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Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer’s disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator., Author summary Loss of nuclear function of TDP-43 and its mislocalization into cytoplasmic inclusions are central features to a suite of neurodegenerative disorders. We screened 2700 Drosophila genes to identify downstream mediators that suppress an age-dependent motor dysfunction phenotype when they are knocked down by RNA interference. We identified both previously implicated pathways and several novel genes whose knock down is sufficient to dramatically and robustly rescue TDP-43 toxicity both in neuronal and glial contexts. Notably, we demonstrate that SF2/SRSF1, which was previously reported as a suppressor of C9orf72 hexanucleotide expansion repeat toxicity, also is a potent suppressor of TDP-43 mediated neurodegeneration.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome., Author summary The genomes of animals and plants contain a vast quantity of so called “junk DNA” that does not provide obvious function to the organism. But it is increasingly clear that “junk DNA” has more important contributions to both normal function and to dysfunction that can cause disease. A great deal of this “junk” is made up by so called “retrotransposons”, which have many similarities to viruses. Many of them are actually evolutionary relatives of retroviruses, and are able to replicate themselves and insert new copies into the host genome. But unlike retroviruses, which are infectious and replicate by moving from one cell to another, retrotransposons replicate within one cell and re-insert their new copies back into the chromosomes of the cell in which they originated. In this publication, we studied replication of gypsy, which is a well known retrotransposon in fruit flies. We found that gypsy has the ability to replicate within a cell, like a retrotransposon and also has the ability to replicate by moving to a new cell like a retrovirus.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-retrotransposons are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) that derive from an exogenous retrovirus that have integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. The fact that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration raises the question of whether their replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.

A hallmark of neurodegenerative disease is focal onset of pathological protein aggregation, followed by progressive spread of pathology to connected brain regions. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), pathology is often associated with aggregation of TAR DNA-binding protein 43 (TDP-43). Although aggregated TDP-43 protein moves between cells, it is not clear whether and how this movement propagates the degeneration. Here, we have established a Drosophila model of human TDP-43 in which we initiated toxic expression of human TDP-43 focally within small groups of glial cells. We found that this focal onset kills adjacent neurons. Surprisingly, we show that this spreading death is caused by an endogenous retrovirus within the glia, which leads to DNA damage and death in adjacent neurons. These findings suggest a possible mechanism by which human retroviruses such as HERV-K might contribute to TDP-43-mediated propagation of neurodegeneration.

Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses., Using gypsy elements in aging fruit flies, this paper describes cellular labeling of endogenous retrovirus replication (CLEVR), an in vivo reporter system that allows the tracing of virus or LTR retrotransposon replication in tissues.

SummaryAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several inherited pathogenic mutations have been identified as causative, the vast majority of cases are sporadic with no family history of disease. Thus, for the majority of ALS cases, a specific causal abnormality is not known and the disease may be a product of multiple inter-related pathways contributing to varying degrees in different ALS patients. Using unsupervised machine learning algorithms, we stratified the transcriptomes of 148 ALS decedent cortex tissue samples into three distinct and robust molecular subtypes. The largest cluster, identified in 61% of patient samples, displayed hallmarks of oxidative and proteotoxic stress. Another 20% of the ALS patient samples exhibited high levels of retrotransposon expression and other signatures of TDP-43 dysfunction. Finally, a third group showed predominant signatures of glial activation (19%). Together these results demonstrate that at least three distinct molecular signatures contribute to ALS disease. While multiple dysregulated components and pathways comprising these clusters have previously been implicated in ALS pathogenesis, unbiased analysis of this large survey demonstrated that sporadic ALS patient tissues can be segregated into distinct molecular subsets.

The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The Drosophila melanogaster MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α′/β′ and α/β) and 7 cell subtypes (γd, γm, α′/β′ap, α′/β′m, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker Fas2 and the α′/β′ class marker trio. Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the Drosophila MB provides a valuable resource for the fly neuroscience community.

Evidence is rapidly mounting that transposable element expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, successful reporters of LINE element mobilization have been in use for some time, but similar strategies have not been developed to report Long Terminal Repeat (LTR) retrotransposons and endogenous retroviruses. We describe Cellular Labeling of Endogenous Virus Replication (CLEVR), which reports replication of the gypsy element in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR-retrotransposons, endogenous retroviruses and exogenous retroviruses.

