A Programmable Ontology Encompassing the Functional Logic of the Drosophila Brain

The Drosophila brain has only a fraction of the number of neurons of higher organisms such as mice. Yet the sheer complexity of its neural circuits recently revealed by large connectomics datasets suggests that computationally modeling the function of fruit fly brain at this scale posits significant challenges. To address these challenges, we present here a programmable ontology that expands the scope of the current Drosophila brain anatomy ontologies to encompass the functional logic of the fly brain. The programmable ontology provides a language not only for defining functional circuit motifs but also for programmatically exploring their functional logic. To achieve this goal, we tightly integrated the programmable ontology with the workflow of the interactive FlyBrainLab computing platform. As part of the programmable ontology, we developed NeuroNLP++, a web application that supports free-form English queries for constructing functional brain circuits fully anchored on the available connectome/synaptome datasets, and the published worldwide literature. In addition, we present a methodology for including a model of the space of odorants into the programmable ontology, and for modeling olfactory sensory circuits of the antenna of the fruit fly brain that detect odorant sources. Furthermore, we describe a methodology for modeling the functional logic of the antennal lobe circuit consisting of massive local feedback loops, a characteristic feature observed across Drosophila brain regions. Finally, using a circuit library, we demonstrate the power of our methodology for interactively exploring the functional logic of the massive number of feedback loops in the antennal lobe.

Perception of Daily Time: Insights from the Fruit Flies

We create mental maps of the space that surrounds us; our brains also compute time—in particular, the time of day. Visual, thermal, social, and other cues tune the clock-like timekeeper. Consequently, the internal clock synchronizes with the external day-night cycles. In fact, daylength itself varies, causing the change of seasons and forcing our brain clock to accommodate layers of plasticity. However, the core of the clock, i.e., its molecular underpinnings, are highly resistant to perturbations, while the way animals adapt to the daily and annual time shows tremendous biological diversity. How can this be achieved? In this review, we will focus on 75 pairs of clock neurons in the Drosophila brain to understand how a small neural network perceives and responds to the time of the day, and the time of the year.

The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology

Nab2 encodes the Drosophila melanogaster member of a conserved family of zinc finger polyadenosine RNA-binding proteins (RBPs) linked to multiple steps in post-transcriptional regulation. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships remain unidentified. Here, we present evidence that Nab2 genetically interacts with Ataxin-2 (Atx2), which encodes a neuronal translational regulator, and that these factors coordinately regulate neuronal morphology, circadian behavior, and adult viability. We then present the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using RNA immunoprecipitation-sequencing (RIP-Seq). Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g., drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g., Arpc2 and tea) promise insight into neuronal functions of, and genetic interactions between, each RBP. Consistent with prior biochemical studies, Nab2-associated neuronal RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. These data support a model where Nab2 functionally opposes Atx2 in neurons, demonstrate Nab2 shares associated neuronal RNAs with Atx2, and reveal Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.

Lipophorin receptors regulate mushroom bodies development and participate in learning, memory, and sleep in flies

Drosophila melanogaster Lipophorin Receptors, LpR1 and LpR2, mediate lipid uptake. The orthologs of these receptors in vertebrates, ApoER2 and VLDL-R, bind Reelin, a glycoprotein not present in flies. These receptors are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain. It is currently unknown whether LpRs play similar roles in the Drosophila brain. We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the Mushroom Bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurites. This effect depends on LpRs and Dab, the Drosophila ortholog of the reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin. Overall, these findings demonstrate that LpRs contribute to MB development and function, supporting the existence of LpR-dependent signaling in Drosophila.

3D reconstruction of cell nuclei in a full Drosophila brain

We reconstructed all cell nuclei in a 3D image of a Drosophila brain acquired by serial section electron microscopy (EM). The total number of nuclei is approximately 133,000, at least 87% of which belong to neurons. Neuronal nuclei vary from several hundred down to roughly 5 cubic micrometers. Glial nuclei can be even smaller. The optic lobes contain more than two times the number of cells than the central brain. Our nuclear reconstruction serves as a spatial map and index to the cells in a Drosophila brain.

