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.

Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory.

Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. Our observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. Our data thus point to an instructional role for energy flux in the execution of demanding higher brain functions.

Role of Drosophila Amyloid Precursor Protein in Memory Formation.

The amyloid precursor protein (APP) is a membrane protein engaged in complex proteolytic pathways. APP and its derivatives have been shown to play a central role in Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by memory decline. Despite a huge effort from the research community, the primary cause of AD remains unclear, making it crucial to better understand the physiological role of the APP pathway in brain plasticity and memory. Drosophila melanogaster is a model system well-suited to address this issue. Although relatively simple, the fly brain is highly organized, sustains several forms of learning and memory, and drives numerous complex behaviors. Importantly, molecules and mechanisms underlying memory processes are conserved from flies to mammals. The fly encodes a single non-essential APP homolog named APP-Like (APPL). Using in vivo inducible RNA interference strategies, it was shown that APPL knockdown in the mushroom bodies (MB)-the central integrative brain structure for olfactory memory-results in loss of memory. Several APPL derivatives, such as secreted and full-length membrane APPL, may play different roles in distinct types of memory phases. Furthermore, overexpression of Drosophila amyloid peptide exacerbates the memory deficit caused by APPL knockdown, thus potentiating memory decline. Data obtained in the fly support the hypothesis that APP acts as a transmembrane receptor, and that disruption of its normal function may contribute to cognitive impairment during early AD.

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.

Amyloid-ß Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the ß-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-ß (Aß) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aß toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway's influence on memory in the adult. We show that transient overexpression of the ß-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, ß-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aß peptide. Because Aß overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aß affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms-APPL loss-of-function and amyloid peptide toxicity-revealing in Drosophila a functional interaction between APPL and amyloid peptide.

The full-length form of the Drosophila amyloid precursor protein is involved in memory formation.

The APP plays a central role in AD, a pathology that first manifests as a memory decline. Understanding the role of APP in normal cognition is fundamental in understanding the progression of AD, and mammalian studies have pointed to a role of secreted APPα in memory. In Drosophila, we recently showed that APPL, the fly APP ortholog, is required for associative memory. In the present study, we aimed to characterize which form of APPL is involved in this process. We show that expression of a secreted-APPL form in the mushroom bodies, the center for olfactory memory, is able to rescue the memory deficit caused by APPL partial loss of function. We next assessed the impact on memory of the Drosophila α-secretase kuzbanian (KUZ), the enzyme initiating the nonamyloidogenic pathway that produces secreted APPLα. Strikingly, KUZ overexpression not only failed to rescue the memory deficit caused by APPL loss of function, it exacerbated this deficit. We further show that in addition to an increase in secreted-APPL forms, KUZ overexpression caused a decrease of membrane-bound full-length species that could explain the memory deficit. Indeed, we observed that transient expression of a constitutive membrane-bound mutant APPL form is sufficient to rescue the memory deficit caused by APPL reduction, revealing for the first time a role of full-length APPL in memory formation. Our data demonstrate that, in addition to secreted APPL, the noncleaved form is involved in memory, raising the possibility that secreted and full-length APPL act together in memory processes.

Circadian modulation of consolidated memory retrieval following sleep deprivation in Drosophila.

OBJECTIVES: Several lines of evidence indicate that sleep plays a critical role in learning and memory. The aim of this study was to evaluate anesthesia resistant memory following sleep deprivation in Drosophila. DESIGN: Four to 16 h after aversive olfactory training, flies were sleep deprived for 4 h. Memory was assessed 24 h after training. Training, sleep deprivation, and memory tests were performed at different times during the day to evaluate the importance of the time of day for memory formation. The role of circadian rhythms was further evaluated using circadian clock mutants. RESULTS: Memory was disrupted when flies were exposed to 4 h of sleep deprivation during the consolidation phase. Interestingly, normal memory was observed following sleep deprivation when the memory test was performed during the 2 h preceding lights-off, a period characterized by maximum wake in flies. We also show that anesthesia resistant memory was less sensitive to sleep deprivation in flies with disrupted circadian rhythms. CONCLUSIONS: Our results indicate that anesthesia resistant memory, a consolidated memory less costly than long-term memory, is sensitive to sleep deprivation. In addition, we provide evidence that circadian factors influence memory vulnerability to sleep deprivation and memory retrieval. Taken together, the data show that memories weakened by sleep deprivation can be retrieved if the animals are tested at the optimal circadian time.

Debra, a protein mediating lysosomal degradation, is required for long-term memory in Drosophila.