Like the famous Collyer’s mansion in NY, our genomes have accumulated vast quantities of sequences that have been referred to as ‘junk DNA’, much of which consists of retrotransposons. A recent literature establishes the phenomenology that many retrotransposons become expressed at progressively higher levels during the course of normal aging. This seems to reflect gradual loss of heterochromatin in old age. In addition, retrotransposons appear to be precociously expressed in brains of younger animals that are experiencing neurodegenerative decline. Although it is difficult to distinguish cause from consequence, several recent studies support the contention that retrotransposon expression, and even perhaps transposition, causally contribute to both the normal deterioration seen with age and to the precipitous decline in some neurodegenerative disorders. This may reflect a two hit model in which normal age-dependent loss of heterochromatin synergizes with a disruption to post transcriptional silencing of RTEs caused by genetic and environmental stress.

Gene interactions are acknowledged to be a likely source of missing heritability in large-scale genetic studies of complex neurological phenotypes. However, involvement of rare variants, de novo mutations, genetic lesions that are not easily detected with commonly used methods and epigenetic factors also are possible explanations. We used a laboratory evolution study to investigate the modulatory effects of background genetic variation on the phenotypic effect size of a null mutation with known impact on olfactory learning. To accomplish this, we first established a population that contained variation at just 23 loci and used selection to evolve suppression of the learning defect seen with null mutations in the rutabaga adenylyl cyclase. We thus biased the system to favor relatively simplified outcomes by choosing a Mendelian trait and by restricting the genetic variation segregating in the population. This experimental design also assures that the causal effects are among the known 23 segregating loci. We observe a robust response to selection that requires the presence of the 23 variants. Analyses of the underlying genotypes showed that interactions between more than two loci are likely to be involved in explaining the selection response, with implications for the missing heritability problem.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD., Author summary Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.

DHP and CRMP proteins comprise a family of structurally similar proteins that perform divergent functions, DHP in pyrimidine catabolism in most organisms and CRMP in neuronal dynamics in animals. In vertebrates, one DHP and five CRMP proteins are products of six genes; however, Drosophila melanogaster has a single CRMP gene that encodes one DHP and one CRMP protein through tissue-specific, alternative splicing of a pair of paralogous exons. The proteins derived from the fly gene are identical over 90% of their lengths, suggesting that unique, novel functions of these proteins derive from the segment corresponding to the paralogous exons. Functional homologies of the Drosophila and mammalian CRMP proteins are revealed by several types of evidence. Loss-of-function CRMP mutation modifies both Ras and Rac misexpression phenotypes during fly eye development in a manner that is consistent with the roles of CRMP in Ras and Rac signaling pathways in mammalian neurons. In both mice and flies, CRMP mutation impairs learning and memory. CRMP mutant flies are defective in circadian activity rhythm. Thus, DHP and CRMP proteins are derived by different processes in flies (tissue-specific, alternative splicing of paralogous exons of a single gene) and vertebrates (tissue-specific expression of different genes), indicating that diverse genetic mechanisms have mediated the evolution of this protein family in animals.

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila : ( i ) DPM neurons were 5HT-antibody immunopositive; ( ii ) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; ( iii ) knocking down the expression of d5HT1A serotonin receptors in α/β MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.

Individuals that experience traumatic events may develop persistent post-traumatic stress disorder (PTSD). Patients with this disorder are commonly treated with exposure therapy, which has had limited long-term success. In experimental neurobiology, fear extinction is a model for exposure therapy. In this behavioral paradigm, animals are repeatedly exposed in a safe environment to the fearful stimulus, which leads to greatly reduced fear. Studying animal models of extinction already has lead to better therapeutic strategies and development of new candidate drugs. Lack of a powerful genetic model of extinction, however, has limited progress in identifying underlying molecular and genetic factors. In this study, we established a robust behavioral paradigm to study the short term effect (acquisition) of extinction in Drosophila melanogaster. We focused on the extinction of olfactory aversive one-day memory with a task that has been the main workhorse for genetics of memory in flies. Using this paradigm, we demonstrate that extinction can inhibit each of two genetically distinct forms of consolidated memory. We then used a series of single-gene mutants with known impact on associative learning, to examine effects on extinction. We find that extinction is intact in each of these mutants, suggesting that extinction learning relies on different molecular mechanisms than does Pavlovian learning.

