Long-term Memory In Drosophila Research Articles

Mrj is a chaperone of the Hsp40 family that regulates Orb2 oligomerization and long-term memory in Drosophila.

Orb2 the Drosophila homolog of cytoplasmic polyadenylation element binding (CPEB) protein forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation is dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A's prion-like oligomerization forms long-term memory but fails to maintain it over time. Since this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here, we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Among these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, Mrj knockout exhibits a deficit in long-term memory and our observations suggest Mrj is needed in mushroom body neurons for the regulation of long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating ribosomes.

Long-Term Memory Formation in Drosophila Depends on the 3'UTR of CPEB Gene orb2.

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.

Circadian photoreceptors are required for light-dependent maintenance of long-term memory in Drosophila

In the fruit fly Drosophila melanogaster, environmental light is required for maintaining long-term memory (LTM). Furthermore, the Pigment dispersing factor (Pdf), which is a circadian neuropeptide, and the neuronal activity of Pdf neurons are essential for light-dependent maintenance of courtship LTM. Since Pdf neurons can sense light directly via circadian photoreceptors [Rhodopsin 7 (Rh7) and Cryptochrome (Cry)], it is possible that Rh7 and Cry in Pdf neurons are involved in the maintenance of LTM. In this study, using a courtship conditioning assay, we demonstrated that circadian photoreceptors in Pdf neurons are required for maintaining courtship LTM.

A neural circuit linking learning and sleep in Drosophila long-term memory

Animals retain some but not all experiences in long-term memory (LTM). Sleep supports LTM retention across animal species. It is well established that learning experiences enhance post-learning sleep. However, the underlying mechanisms of how learning mediates sleep for memory retention are not clear. Drosophila males display increased amounts of sleep after courtship learning. Courtship learning depends on Mushroom Body (MB) neurons, and post-learning sleep is mediated by the sleep-promoting ventral Fan-Shaped Body neurons (vFBs). We show that post-learning sleep is regulated by two opposing output neurons (MBONs) from the MB, which encode a measure of learning. Excitatory MBONs-γ2α’1 becomes increasingly active upon increasing time of learning, whereas inhibitory MBONs-β’2mp is activated only by a short learning experience. These MB outputs are integrated by SFS neurons, which excite vFBs to promote sleep after prolonged but not short training. This circuit may ensure that only longer or more intense learning experiences induce sleep and are thereby consolidated into LTM.

Differential conditioning produces merged long-term memory in Drosophila.

Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS-). Here, we show that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS-. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and -unpaired CS-, and be retrieved by either CS+ or CS-. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves αβ Kenyon cells and corresponding approach-directing mushroom body output neurons, in which similar-amplitude long-term depression of responses to CS+ and CS- seems to signal the mLTM. Our results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences.

Magnesium efflux from Drosophila Kenyon cells is critical for normal and diet-enhanced long-term memory.

Dietary magnesium (Mg2+) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. Here we show that Mg2+ feeding also enhances long-term memory in Drosophila. Normal and Mg2+-enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg2+-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon cells (KCs) is altered in memory-defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of KC Mg2+. We propose that regulated neuronal Mg2+ efflux is critical for normal and Mg2+-enhanced memory.

Environmental Light Is Required for Maintenance of Long-Term Memory in Drosophila.

Long-term memory (LTM) is stored as functional modifications of relevant neural circuits in the brain. A large body of evidence indicates that the initial establishment of such modifications through the process known as memory consolidation requires learning-dependent transcriptional activation and de novo protein synthesis. However, it remains poorly understood how the consolidated memory is maintained for a long period in the brain, despite constant turnover of molecular substrates. Using the Drosophila courtship conditioning assay of adult males as a memory paradigm, here, we show that in Drosophila, environmental light plays a critical role in LTM maintenance. LTM is impaired when flies are kept in constant darkness (DD) during the memory maintenance phase. Because light activates the brain neurons expressing the neuropeptide pigment-dispersing factor (Pdf), we examined the possible involvement of Pdf neurons in LTM maintenance. Temporal activation of Pdf neurons compensated for the DD-dependent LTM impairment, whereas temporal knockdown of Pdf during the memory maintenance phase impaired LTM in light/dark cycles. Furthermore, we demonstrated that the transcription factor cAMP response element-binding protein (CREB) is required in the memory center, namely, the mushroom bodies (MBs), for LTM maintenance, and Pdf signaling regulates light-dependent transcription via CREB. Our results demonstrate for the first time that universally available environmental light plays a critical role in LTM maintenance by activating the evolutionarily conserved memory modulator CREB in MBs via the Pdf signaling pathway.SIGNIFICANCE STATEMENT Temporary memory can be consolidated into long-term memory (LTM) through de novo protein synthesis and functional modifications of neuronal circuits in the brain. Once established, LTM requires continual maintenance so that it is kept for an extended period against molecular turnover and cellular reorganization that may disrupt memory traces. How is LTM maintained mechanistically? Despite the critical importance of LTM maintenance, its molecular and cellular underpinnings remain elusive. This study using Drosophila is significant because it revealed for the first time in any organism that universally available environmental light plays an essential role in LTM maintenance. Interestingly, light does so by activating the evolutionarily conserved transcription factor cAMP response element-binding protein via peptidergic signaling.

Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila.

Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. Here, we show that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, we provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation.

The ERM protein Moesin is essential for neuronal morphogenesis and long-term memory in Drosophila

Moesin is a cytoskeletal adaptor protein that plays an important role in modification of the actin cytoskeleton. Rearrangement of the actin cytoskeleton drives both neuronal morphogenesis and the structural changes in neurons that are required for long-term memory formation. Moesin has been identified as a candidate memory gene in Drosophila, however, whether it is required for memory formation has not been evaluated. Here, we investigate the role of Moesin in neuronal morphogenesis and in short- and long-term memory formation in the courtship suppression assay, a model of associative memory. We found that both knockdown and overexpression of Moesin led to defects in axon growth and guidance as well as dendritic arborization. Moreover, reduction of Moesin expression or expression of a constitutively active phosphomimetic in the adult Drosophila brain had no effect on short term memory, but prevented long-term memory formation, an effect that was independent of its role in development. These results indicate a critical role for Moesin in both neuronal morphogenesis and long-term memory formation.

Metal Binding Properties of the N-Terminus of the Functional Amyloid Orb2.

The cytoplasmic polyadenylation element binding protein (CPEB) homologue Orb2 is a functional amyloid that plays a key regulatory role for long-term memory in Drosophila. Orb2 has a glutamine, histidine-rich (Q/H-rich) domain that resembles the Q/H-rich, metal binding domain of the Hpn-like protein (Hpnl) found in Helicobacter pylori. In the present study, we used chromatography and isothermal titration calorimetry (ITC) to show that the Q/H-rich domain of Orb2 binds Ni2+ and other transition metals ions with μM affinity. Using site directed mutagenesis, we show that several histidine residues are important for binding. In particular, the H61Y mutation, which was previously shown to affect the aggregation of Orb2 in cell culture, completely inhibited metal binding of Orb2. Finally, we used thioflavin T fluorescence and electron microscopy images to show that Ni2+ binding induces the aggregating of Orb2 into structures that are distinct from the amyloid fibrils formed in the absence of Ni2+. These data suggest that transition metal binding might be important for the function of Orb2 and potentially long-term memory in Drosophila.

Neural circuits for long-term water-reward memory processing in thirsty Drosophila

The intake of water is important for the survival of all animals and drinking water can be used as a reward in thirsty animals. Here we found that thirsty Drosophila melanogaster can associate drinking water with an odour to form a protein-synthesis-dependent water-reward long-term memory (LTM). Furthermore, we found that the reinforcement of LTM requires water-responsive dopaminergic neurons projecting to the restricted region of mushroom body (MB) β′ lobe, which are different from the neurons required for the reinforcement of learning and short-term memory (STM). Synaptic output from α′β′ neurons is required for consolidation, whereas the output from γ and αβ neurons is required for the retrieval of LTM. Finally, two types of MB efferent neurons retrieve LTM from γ and αβ neurons by releasing glutamate and acetylcholine, respectively. Our results therefore cast light on the cellular and molecular mechanisms responsible for processing water-reward LTM in Drosophila.

Identification and Structural Characterization of the N-terminal Amyloid Core of Orb2 isoform A

Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, we recorded solid-state NMR and EPR data on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel β-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.

Long-Term Memory in Drosophila Is Influenced by Histone Deacetylase HDAC4 Interacting with SUMO-Conjugating Enzyme Ubc9.

HDAC4 is a potent memory repressor with overexpression of wild type or a nuclear-restricted mutant resulting in memory deficits. Interestingly, reduction of HDAC4 also impairs memory via an as yet unknown mechanism. Although histone deacetylase family members are important mediators of epigenetic mechanisms in neurons, HDAC4 is predominantly cytoplasmic in the brain and there is increasing evidence for interactions with nonhistone proteins, suggesting HDAC4 has roles beyond transcriptional regulation. To that end, we performed a genetic interaction screen in Drosophila and identified 26 genes that interacted with HDAC4, including Ubc9, the sole SUMO E2-conjugating enzyme. RNA interference-induced reduction of Ubc9 in the adult brain impaired long-term memory in the courtship suppression assay, a Drosophila model of associative memory. We also demonstrate that HDAC4 and Ubc9 interact genetically during memory formation, opening new avenues for investigating the mechanisms through which HDAC4 regulates memory formation and other neurological processes.

Molecular Basis of Orb2 Amyloidogenesis and Blockade of Memory Consolidation.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.

Insulin signaling is acutely required for long-term memory in Drosophila.

Memory formation has been shown recently to be dependent on energy status in Drosophila. A well-established energy sensor is the insulin signaling (InS) pathway. Previous studies in various animal models including human have revealed the role of insulin levels in short-term memory but its role in long-term memory remains less clear. We therefore investigated genetically the spatial and temporal role of InS using the olfactory learning and long-term memory model in Drosophila. We found that InS is involved in both learning and memory. InS in the mushroom body is required for learning and long-term memory whereas long-term memory specifically is impaired after InS signaling disruption in the ellipsoid body, where it regulates the level of p70s6k, a downstream target of InS and a marker of protein synthesis. Finally, we show also that InS is acutely required for long-term memory formation in adult flies.

