學(xué)習(xí)和記憶是大腦的基本功能，長時(shí)記憶的形成被認(rèn)為需要神經(jīng)元活動誘導(dǎo)下的蛋白質(zhì)合成。以往的研究表明，m⁶A參與調(diào)節(jié)神經(jīng)功能，包括小鼠中腦多巴胺能信號傳導(dǎo)、飛蟲飛行行為、成年小鼠神經(jīng)生成和軸突再生。在大腦發(fā)育、行為體驗(yàn)和記憶形成過程中觀察到^m6A的上調(diào)，這表明m⁶A的積累和大腦活動之間存在相關(guān)性。而YTHDF家族蛋白可以與mRNA上的m⁶A結(jié)合，從而調(diào)控mRNA的翻譯，同時(shí)前期實(shí)驗(yàn)已證實(shí)YTDHF1在小鼠海馬體中高表達(dá)。因此也提示了學(xué)習(xí)和記憶過程可能會受到m⁶A及其結(jié)合蛋白Ythdf1翻譯效應(yīng)的影響。
 
研究團(tuán)隊(duì)利用CRISPR/Cas9技術(shù)，培育出了Ythdf1 敲除小鼠 (Ythdf1-KO)。Morris水迷宮實(shí)驗(yàn)顯示與野生型小鼠對比，Ythdf1-KO小鼠的空間記憶能力明顯降低。
 
另外通過經(jīng)典的條件恐懼測試實(shí)驗(yàn)，在經(jīng)過電擊刺激的訓(xùn)練之后，Ythdf1-KO小鼠在實(shí)驗(yàn)間歇期（ITI）freezing的表現(xiàn)相對較少，而聽覺相關(guān)的恐懼反應(yīng)（與杏仁核功能相關(guān)）（即freezing）的表現(xiàn)沒有改變，由此也提示了 Ythdf1的缺失擾亂了小鼠海馬正常的空間學(xué)習(xí)記憶能力。具體實(shí)驗(yàn)數(shù)據(jù)如下圖所示。
  Figure 1.Impaired spatial learning and memory in Ythdf1-KO mice.
a, b, Representative images of Ythdf1 immunostaining (a) and Hoechst (b) in the control and Ythdf1-KO hippocampus. DG, dentate gyrus; P30/P120, postnatal day 30/120. c, d, Learning curves of control (blue) and Ythdf1-KO (red) mice in Morris water maze (MWM) tests in visible (c) and hidden (d) platform training. e, Quadrant time (%) (left) and representative swimming paths (right) of control and Ythdf1-KO mice in the MWM probe test. The red dash line represents the chance level (25%). f, Learning curves of control (blue) and Ythdf1-KO mice (red) for contextual fear conditioning (FC) in moderate (left) or strong (right) training sessions. Base, baseline; ITI, inter-trial interval. g, h, Contextual fear memory assessed 24 hours (g) or 2 hours (h) after the indicated FC. P values, two-way ANOVA with two tailed t-test (relative to “Target” or between genotypes) (e), two-way repeated measures ANOVA with post hoc test (c, d, f), and two-tailed t-test (g, h). Numbers in bars, numbers of mice. Error bars, mean ± s.e.m.
 
為了探究Ythdf1的作用機(jī)制，通過電生理實(shí)驗(yàn)，研究團(tuán)隊(duì)發(fā)現(xiàn)相較于對照組，Ythdf1-KO小鼠 CA1神經(jīng)元的mEPSCs的振幅和頻率顯著降低 (Fig. 2a-b)。配對脈沖比(PPRs)分析也顯示Ythdf1-KO CA1神經(jīng)元突觸前釋放概率降低((Extended Data Fig. 4a-b),)，由此也證實(shí)了Ythdf1缺失造成了基礎(chǔ)突觸傳遞功能的缺陷。同時(shí)形態(tài)學(xué)染色結(jié)果顯示，Ythdf1-KO CA1神經(jīng)元降低了樹突棘密度，但并未改變棘的大小 (Extended Data Fig. 4c-d)。
  Figure 2. Deficient basal transmission and plasticity in Ythdf1-KO hippocampal synapses.
a,b, Representative traces (a) and quantification of amplitude (b, left) and frequency (b, right) of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in control and Ythdf1-KO hippocampal CA1 neurons. c, d, Summary plots (c) and average amplitude (d) of long-term potentiation (LTP) induced by 2 × high frequency stimulation (HFS) in the CA1 region of control and Ythdf1-KO acute slices. fEPSP, field excitatory postsynaptic potential. e, f, Summary plots (e) and average amplitude (f) of late phase LTP induced by 4 × HFS. Top panels, sample traces taken at time points 1 and 2 indicated above the summary plots; scale bars, 10 ms (horizontal) and 0.2 mV (vertical) (c, e). g, h, Representative western blots (g) and quantification (h) of a number of LTP-related proteins in the control and Ythdf1-KO hippocampal postsynaptic density (PSD) fraction. P values, Kolmogorov-Smirnov test for cumulative distributions followed by comparisons with Mann–Whitney U test (b) and two-tailed t-test (d, f, h). Numbers in bars, numbers of neurons/mice (b), slices/mice (d, f), and mice (h). Error bars, mean ± s.e.m
 
