experiment. BDNF (200 ng/ml) was applied 3 minutes after 12TBS stimulation and successfully rescued L-LTP impairment. ZIP was applied at 1 hour after stimulation and completely abolished L-LTP. (B) Quantification of the initial slope from the last 5 minutes of recording. (C) PKMζ protein level of hippocampal CA1 derived from WT mice at 1 hour and 3 hours after 12TBS stimulation. Tubulin was used as loading control. The 12 TBS group was normalized against control group. The 12 TBS plus BDNF and anisomycin treatment groups were normalized against that without BDNF treatment. Representative blots are shown on top of the quantification of data (3–5 slices per treatment, n = 3 independent experiments).