Summary Background A common feature of memory and its underlying synaptic plasticity is that each can be dissected into short-lived forms involving modification or trafficking of existing proteins and long-term forms that require new gene expression. An underlying assumption of this cellular view of memory consolidation is that these different mechanisms occur within a single neuron. At the neuroanatomical level, however, different temporal stages of memory can engage distinct neural circuits, a notion that has not been conceptually integrated with the cellular view. Results Here, we investigated this issue in the context of aversive Pavlovian olfactory memory in Drosophila . Previous studies have demonstrated a central role for cAMP signaling in the mushroom body (MB). The Ca 2+ -responsive adenylyl cyclase RUTABAGA is believed to be a coincidence detector in γ neurons, one of the three principle classes of MB Kenyon cells. We were able to separately restore short-term or long-term memory to a rutabaga mutant with expression of rutabaga in different subsets of MB neurons. Conclusions Our findings suggest a model in which the learning experience initiates two parallel associations: a short-lived trace in MB γ neurons, and a long-lived trace in α/β neurons.

Unlike most organ systems, which have evolved to maintain homeostasis, the brain has been selected to sense and adapt to environmental stimuli by constantly altering interactions in a gene network that functions within a larger neural network. This unique feature of the central nervous system provides a remarkable plasticity of behavior, but also makes experimental investigations challenging. Each experimental intervention ramifies through both gene and neural networks, resulting in unpredicted and sometimes confusing phenotypic adaptations. Experimental dissection of mechanisms underlying behavioral plasticity ultimately must accomplish an integration across many levels of biological organization, including genetic pathways acting within individual neurons, neural network interactions which feed back to gene function, and phenotypic observations at the behavioral level. This dissection will be more easily accomplished for model systems such as Drosophila, which, compared with mammals, have relatively simple and manipulable nervous systems and genomes. The evolutionary conservation of behavioral phenotype and the underlying gene function ensures that much of what we learn in such model systems will be relevant to human cognition. In this essay, we have not attempted to review the entire Drosophila memory field. Instead, we have tried to discuss particular findings that provide some level of intellectual synthesis across three levels of biological organization: behavior, neural circuitry and biochemical pathways. We have attempted to use this integrative approach to evaluate distinct mechanistic hypotheses, and to propose critical experiments that will advance this field.

Background: Memory formation after olfactory learning in Drosophila displays behavioral and molecular properties similar to those of other species. Particularly, long-term memory requires CREB-dependent transcription, suggesting the regulation of "downstream" genes. At the cellular level, long-lasting synaptic plasticity in many species also appears to depend on CREB-mediated gene transcription and subsequent structural and functional modification of relevant synapses. To date, little is known about the molecular-genetic mechanisms that contribute to this process during memory formation. Results: We used two complementary strategies to identify these genes. From DNA microarrays, we identified 42 candidate memory genes that appear to be transcriptionally regulated in normal flies during memory formation. Via mutagenesis, we have independently identified 60 mutants with defective long-term memory and have defined molecular lesions for 58 of these. The pumilio translational repressor was found from both approaches, along with six additional genes with established roles in local control of mRNA translation. In vivo disruptions of four genes— staufen , pumilio , oskar , and eIF-5C —yield defective memory. Conclusions: Convergent findings from our behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio / staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification.

T. Dobzhansky (1973) has been credited with saying: 'nothing in biology makes sense, except in the light of evolution'. The evolutionary conservation of gene function, as well as remarkable conservation of elemental behavioral mechanisms, guarantees that much of what we learn in one organism will inform our understanding of behavior in all animals, including humans. This insight has permitted behavior-geneticists to choose organisms based on experimental tractability for a given scientific question. IBANGS as a society has clearly embraced this Dobzhanskian worldview. As a result, the intellectual synergy of cross-species behavior-genetic analysis was palpable at the IBANGS meeting in Tours, France.

Summary Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist., Highlights ► Orb2A and Orb2B function by distinct mechanisms in long-term memory ► Orb2A function requires its glutamine-rich domain but not its RNA-binding domain ► Orb2B function requires the RNA-binding domain but not its glutamine-rich domain ► Neuronal activation induces Orb2 heteromers dependent upon Orb2A’s glutamine-rich domain, RNA binding proteins at synapses may underlie plasticity, but their role is not well understood. Krüttner et al. show here that Orb2 isoforms affect memory by distinct mechanisms: through its RNA-binding domain or by forming heterocomplexes in a manner dependent on its glutamine-rich region and neuronal activity.

