miR-137 regulates PTP61F, affecting insulin signaling, metabolic homeostasis, and starvation resistance in Drosophila.

miR-137 is a highly conserved brain-enriched microRNA (miRNA) that has been associated with neuronal function and proliferation. Here, we show that Drosophila miR-137 null mutants display increased body weight with enhanced triglyceride content and decreased locomotor activity. In addition, when challenged by nutrient deprivation, miR-137 mutants exhibit reduced motivation to feed and prolonged survival. We show through genetic epistasis and rescue experiments that this starvation resistance is due to a disruption in insulin signaling. Our studies further show that miR-137 null mutants exhibit a drastic reduction in levels of the phosphorylated/activated insulin receptor, InR (InR-P). We investigated if this is due to the predicted miR-137 target, Protein Tyrosine Phosphatase 61F (PTP61F), ortholog of mammalian TC-PTP/PTP1B, which are known to dephosphorylate InR-P. Indeed, levels of an endogenously tagged GFP-PTP61F are significantly elevated in miR-137 null mutants, and we show that overexpression of PTP61F alone is sufficient to mimic many of the metabolic phenotypes of miR-137 mutants. Finally, we knocked-down elevated levels of PTP61F in the miR-137 null mutant background and show that this rescues levels of InR-P, restores normal body weight and triglyceride content, starvation sensitivity, as well as attenuates locomotor and starvation-induced feeding defects. Our study supports a model in which miR-137 is critical for dampening levels of PTP61F, thereby maintaining normal insulin signaling and energy homeostasis.

Linking aß42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model.

Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly. ß-amyloid (Aß) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aß has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. Here, we show that overexpression of human Aß42 in a Drosophila model indeed induces increased neuronal activity. We found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, we find that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aß42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, we transgenically over-expressed Kv4 to near wild-type levels in Aß42-expressing animals. We show that restoration of Kv4 attenuated age-dependent learning and locomotor deficits, slowed the onset of neurodegeneration, and partially rescued premature death seen in Aß42-expressing animals. We conclude that Aß42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aß42-Drosophila model, and possibly in AD.

SIDL interacts with the dendritic targeting motif of Shal (K(v)4) K+ channels in Drosophila.

Shal K(+) (K(v)4) channels in mammalian neurons have been shown to be localized exclusively to somato-dendritic regions of neurons, where they function as key determinants of dendritic excitability. To gain insight into the mechanisms underlying dendritic localization of K(v)4 channels, we use Drosophila melanogaster as our model system. We show that Shal K(+) channels display a conserved somato-dendritic localization in vivo in Drosophila. From a yeast-2-hybrid screen, we identify the novel interactor, SIDL (for Shal Interactor of Di-Leucine Motif), as the first target protein reported to bind the highly conserved di-leucine motif (LL-motif) implicated in dendritic targeting. We show that SIDL is expressed primarily in the nervous system, co-localizes with GFP-Shal channels in neurons, and interacts specifically with the LL-motif of Drosophila and mouse Shal channels. We disrupt the Shal-SIDL interaction by mutating the LL-motif on Shal channels, and show that Shal K(+) channels are then mislocalized to some, but not all, axons in vivo. These results suggest that there are multiple mechanisms underlying Shal K(+) channel targeting, one of which depends on the LL-motif. The identification of SIDL may provide the first step for future investigation into the molecular machinery regulating the LL-motif-dependent targeting of K(+) channels.

Fast inactivation of Shal (K(v)4) K+ channels is regulated by the novel interactor SKIP3 in Drosophila neurons.

Shal K+ (K(v)4) channels across species carry the major A-type K+ current present in neurons. Shal currents are activated by small EPSPs and modulate post-synaptic potentials, backpropagation of action potentials, and induction of LTP. Fast inactivation of Shal channels regulates the impact of this post-synaptic modulation. Here, we introduce SKIP3, as the first protein interactor of Drosophila Shal K+ channels. The SKIP gene encodes three isoforms with multiple protein-protein interaction domains. SKIP3 is nervous system specific and co-localizes with Shal channels in neuronal cell bodies, and in puncta along processes. Using a genetic deficiency of SKIP, we show that the proportion of neurons displaying a very fast inactivation, consistent with Shal channels exclusively in a "fast" gating mode, is increased in the absence of SKIP3. As a scaffold-like protein, SKIP3 is likely to lead to the identification of a novel regulatory complex that modulates Shal channel inactivation.

