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Atg9 resides on a unique population of small vesicles in presynaptic nerve terminals

ABSTRACT: In neurons, autophagosomes are mainly initiated in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing Atg9, a lipid scramblase essential for autophagocytosis. Here, we show that Atg9 vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of Atg9 vesicles immunoisolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that Atg9-vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that Atg9-vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.

INSTRUMENT(S): Q Exactive HF-X, Q Exactive HF

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

SUBMITTER: Aleksandar Chernev

LAB HEAD: Henning Urlaub

PROVIDER: PXD041702 | Pride | 2024-01-17

REPOSITORIES: Pride

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Source:

			Action	DRS
	29032019_rattus_uniprot.fasta	Fasta
	MQ_ATG9_IPvsIgG_mqpar.xml	Xml
	MQ_ATG9_IPvsIgG_search.zip	Other
	MQ_ATG9_IPvsIgG_txt.zip	Other
	MQ_ATG9_Input_mqpar.xml	Xml

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Project description: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.

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Atg9 resides on a unique population of small vesicles in presynaptic nerve terminals

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