A central goal of neuroscience is to understand how neural circuits encode memory and guide behavior changes. Many of the molecular mechanisms underlying memory are conserved from flies to mammals, and Drosophila has been used extensively to study memory processes. To identify new genes involved in long-term memory, we screened Drosophila enhancer-trap P(Gal4) lines showing Gal4 expression in the mushroom bodies, a specialized brain structure involved in olfactory memory. This screening led to the isolation of a memory mutant that carries a P-element insertion in the debra locus. debra encodes a protein involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome. To study debra's role in memory, we achieved debra overexpression, as well as debra silencing mediated by RNA interference. Experiments conducted with a conditional driver that allowed us to specifically restrict transgene expression in the adult mushroom bodies led to a long-term memory defect. Several conclusions can be drawn from these results: i) debra levels must be precisely regulated to support normal long-term memory, ii) the role of debra in this process is physiological rather than developmental, and iii) debra is specifically required for long-term memory, as it is dispensable for earlier memory phases. Drosophila long-term memory is the only long-lasting memory phase whose formation requires de novo protein synthesis, a process underlying synaptic plasticity. It has been shown in several organisms that regulation of proteins at synapses occurs not only at translation level of but also via protein degradation, acting in remodeling synapses. Our work gives further support to a role of protein degradation in long-term memory, and suggests that the lysosome plays a role in this process.

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila.

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. Here, we took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, we show that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms.

Drosophila amyloid precursor protein-like is required for long-term memory.

The amyloid precursor protein (APP) plays an important role in Alzheimer's disease (AD), a progressive neurodegenerative pathology that first manifests as a decline of memory. While the main hypothesis for AD pathology centers on the proteolytic processing of APP, very little is known about the physiological function of the APP protein in the adult brain. Likewise, whether APP loss of function contributes to AD remains unclear. Drosophila has been used extensively as a model organism to study neuronal function and pathology. In addition, many of the molecular mechanisms underlying memory are thought to be conserved from flies to mammals, prompting us to study the function of APPL, the fly APP ortholog, during associative memory. It was previously shown that APPL expression is highly enriched in the mushroom bodies (MBs), a specialized brain structure involved in olfactory memory. We analyzed memory in flies in which APPL expression has been silenced specifically and transiently in the adult MBs. Our results show that in adult flies, APPL is not required for learning but is specifically involved in long-term memory, a long lasting memory whose formation requires de novo protein synthesis and is thought to require synaptic structural plasticity. These data support the hypothesis that disruption of normal APP function may contribute to early AD cognitive impairment.

CTD kinase I is required for the integrity of the rDNA tandem array.

The genomic stability of the rDNA tandem array is tightly controlled to allow sequence homogenization and to prevent deleterious rearrangements. In this report, we show that the absence of the yeast CTD kinase I (CTDK-I) complex in null mutant strains leads to a decrease in the number of tandem rDNA repeats. Reintroduction of the missing gene induces an increase of rDNA repeats to reach a copy number similar to that of the original strain. Interestingly, while expansion is dependent on Fob1, a protein required for replication fork blocking activity in rDNA, contraction occurs in the absence of Fob1. Furthermore, silencing of class II genes at the rDNA, a process connected to rDNA stability, is not affected. Ctk1, the kinase subunit of the CTDK-I complex is involved in various steps of mRNA synthesis. In addition, we have recently shown that Ctk1 is also implicated in rRNA synthesis. The results suggest that the RNA polymerase I transcription defect occurring in a ctk1 mutant strain causes rDNA contraction.

CTD kinase I is involved in RNA polymerase I transcription.

RNA polymerase II carboxy terminal domain (CTD) kinases are key elements in the control of mRNA synthesis. Yeast CTD kinase I (CTDK-I), is a non-essential complex involved in the regulation of mRNA synthesis at the level of transcription elongation, pre-mRNA 3' formation and nuclear export. Here, we report that CTDK-I is also involved in ribosomal RNA synthesis. We show that CTDK-I is localized in part in the nucleolus. In its absence, nucleolar structure and RNA polymerase I transcription are affected. In vitro experiments show an impairment of the Pol I transcription machinery. Remarkably, RNA polymerase I co-precipitates from cellular extracts with Ctk1, the kinase subunit of the CTDK-I complex. In vitro analysis further demonstrates a direct interaction between RNA polymerase I and Ctk1. The results suggest that CTDK-I might participate in the regulation of distinct nuclear transcriptional machineries, thus playing a role in the adaptation of the global transcriptional response to growth signalling.