Parallel processing of olfactory memories in Drosophila

One of the hallmarks of both memory and the underlying synaptic plasticity is that they each rely on short-lived and longer-lived forms. Short-lived memory is thought to rely on modification to existing proteins, whereas long-term memory requires induction of new gene expression. The most common view is that these two processes rely on signaling mechanisms within the same neurons. We recently demonstrated a dissection of the signaling requirements for short and long-lived memory into distinct sets of neurons. Using an aversive olfactory conditioning task in Drosophila, we found that cAMP signaling in different neuron cell types is sufficient to support short or long-term memory independently

Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders

Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases

YfmK is a Novel Nε-lysine Acetyltransferase that Directly Acetylates the Histone-like Protein HBsu in Bacillus Subtilis

Recently, Ne-lysine acetylation was realized to be a prevalent bacterial post-translational modification (PTM), contrary to the historical notion that this was a rare occurrence. Acetylation can impact protein function in multiple ways, by modifying conformation, interactions, subcellular localization or activity. In bacteria, hundreds of proteins are known to be acetylated, including those involved essential processes such as DNA replication, nucleoid organization, translation, cell shape, central carbon metabolism, and even several virulence factors. Despite the growing recognition that numerous proteins are acetylated, the biological significance of the vast majority of these modifications in any bacteria remains largely unknown. Previously, we characterized the Bacillus subtilis acetylome and found that the essential histone-like protein HBsu contains seven novel acetylation sites in vivo. HBsu is a bacterial nucleoid-associated protein, which is largely responsible for chromosome compaction and the coordination of DNA processes. Despite the lack of sequence or structural homology, it is generally considered to be a functional homolog of eukaryotic histones. We investigated whether acetylation is a regulatory component of the function of HBsu in nucleoid compaction. Using mutations that mimic the acetylated and unacetylated forms of the protein, we showed that the inability to acetylate key HBsu lysine residues results in a more compacted nucleoid. We further investigated the mechanism of HBsu acetylation. By screening knockouts of the ~50 putative Gcn5-N-acetyltransferase (GNAT)-domain encoding genes in B. subtilis for their effects on DNA compaction, five candidates were identified that may encode transacetylases acting on HBsu. Genetic bypass experiments demonstrated that two of these, YfmK and YdgE, can acetylate HBsu and their potential sites of action on HBsu were identified. Additionally, purified YfmK was able to directly acetylate HBsu in vitro,suggesting that it is the second identified protein acetyltransferase in B. subtilis. We propose that at least one physiological function of the acetylation of HBsu at key lysine residues is to regulate nucleoid compaction, analogously to the role of histone acetylation in eukaryotes. With the alarming rise in antibiotic resistance, the need to develop novel therapeutics is critical. Bacterial protein acetylation represents a world of untapped potential that may uncover new drug targets to replace or supplement our antiquated antibiotic arsenal. With proper study, the enzymes involved in regulation (i.e. acetylases and deacetylases) or the acetylated form of a key protein (i.e. virulence factors, essential genes, etc.) may provide valuable, druggable targets. The targeting of bacterial protein acetylation is a practical option, as targeting enzymes involved in acetylation regulation has been successful in treatment of certain cancers, latent viral and fungal infections

Expression of pheromone binding proteins during antennal development in the gypsy moth Lymantria dispar

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

MicroRNA-276a Functions in Ellipsoid Body and Mushroom Body Neurons for Naive and Conditioned Olfactory Avoidance in Drosophila

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 in Drosophila that functions in distinct circuits for naive odor responses and conditioned odor memory. Drosophila olfactory 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

Sequencing the Connectome

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC ("barcoding of individual neuronal connections"), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale--sequencing billions of nucleotides per day is now routine--is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research

The role of transposable elements in health and diseases of the central nervous system

First discovered in maize by Barbara McClintock in the 1940s, transposable elements (TEs) are DNA sequences that in some cases have the ability to move along chromosomes or "transpose" in the genome. This revolutionary finding was initially met with resistance by the scientific community and viewed by some as heretical. A large body of knowledge has accumulated over the last 60 years on the biology of TEs. Indeed, it is now known that TEs can generate genomic instability and reconfigure gene expression networks both in the germline and somatic cells. This review highlights recent findings on the role of TEs in health and diseases of the CNS, which were presented at the 2013 Society for Neuroscience meeting. The work of the speakers in this symposium shows that TEs are expressed and active in the brain, challenging the dogma that neuronal genomes are static and revealing that they are susceptible to somatic genomic alterations. These new findings on TE expression and function in the CNS have major implications for understanding the neuroplasticity of the brain, which could hypothetically have a role in shaping individual behavior and contribute to vulnerability to disease

Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS

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

Nuclear Transcriptomes of the Seven Neuronal Cell Types That Constitute the Drosophila Mushroom Bodies

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 (gamma, alpha'/beta' and alpha/beta) and 7 cell subtypes (gammad, gammam, alpha'/beta'ap, alpha'/beta'm, alpha/betap, alpha/betas and alpha/betac) 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 alpha/beta class marker Fas2 and the alpha'/beta' 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