The Mobile Genetic Elements and Genome Evolution conference was hosted by Keystone Symposia in Santa Fe, NM USA, 9 March through 14 March 2014. The goal of this conference was to bring together scientists from around the world who study transposable elements in diverse organisms and researchers who study the impact these elements have on genome evolution. The meeting included over 200 scientists who participated through poster presentations, short talks selected from abstracts, and invited speakers. The talks were organized into eight sessions and two workshops. The topics varied from diverse mechanisms of mobilization to the evolution of genomes and their defense strategies against transposable elements.

The Drosophila memory gene amnesiac is expressed in neurons that project to mushroom body axons. Blockade of synaptic transmission in the amnesiac-expressing cells disrupts memory, but not learning, suggesting presynaptic and postsynaptic sites for memory formation.

MicroRNA (miRNA)-mediated gene regulation plays a key role in brain development and function. But there are few cases in which the roles of individual miRNAs have been elucidated in behaving animals. We report a miR-276a::DopR regulatory module inDrosophilathat functions in distinct circuits for naive odor responses and conditioned odor memory.Drosophilaolfactory aversive memory involves convergence of the odors (conditioned stimulus) and the electric shock (unconditioned stimulus) in mushroom body (MB) neurons. Dopamine receptor DopR mediates the unconditioned stimulus inputs onto MB. Distinct dopaminergic neurons also innervate ellipsoid body (EB), where DopR function modulates arousal to external stimuli. We demonstrate that miR-276a is required in MB neurons for memory formation and in EB for naive responses to odors. Both roles of miR-276a are mediated by tuning DopR expression. The dual role of this miR-276a::DopR genetic module in these two neural circuits highlights the importance of miRNA-mediated gene regulation within distinct circuits underlying both naive behavioral responses and memory.

In Drosophila, the primary determinant of anterior pattern is the gradient morphogen bicoid (bcd), a homeodomain protein that binds DNA and transcriptionally activates target genes at different threshold concentrations. Here we present evidence that bcd also binds RNA and acts as a translational repressor to generate an opposing gradient of the homeodomain protein caudal (cad). RNA binding by bcd seems to involve direct interactions between the bcd homeodomain and discrete target sequences within the 3' untranslated region of the cad messenger RNA and to block the initiation of cad translation.

We report the surprising finding that several transposable elements are highly active in Drosophila brain during normal aging. We also show that mutations in Drosophila Argonaute 2 (dAgo2) exhibit exacerbated transposon expression in brain, progressive and age-dependent memory impairment and shortened lifespan. These findings suggest that transposon activation may contribute to age-dependent loss of neuronal function.

Background: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants. Methods: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure. Results: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects. Conclusions: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

Surgical, pharmacological and genetic lesion studies have revealed distinct anatomical sites involved with different forms of learning. Studies of patients with localized brain damage and work in rodent model systems, for example, have shown that the hippocampal formation participates in acquisition of declarative tasks but is not the site of their long-term storage1,2. Such lesions are usually irreversible, however, which has limited their use for dissecting the temporal processes of acquisition, storage and retrieval of memories3,4. Studies in bees and flies have similarly revealed a distinct anatomical region of the insect brain, the mushroom body, that is involved specifically in olfactory associative learning5,6. We have used a temperature-sensitive dynamin transgene, which disrupts synaptic transmission reversibly and on the time-scale of minutes7, to investigate the temporal requirements for ongoing neural activity during memory formation. Here we show that synaptic transmission from mushroom body neurons is required during memory retrieval but not during acquisition or storage. We propose that the hebbian processes underlying olfactory associative learning reside in mushroom body dendrites or upstream of the mushroom body and that the resulting alterations in synaptic strength modulate mushroom body output during memory retrieval.

To understand how Mycobacterium tuberculosis survives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about sigma factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that sigma A is the essential housekeeping sigma factor and the alternative sigma factor sigma B, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in sigma A prevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatis and M. tuberculosis that are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosis gene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosis genes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosis grown in vitro with those found in bacteria growing in human macrophages.

Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage., Author Summary The Drosophila Pumilio (Pum) protein was originally identified as a translational control factor for embryo patterning. Subsequent studies have identified Pum's role in multiple biological processes, including the maintenance of germline stem cell, the proliferation and migration of primordial germ cells, olfactory leaning and memory, and synaptic plasticity. Pum is highly conserved across phyla, i.e., from worm to human; however, the mRNA targets of Pum within each tissue and organism are largely unknown. On the other hand, the prediction of RNA binding sites remains a hard question in the computational field. We were interested in finding Pum targets in the nervous system using fruit flies as a model organism. To accomplish this, we used the few Pum binding sequences that had previously been shown in vivo as “training sequences” to construct bioinformatic models of the Pum binding site. We then predicted a few Pum mRNA targets among the genes known to function in neuronal synapses. We then used a combination of “golden standards” to verify these predictions: a biochemical assay called gel shifts, and in vivo functional assays both in embryo and neurons. With these approaches, we successfully confirmed one of the targets as Dlg, which is the Drosophila ortholog of human PSD95. Therefore, we present a complete story from computational study to real biological functions.

We have identified 2 olfactory specific proteins in the gypsy moth Lymantria dispar that are uniquely associated with the male antennae, the principal olfactory organs of this animal. These proteins were the major soluble protein components of the olfactory sensilla, present in equivalent amounts. Both proteins comigrated on SDS-PAGE, showing an apparent molecular mass of 15,000 Da but migrated separately on non-SDS- PAGE, indicating differences in net charge. N-terminal amino acid sequence analysis showed that the 2 proteins share 50% identity, indicating that they are genetically distinct homologs. Both proteins bound the L. dispar sexpheromone, associated with antisera prepared against the previously identified phermone-binding protein (PBP) of the moth Antheraea polyphemus, and shared sequence identity with the A. polyphemus PBP. These 2 proteins are therefore identified as L. dispar PBPs and are termed PBP1 and PBP2 based on their migration differences on non-SDS-PAGE. It is estimated that PBP1 and PBP2 are present in the sensilla lumen at a combined concentration of 13.4 mM. The expression of the L. dispar PBPs was examined during the 11 d development of the adult antenna. PBP1 and PBP2 were first detected by non-SDS-PAGE analysis and Coomassie blue staining 3 d before adult eclosion, on day A-3. Levels increased, reaching a plateau on day A-1 that continued into adult life. In vivo labeling studies indicated that the rate of PBP synthesis increased from A-3 to a plateau on A-2, where it remained into adult life. In vitro translations of antennal mRNAs indicated that translatable PBP mRNA was available at a very low level on day A-4, increased slightly on A-3 and dramatically on A-2, and remained at a high level into adult life. PBP mRNA represented the major translatable mRNA in the antenna during this period. It was estimated that the PBPs undergo a combined steady-state turnover of 8 x 10(7) molecules/hr/sensillum. Cursory in vivo and in vitro translation studies of antennal mRNA from A. polyphemus and Manduca sexta showed similar temporal patterns of PBP expression, suggesting that the L. dispar observations are general.

This article has been retracted at the request of the authors. Please see http://www.elsevier.com/locate/withdrawalpolicy. The Publisher apologizes for any inconvenience this may cause. Reason: Our identification of the radish gene as a phospholipase-A2 was based on two lines of evidence: first, we reported that a transposon insertion (C133) in a phospholipase-A2 gene failed to complement the memory defect of the original radish1 allele. Second, we showed that transgenic expression of the PLA2 cDNA within the C133 enhancer trap expression pattern rescued the memory defect of C133 mutant animals. After publication, we learned via personal communication with William Quinn at MIT that his laboratory had evidence that radish1 encoded a different gene. We then re-examined the phenotype of the C133 strain and were unable to reproduce our original finding that the transposon insertion causes a memory defect. After multiple attempts at behavioral characterization with the C133 transposon crossed into a variety of genetic backgrounds, we have come to the conclusion that our original claim was incorrect. The original genetic background upon which we obtained the C133 insertion does indeed exhibit defective anesthesia-resistant memory, but this memory defect is not caused by the C133 transposon insertion in the PLA2. Although we do not fully understand how this error occurred, our recent experiments force us to conclude that C133 is not allelic to radish1, and that the C133 P-element insertion was not responsible for the mutant phenotype we saw. This error directly undermines the conclusion that radish encodes a PLA2 but does not impact the neuro-anatomical studies of the C133 expression pattern. Although we have communicated these findings to Quinn (1) and to many other colleagues, we felt it was essential to make this error known more broadly. We deeply regret any confusion that this has caused to the field. (1) Folkers, E., Waddel, S., and Quinn W.G. (2006) The Drosophila radish gene encodes a protein required for anesthesia-resistant memory. Proc. Natl. Acad. Sci. USA 103 (46): 17496-17500.