TAMI-43. HYPOXIC REGULATION OF GLIOBLASTOMA TUMOR GROWTH THROUGH L(3)MBTL1

Glioblastoma (GBM) is the deadliest and most common of all primary brain tumors. Drosophila brain tumor models have uncovered signaling pathways regulating tumor growth that are highly conserved in GBM. Our search for a novel tumor suppressor using Drosophila led to Lethal (3) malignant brain tumor [l(3)mbt], temperature-sensitive mutants of which cause neuroepithelial tumor-like overproliferation in optic lobes. dL(3)MBT and its human orthologs L3MBTL1-L3MBTL4 all harbor Malignant Brain Tumor (MBT) domains that recognize methylated lysines on histone tails. Like dL(3)MBT, hL(3)MBTL1 acts as a chromatin compaction factor that represses transcription and inhibits cytokinesis in GBM cell lines. The highly hypoxic tumor microenvironment (TME) in GBM drives its progression, recurrence, and therapeutic resistance. However, it remains unclear if L(3)MBTL1 is regulated by TME cues to promote GBM growth. Based on this knowledge gap and our preliminary data, we hypothesize that hypoxia directly regulates L(3)MBTL1 in favor of GBM growth. Analysis of TCGA data for IDH-wildtype gliomas revealed that L3MBTL1 gene expression is downregulated in GBM, which are necrotic and severely hypoxic, compared to histologic grade 2/3 gliomas, which do not contain necrosis, indicating that hypoxia could potentially suppress L3MBTL1 to enhance glioma progression. TCGA data also revealed a number of HIF pathway and hypoxia-inducible genes strongly correlating with L3MBTL1 expression, including HIF1a and VHL. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to hypoxia (1% O2 for 24hrs) and found that L3MBTL1 protein levels were suppressed compared to normoxia (21%). Under these same conditions, we found more rapid cell proliferation under hypoxia. Exploration of hypoxic TME regulation of the novel tumor suppressor L3MBTL1 in glioma progression has the potential to uncover novel mechanisms involving epigenetic modulation and potentially new therapeutic strategies.

Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila

Parkinson’s disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of alpha-Synuclein (aSyn) protein. The process of aSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on aSyn conformation remains poorly understood. To study aSyn pathogenic modifications in response to PQ, we exposed Drosophila expressing human aSyn to a chronic PQ protocol. We first showed that PQ exposure and aSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of aSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble aSyn in highly denaturating buffer but did not increase aSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of aSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, our results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on aSyn driven pathology.

Identification of Ultrastructural Signatures of Sleep and Wake in the Fly Brain

The cellular consequences of sleep loss are poorly characterized. In the pyramidal neurons of mouse frontal cortex we found that mitochondria and secondary lysosomes occupy a larger proportion of the cytoplasm after chronic sleep restriction compared to sleep, consistent with increased cellular burden due to extended wake. For each morphological parameter the within-animal variance was high, suggesting that the effects of sleep and sleep loss vary greatly among neurons. However, the analysis was based on 4-5 mice/group and a single section/cell. Here, we applied serial block-face scanning electron microscopy to identify signatures of sleep and sleep loss in the Drosophila brain. Stacks of images were acquired and used to obtain full 3D reconstructions of the cytoplasm and nucleus of 263 Kenyon cells from adult flies collected after a night of sleep (S) or after 11 hours (SD11) or 35 hours (SD35) of sleep deprivation (9 flies/group). Relative to S flies, SD35 flies showed increased density of dark clusters of chromatin and of Golgi apparata and a trend increase in the percent of cell volume occupied by mitochondria, consistent with increased need for energy and protein supply during extended wake. Logistic regression models could assign each neuron to the correct experimental group with good accuracy, but in each cell nuclear and cytoplasmic changes were poorly correlated, and within-fly variance was substantial in all experimental groups. Together, these results support the presence of ultrastructural signatures of sleep and sleep loss but underscore the complexity of their effects at the single-cell level.