Presynaptic regulators in memory formation.

The intricate molecular and structural sequences guiding the formation and consolidation of memories within neuronal circuits remain largely elusive. In this study, we investigate the roles of two pivotal presynaptic regulators, the small GTPase Rab3, enriched at synaptic vesicles, and the cell adhesion protein Neurexin-1, in the formation of distinct memory phases within the Drosophila mushroom body Kenyon cells. Our findings suggest that both proteins play crucial roles in memory-supporting processes within the presynaptic terminal, operating within distinct plasticity modules. These modules likely encompass remodeling and maturation of existing active zones (AZs), as well as the formation of new AZs.

Drosophila Middle-Term Memory: Amnesiac is Required for PKA Activation in the Mushroom Bodies, a Function Modulated by Neprilysin 1.

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga (Rut) adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G-protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). Here, we examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were conducted with both sexes, or with either sex. Our data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, we searched for an interaction between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. We show that when Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together, the results suggest that Nep1 targets Amnesiac degradation to terminate its signaling function. Our work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory (MTM) formation, a function modulated by Nep1.SIGNIFICANCE STATEMENT The Drosophila amnesiac gene encodes a secreted neuropeptide whose expression is required for specific memory phases in the mushroom bodies (MB), the olfactory memory center. Here, we show that Amnesiac is required for PKA activation resulting from coincidence detection, a mechanism by which the MB integrate two spatially distinct stimuli to encode associative memory. Furthermore, our results uncover a functional relationship between Amnesiac and Neprilysin 1 (Nep1), a membrane peptidase involved in memory and expressed in the MB. These results suggest that Nep1 modulates Amnesiac levels. We propose that on conditioning, Amnesiac release from the MB allows, via an autocrine process, the sustaining of PKA activation-mediating memory, which subsequently is inactivated by Nep1 degradation.

Amnesiac Is Required in the Adult Mushroom Body for Memory Formation.

It was proposed that the Drosophila amnesiac gene (amn) is required for consolidation of aversive memory in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the fly center for olfactory learning and memory (Waddell et al., 2000). Yet, a conditional analysis showed that it was not possible to rescue the memory deficit of amnX8 null mutant flies when amn expression was restored only in the adult (DeZazzo et al., 1999), which led the authors to suggest that amn might be involved in the development of brain structures that normally promote adult olfactory memory. To further investigate temporal and spatial requirements of Amnesiac (AMN) peptide in memory, we used RNA interference in combination with conditional drivers. Experiments were conducted either in both sexes, or in either sexes. Our data show that acute modulation of amn expression in adult DPM neurons does not impact memory. We further show that amn expression is required for normal development of DPM neurons. Detailed enhancer trap analyses suggest that amn transcription unit contains two distinct enhancers, one specific of DPM neurons, and the other specific of α/ß MB neurons. This prompted us to investigate extensively the role of AMN in the adult MB. Together, our results demonstrate that amn is acutely required in adult α/ß MB neurons for middle-term and long-term memory. The data thus establish that amn plays two distinct roles. Its expression is required in DPM neurons for their development, and in adult MB for olfactory memory.SIGNIFICANCE STATEMENT The Drosophila amnesiac gene encodes a neuropeptide whose expression was proposed to be required for consolidation of aversive memory in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the olfactory memory center. Here, we investigated amnesiac temporal and spatial requirement using conditional tools that allowed us to manipulate its expression in selected neurons. This work leads to a complete reassessment of the role of amnesiac in brain development and memory. We show that amnesiac is required for two distinct processes: for normal development of DPM neurons, and in adult MB for memory.

Drosophila Neprilysin 1 Rescues Memory Deficits Caused by Amyloid-ß Peptide.

Neprilysins are Type II metalloproteinases known to degrade and inactivate a number of small peptides, in particular the mammalian amyloid-ß peptide (Aß). In Drosophila, several neprilysins expressed in the brain are required for middle-term (MTM) and long-term memory (LTM) in the dorsal paired medial (DPM) neurons, a pair of large neurons that broadly innervate the mushroom bodies (MB), the center of olfactory memory. These data indicate that one or several peptides need to be degraded for MTM and LTM. We have previously shown that the fly amyloid precursor protein (APPL) is required for memory in the MB. We show here that APPL is also required in adult DPM neurons for MTM and LTM formation. This finding prompted us to search for an interaction between neprilysins and Drosophila Aß (dAß), a cleavage product of APPL. To find out whether dAß was a neprilysin's target, we used inducible drivers to modulate neprilysin 1 (Nep1) and dAß expression in adult DPM neurons. Experiments were conducted either in both sexes or in females. We show that Nep1 inhibition makes dAß expression detrimental to both MTM and LTM. Conversely, memory deficits displayed by dAß-expressing flies are rescued by Nep1 overexpression. Consistent with behavioral data, biochemical analyses confirmed that Nep1 degrades dAß. Together, our findings establish that Nep1 and dAß expressed in DPM neurons are functionally linked for memory processes, suggesting that dAß is a physiological target for Nep1.SIGNIFICANCE STATEMENT Neprilysins are endopeptidases known to degrade a number of small peptides and in particular the amyloid peptide. We previously showed that all four neprilysins expressed in the Drosophila brain are involved in specific phases of olfactory memory. Here we show that an increase in the level of the neprilysin 1 peptidase overcomes memory deficits induced by amyloid peptide in young flies. Together, the data reveal a functional interaction between neprilysin 1 and amyloid peptide, suggesting that neprilysin 1 degrades amyloid peptide. These findings raise the possibility that, under nonpathological conditions, mammalian neprilysins degrade amyloid peptide to ensure memory formation.

Drosophila Neprilysins Are Involved in Middle-Term and Long-Term Memory.

UNLABELLED: Neprilysins are type II metalloproteinases known to degrade and inactivate a number of small peptides. Neprilysins in particular are the major amyloid-ß peptide-degrading enzymes. In mouse models of Alzheimer's disease, neprilysin overexpression improves learning and memory deficits, whereas neprilysin deficiency aggravates the behavioral phenotypes. However, whether these enzymes are involved in memory in nonpathological conditions is an open question. Drosophila melanogaster is a well suited model system with which to address this issue. Several memory phases have been characterized in this organism and the neuronal circuits involved are well described. The fly genome contains five neprilysin-encoding genes, four of which are expressed in the adult. Using conditional RNA interference, we show here that all four neprilysins are involved in middle-term and long-term memory. Strikingly, all four are required in a single pair of neurons, the dorsal paired medial (DPM) neurons that broadly innervate the mushroom bodies (MBs), the center of olfactory memory. Neprilysins are also required in the MB, reflecting the functional relationship between the DPM neurons and the MB, a circuit believed to stabilize memories. Together, our data establish a role for neprilysins in two specific memory phases and further show that DPM neurons play a critical role in the proper targeting of neuropeptides involved in these processes. SIGNIFICANCE STATEMENT: Neprilysins are endopeptidases known to degrade a number of small peptides. Neprilysin research has essentially focused on their role in Alzheimer's disease and heart failure. Here, we use Drosophila melanogaster to study whether neprilysins are involved in memory. Drosophila can form several types of olfactory memory and the neuronal structures involved are well described. Four neprilysin genes are expressed in adult Drosophila Using conditional RNA interference, we show that all four are specifically involved in middle-term memory (MTM) and long-term memory (LTM) and that their expression is required in the mushroom bodies and also in a single pair of closely connected neurons. The data show that these two neurons play a critical role in targeting neuropeptides essential for MTM and LTM.

An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

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.

Transient active zone remodeling in the Drosophila mushroom body supports memory.

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.