CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity

Background Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. Methods Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or AQ2glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. Results Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. Conclusions We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment. RNAs precipitated by CPEB2 IgG and control IgG

研究背景 突触局部翻译对于快速重塑突触蛋白质组以维持长期可塑性与记忆至关重要。尽管记忆相关局部翻译的调控机制已在突触后/树突区域得到广泛阐明，但目前尚无直接证据表明轴突内何种RNA结合蛋白（RNA-binding protein, RBP）能够调控靶标特异性mRNA翻译，进而促进长时程增强（long-term potentiation, LTP）与记忆形成。本团队此前报道，胞质多聚腺苷酸化元件结合蛋白2（cytoplasmic polyadenylation element binding protein 2, CPEB2）介导的翻译调控对突触后可塑性与记忆具有重要作用。本研究旨在探究CPEB2是否通过调控轴突翻译以支持突触前可塑性。

研究方法 我们对泛神经元/胶质细胞或AQ2谷氨酸能神经元特异性敲除CPEB2的小鼠开展行为学与电生理检测。通过记录海马Schaffer侧支（Schaffer collateral, SC）-CA1及颞氨通路（temporoammonic, TA）-CA1通路的突触传递与4组高频刺激诱发的LTP，以监测突触功能。我们采用RNA免疫沉淀（RNA immunoprecipitation, RIP）结合生物信息学分析，筛选与学习相关的CPEB2结合轴突RNA候选靶点，并通过蛋白质印迹法与荧光素酶报告基因实验对候选靶点进行验证。将表达Cre重组酶的腺相关病毒（adeno-associated viruses, AAV）立体定位注射至TA环路的突触前或突触后区域，以敲除Cpeb2，进一步开展电生理研究。我们通过生化分离的突触小体以及微流控平台培养的轴突切断神经元，检测轴突蛋白质合成与FM4-64FX标记的突触囊泡。

研究结果 对海马CA1神经元的电生理分析显示，CPEB2敲除的SC与TA传入神经元存在兴奋性异常与囊泡释放概率改变。因此，我们将CPEB2免疫沉淀得到的转录组与成年皮层中学习诱导的轴突翻译组进行交叉比对，以筛选可能受CPEB2调控的轴突靶点。我们验证了编码囊泡谷氨酸转运体2（vesicular glutamate transporter 2, VGLUT2）的Slc17a6可被CPEB2介导翻译上调。在表达VGLUT2的谷氨酸能神经元中条件性敲除CPEB2，会损伤小鼠海马依赖性记忆的巩固过程。在以VGLUT2为标记的TA传入神经元中特异性敲除突触前Cpeb2，足以削弱依赖蛋白质合成的LTP。此外，通过CPEB2缺失或环己酰亚胺阻断活动依赖性轴突Slc17a6翻译，会减少含VGLUT2的突触囊泡的可释放池。

研究结论 我们共筛选得到272个学习后轴突翻译发生改变的CPEB2结合转录本，并建立了CPEB2介导的VGLUT2轴突合成与依赖翻译的突触前LTP之间的因果关联。本研究结果拓展了我们对突触前区域内记忆相关翻译调控机制的认知。由CPEB2 IgG与对照IgG沉淀得到的RNA