Cholinergic Homeostatic Synaptic Plasticity Drives the Progression of Aß-Induced Changes in Neural Activity.

Homeostatic synaptic plasticity (HSP) is the ability of neurons to exert compensatory changes in response to altered neural activity. How pathologically induced activity changes are intertwined with HSP mechanisms is unclear. We show that, in cholinergic neurons from Drosophila, beta-amyloid (Aß) peptides Aß40 and Aß42 both induce an increase in spontaneous activity. In a transgenic line expressing Aß42, we observe that this early increase in spontaneous activity is followed by a dramatic reduction in spontaneous events, a progression that has been suggested to occur in cholinergic brain regions of mammalian models of Alzheimer's disease. We present evidence that the early enhancement in synaptic activity is mediated by the Drosophila α7 nicotinic acetylcholine receptor (nAChR) and that, later, Aß42-induced inhibition of synaptic events is a consequence of Dα7-dependent HSP mechanisms induced by earlier hyperactivity. Thus, while HSP may initially be an adaptive response, it may also drive maladaptive changes and downstream pathologies.

Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila.

BACKGROUND: Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K(+) current (I(A)), encoded by the highly conserved Shal/K(v)4 gene, in neuronal firing patterns and repetitive behaviors. While I(A) is present in nearly all neurons across species, elimination of I(A) has been complicated in mammals because of multiple genes underlying I(A), and/or electrical remodeling that occurs in response to affecting one gene. METHODOLOGY/PRINCIPAL FINDINGS: In Drosophila, the single Shal/K(v)4 gene encodes the predominant I(A) current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK(v)4), we show that I(A) is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK(v)4 neurons display multiple defects during prolonged stimuli. DNK(v)4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K(v)4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K(v)4 function. Further, knock-out of Shal/K(v)4 function specifically in motoneurons significantly affects the locomotion behaviors tested. CONCLUSIONS/SIGNIFICANCE: Based on our results, Shal/K(v)4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K(v)4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors.

Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors.

Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD-TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking.

Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2 are differentially expressed and the active DmLOXL-1 influences gene expression and development.

Mammalian lysyl oxidase (LOX) is essential for the catalysis of lysyl-derived cross-links in fibrillar collagens and elastin in the extracellular matrix and has also been implicated in cell motility, differentiation, and tumor cell invasion. The active LOX has been shown to translocate to the nuclei of smooth muscle cells and regulate chromatin structure and transcription. It is difficult to interpret the role of the LOX protein as it is co-expressed with other members of the LOX amine oxidase family in most mammalian cells. To investigate the function of the LOX proteins, we have characterized the Drosophila lysyl oxidases Dmloxl-1 and Dmloxl-2. We present the gene, domain structure, and expression pattern of Dmloxl-1 and Dmloxl-2 during development. In early development, only Dmloxl-1 was expressed, which allowed functional studies. We have expressed Dmloxl-1 in S2 cells and determined that it is a catalytically active enzyme, inhibited by beta-amino-proprionitrile (BAPN), a specific LOX inhibitor. We localized DmLOXL-1 in the nuclei in embryos and in adult salivary gland cells in the nuclei, cytoplasm, and cell surface, using immunostaining and a DmLOXL-1 antibody. To address the biological function of Dmloxl-1, we raised larvae under BAPN inhibitory conditions and over-expressed Dmloxl-1 in transgenic Drosophila. DmLOXL-1 inhibition resulted in developmental delay and a shift in sex ratio; over-expression in the w(m4) variegating strain increased drosopterin production, demonstrating euchromatinization. Our previous data on the transcriptional down-regulation of seven ribosomal genes and the glue gene under inhibitory conditions and the current results collectively support a nuclear role for Dmloxl-1 in euchromatinization and gene regulation.