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.

Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons, it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity, i.e., decreased facilitation and post-tetanic potentiation, but increased long-term potentiation in male synapsin triple knock-out (KO) mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from KO animals. Our results indicate that all synapsin isoforms are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse.

Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved in SNARE complex assembly, and controls multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of αRIMs, providing a link to αRIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2α zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2α ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by αRIMs and calmodulin.

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.

Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved SNARE complex assembly, and control multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of αRIMs, providing a link to αRIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2α zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2α ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by αRIMs and calmodulin.

Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. We generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. We used a protein kinase A (PKA) sensor to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, our results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Presynaptic plasticity is an activity-dependent change in the neurotransmitter release and plays a key role in dynamic modulation of synaptic strength. Particularly, presynaptic potentiation mediated by cyclic adenosine monophosphate (cAMP) is widely seen across the animals and thought to contribute to learning and memory. Hippocampal mossy fiber-CA3 pyramidal cell synapses have been used as a model because of robust presynaptic potentiation in short- and long-term forms. Moreover, direct presynaptic recordings from large mossy fiber terminals allow one to dissect the potentiation mechanisms. Recently, super-resolution microscopy and flash-and-freeze electron microscopy have revealed the localizations of release site molecules and synaptic vesicles during the potentiation at a nanoscale, identifying the molecular mechanisms of the potentiation. Incorporating these growing knowledges, we try to present plausible mechanisms underlying the cAMP-mediated presynaptic potentiation.

Modulation of presynaptic short-term plasticity induced by actin polymerization was studied in rat hippocampal slices using the paired-pulse paradigm. Schaffer collaterals were stimulated with paired pulses with a 70-ms interstimulus interval every 30 s before and during perfusion with jasplakinolide, an activator of actin polymerization. Jasplakinolide application resulted in the increase in the amplitudes of CA3-CA1 responses (potentiation) accompanied by a decrease in the paired-pulse facilitation, suggesting induction of presynaptic modifications. Jasplakinolide-induced potentiation depended on the initial paired-pulse rate. These data indicate that the jasplakinolide-mediated changes in actin polymerization increased the probability of neurotransmitter release. Less typical for CA3-CA1 synapses responses, such as a very low paired-pulse ratio (close to 1 or even lower) or even paired-pulse depression, were affected differently. Thus, jasplakinolide caused potentiation of the second, but not the first response to the paired stimulus, which increased the paired-pulse ratio from 0.8 to 1.0 on average, suggesting a negative impact of jasplakinolide on the mechanisms promoting paired-pulse depression. In general, actin polymerization facilitated potentiation, although the patterns of potentiation differed depending on the initial synapse characteristics. We conclude that in addition to the increase in the neurotransmitter release probability, jasplakinolide induced other actin polymerization-dependent mechanisms, including those involved in the paired-pulse depression.

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. We here provide evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). Our data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories.