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Abstract：
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 glutamatergic 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.
METHODS: Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic 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.
摘要：
背景：突触的局部翻译对于快速重塑突触蛋白质组以维持长期可塑性和记忆很重要。虽然记忆相关的局部翻译的调节机制已在突触后/树突区域得到广泛阐明，没有直接证据表明轴突中的RNA结合蛋白(RBP)控制靶特异性mRNA的翻译,从而促进长时程增强(LTP)和记忆.我们先前报道，由胞质聚腺苷酸化元件结合蛋白2（CPEB2）控制的翻译对于突触后可塑性和记忆很重要。这里,我们调查了CPEB2是否调节轴突平移以支持突触前可塑性。
方法:在具有CPEB2的泛神经元/神经胶质细胞或谷氨酸能神经元特异性敲除的小鼠中进行行为和电生理学评估。电记录海马Schaffer侧支（SC）-CA1和颞氨（TA）-CA1途径，以监测4列高频刺激引起的突触传递和LTP。RNA免疫沉淀，结合生物信息学分析，用于揭示与学习相关的CPEB2结合轴突RNA候选物，通过Western印迹和荧光素酶报告基因检测进一步验证。将表达Cre重组酶的腺相关病毒立体定向递送至TA回路的突触前或突触后区域以消融Cpeb2用于进一步的电生理研究。在微流体平台上培养的生化分离的突触小体和轴突化神经元用于测量轴突蛋白合成和FM4-64FX负载的突触小泡。
结果：海马CA1神经元的电生理分析检测到CPEB2耗尽的SC和TA传入的异常兴奋性和囊泡释放概率，因此,我们将CPEB2免疫沉淀的转录组与成人皮质中学习诱导的轴突翻译组交叉比较,以鉴定可能受CPEB2调节的轴突靶标.我们验证了Slc17a6，编码囊泡谷氨酸转运蛋白2（VGLUT2），由CPEB2翻译上调。在表达VGLUT2的谷氨酸能神经元中CPEB2的条件性敲除会损害小鼠海马依赖性记忆的巩固。在VGLUT2主导的TA传入中，突触前特异性的Cpeb2消融足以减弱蛋白质合成依赖性LTP。此外，CPEB2缺乏症或环己酰亚胺阻断活性诱导的轴突Slc17a6翻译减少了含VGLUT2的突触小泡的可释放池。
结论：我们确定了272个CPEB2结合转录本，其轴突翻译在学习后发生改变，并在CPEB2驱动的轴突合成VGLUT2和突触前翻译依赖性LTP之间建立了因果关系。这些发现扩展了我们对突触前室中与记忆相关的翻译控制机制的理解。
方法:在具有CPEB2的泛神经元/神经胶质细胞或谷氨酸能神经元特异性敲除的小鼠中进行行为和电生理学评估。电记录海马Schaffer侧支（SC）-CA1和颞氨（TA）-CA1途径，以监测4列高频刺激引起的突触传递和LTP。RNA免疫沉淀，结合生物信息学分析，用于揭示与学习相关的CPEB2结合轴突RNA候选物，通过Western印迹和荧光素酶报告基因检测进一步验证。将表达Cre重组酶的腺相关病毒立体定向递送至TA回路的突触前或突触后区域以消融Cpeb2用于进一步的电生理研究。在微流体平台上培养的生化分离的突触小体和轴突化神经元用于测量轴突蛋白合成和FM4-64FX负载的突触小泡。
结果：海马CA1神经元的电生理分析检测到CPEB2耗尽的SC和TA传入的异常兴奋性和囊泡释放概率，因此,我们将CPEB2免疫沉淀的转录组与成人皮质中学习诱导的轴突翻译组交叉比较,以鉴定可能受CPEB2调节的轴突靶标.我们验证了Slc17a6，编码囊泡谷氨酸转运蛋白2（VGLUT2），由CPEB2翻译上调。在表达VGLUT2的谷氨酸能神经元中CPEB2的条件性敲除会损害小鼠海马依赖性记忆的巩固。在VGLUT2主导的TA传入中，突触前特异性的Cpeb2消融足以减弱蛋白质合成依赖性LTP。此外，CPEB2缺乏症或环己酰亚胺阻断活性诱导的轴突Slc17a6翻译减少了含VGLUT2的突触小泡的可释放池。
结论：我们确定了272个CPEB2结合转录本，其轴突翻译在学习后发生改变，并在CPEB2驱动的轴突合成VGLUT2和突触前翻译依赖性LTP之间建立了因果关系。这些发现扩展了我们对突触前室中与记忆相关的翻译控制机制的理解。