為了進(jìn)一步探究Ythdf1是否能夠調(diào)控長時(shí)程的突觸可塑性，通過一種主流的闡釋學(xué)習(xí)記憶的分子機(jī)制——長時(shí)程增強(qiáng)效應(yīng)(Long Term Potentiation ，LTP)，即通過給與海馬CA1區(qū)高頻刺激，記錄Ythdf1-KO和對照組小鼠（野生型）的LTP，結(jié)果顯示，Ythdf1-KO未能顯示正常水平的突觸后電位。同時(shí)Ythdf1的缺失顯著降低了海馬神經(jīng)元突觸后致密物中參與LTP的關(guān)鍵蛋白質(zhì)的水平 (Fig. 2g-h; Extended Data Fig. 4e)。以上實(shí)驗(yàn)結(jié)果顯示，Ythdf1的缺失損害了海馬區(qū)神經(jīng)元的突觸傳導(dǎo)功能，由此導(dǎo)致學(xué)習(xí)和記憶能力的下降。
  Extended Data Figure 4 |. Paired-pulse ratios (PPR), spine morphology, and total protein levels of various LTP-related genes in Ythdf1-KO mouse hippocampus, related to Figure 2.
a, b, PPR with different inter-stimulus intervals in CA1 neurons from wild-type control and Ythdf1-KO mice. c, d, Representative images of Lucifer Yellow staining (c) and statistical analyses of spine density (d, left) and spine size (d, right) in CA1 neurons from adult control and Ythdf1-KO brain. e, Uncropped western blot images for Fig. 2g. f, Total protein levels of a set of LTP-related genes in control and Ythdf1-KO mouse hippocampus. For gel source data, see Supplementary Figure 1. P values, two-way repeated measures ANOVA with post hoc two-tailed t-test (a) and two tailed t-test (b, d, f). Numbers in bars, numbers of slices (b), neurons/mice (d, left), spines (d, right), or mice (f). Error bars, mean ± s.e.m.
 
為了證實(shí)小鼠學(xué)習(xí)記憶功能的下降是由海馬區(qū)Ythdf1缺失導(dǎo)致的，研究團(tuán)隊(duì)針對Ythdf1-KO成年小鼠重表達(dá)了海馬區(qū)Ythdf1，Morris水迷宮實(shí)驗(yàn)和情景相關(guān)條件恐懼實(shí)驗(yàn)證實(shí)，重表達(dá)Ythdf1的Ythdf1-KO小鼠學(xué)習(xí)記憶能力大幅度提高。同時(shí)KCl去極化刺激實(shí)驗(yàn)和電痙攣療法實(shí)驗(yàn)顯示， Ythdf1介導(dǎo)的蛋白質(zhì)合成功能，可在神經(jīng)元的刺激下趨于正�；�。
 
此項(xiàng)研究將m⁶A甲基化這一生物體普遍存在的修飾機(jī)制研究引入到解決神經(jīng)科學(xué)領(lǐng)域關(guān)鍵科學(xué)問題中，對于開啟和深入闡明蛋白翻譯調(diào)控在突觸可塑性中的作用有著重要的意義。
 
就記憶功能而言，可分為短時(shí)（工作記憶）和長時(shí)記憶。Anderson（1976）將長時(shí)記憶分為陳述性記憶或非陳述性記憶。陳述性記憶涉及的主要腦結(jié)構(gòu)是海馬以及其他顳葉內(nèi)側(cè)結(jié)構(gòu)。非陳述性記憶促成了運(yùn)動技能和習(xí)慣的獲得，并通過新紋狀體和小腦實(shí)現(xiàn)。此外，杏仁核介導(dǎo)情緒記憶，并被證明參與記憶的整合。