Delayed Dopamine Signaling of Energy Level Builds Appetitive Long-Term Memory in Drosophila

Sensory cues relevant to a food source, such as odors, can be associated with post-ingestion signals related either to food energetic value or toxicity. Despite numerous behavioral studies, a global understanding of the mechanisms underlying these long delay associations remains out of reach. Here, we demonstrate in Drosophila that the long-term association between an odor and a nutritious sugar depends on delayed post-ingestion signaling of energy level. We show at the neural circuit level that the activity of two pairs of dopaminergic neurons is necessary and sufficient to signal energy level to the olfactory memory center. Accordingly, we have identified in these dopaminergic neurons a delayed calcium trace that correlates with appetitive long-term memory formation. Altogether, these findings demonstrate that the Drosophila brain remembers food quality through a two-step mechanism that consists of the integration of olfactory and gustatory sensory information and then post-ingestion energetic value.

Spatio-temporal in vivo recording of dCREB2 dynamics in Drosophila long-term memory processing

CREB (cAMP response element-binding protein) is an evolutionarily conserved transcription factor, playing key roles in synaptic plasticity, intrinsic excitability and long-term memory (LTM) formation. The Drosophila homologue of mammalian CREB, dCREB2, is also important for LTM. However, the spatio-temporal nature of dCREB2 activity during memory consolidation is poorly understood. Using an in vivo reporter system, we examined dCREB2 activity continuously in specific brain regions during LTM processing. Two brain regions that have been shown to be important for Drosophila LTM are the ellipsoid body (EB) and the mushroom body (MB). We found that dCREB2 reporter activity is persistently elevated in EB R2/R4m neurons, but not neighboring R3/R4d neurons, following LTM-inducing training. In multiple subsets of MB neurons, dCREB2 reporter activity is suppressed immediately following LTM-specific training, and elevated during late windows. In addition, we observed heterogeneous responses across different subsets of neurons in MB αβ lobe during LTM processing. All of these changes suggest that dCREB2 functions in both the EB and MB for LTM formation, and that this activity contributes to the process of systems consolidation.

Molecule of the month: Synaptic plasticity – Protein miRNA interactions

miRNAs (microRNAs) are small RNAs that are transcribed from intron and other non-coding RNAs. miRNAs are utilized by the cell's molecular biological machinery to help control transcription [1]. There are more than 500 known miRNAs involved in neuronal function including development, transport, and neuronal plasticity. [2] However, what of the neuronal proteins that interacts with these miRNAs? In this month's Editorial, we examine a few protein-protein interactions of proteins that interact with the miRNAs and demonstrate additional network pathways in which these proteins function in neurons. The protein product Atx2 (Ataxin-2 protein) is implicated in the human neurodegenerative disease, spinocerebellar ataxia. In studies in the brains of the fruit fly, Drosophila, Atx2 was identified as a miRNA pathway component. Both miRNA and Atx2 components were involved in long-term plasticity of synapses in these studies. Interestingly, the control of translation occurs locally in proximity to the involved synapses. In addition, Atx2 interacts with the Me31B family DEAD box helicases - DHH1 – (also termed DDX helicases) that interact with Argonaute (Ago) and affect function of miRNAs [1, 3] (Figure 1). Figure 1 Protein interactions with Atx2, Ago, and DDX. In this figure, line-colors and various interactions with other genes are red Down-regulation, green Up-regulation, beige Regulation, purple Co-expression, brown Physical Interaction, turquoise dotted Predicted ... The contributions of miRNAs to synaptic plasticity are extensive study. For example, CREB (cAMP responsive element binding protein), MECP2 (methyl CpG binding protein 2), and FMRP (fragile X mental retardation protein) interact with miRNAs in their role as mediators of synaptic plasticity within dendrites. The RISC machinery is utilized post-DICER action, producing the miRNAs in situ as above. Post-translation inhibition occurs for these proteins following bursts of synaptic activity mediated by the rapid increase of calcium levels. These activities occur in mouse forebrain and are involved in longterm memory in Drosophila [5] (Figure 2). Figure 2 Protein interactions with CREB, MECP2, and FMRP. In this figure, line-colors and various interactions with other genes are red Down-regulation, green Up-regulation, beige Regulation, purple Co-expression, brown Physical Interaction, turquoise dotted Predicted ... The two figures illustrate various gene interactions, up to 100 [4]. It is left as a puzzle for the interested reader to identify the various genes and their functions in the figures [6].

Mushroom-Body Memories: An Obituary Prematurely Written?

Studies on insect olfactory learning have established the mushroom bodies as key brain structures for the formation of long-term memory (LTM). Two new neurons in the fly brain are reported now as essential sites for LTM formation, while mushroom bodies are claimed to be unnecessary to this end.

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.