Junhai Han

The Fly CAMTA Transcription Factor Potentiates Deactivation of Rhodopsin, a G Protein-Coupled Light Receptor

Control of membrane-receptor activity is required not only for the accuracy of sensory responses, but also to protect cells from excitotoxicity. Here we report the isolation of two noncomplementary fly mutants with slow termination of photoresponses. Genetic and electrophysiological analyses of the mutants revealed a defect in the deactivation of rhodopsin, a visual G protein-coupled receptor (GPCR). The mutant gene was identified as the calmodulin-binding transcription activator (dCAMTA). The known rhodopsin regulator Arr2 does not mediate this visual function of dCAMTA. A genome-wide screen identified five dCAMTA target genes. Of these, overexpression of the F box gene dFbxl4 rescued the mutant phenotypes. We further showed that dCAMTA is stimulated in vivo through interaction with the Ca(2+) sensor calmodulin. Our data suggest that calmodulin/CAMTA/Fbxl4 may mediate a long-term feedback regulation of the activity of Ca(2+)-stimulating GPCRs, which could prevent cell damage due to extra Ca(2+) influx.

Neuroligin 2 Is Required for Synapse Development and Function at the Drosophila Neuromuscular Junction

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function.

TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA‐binding protein 43 (TDP‐43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP‐43 is a multi‐functional protein involved in RNA processing and a large number of TDP‐43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP‐43‐linked neurodegeneration remain elusive. In this study, we found that loss of TDP‐43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy–lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP‐43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP‐43‐depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP‐43‐mediated neurodegeneration.

Drosophila neuroligin 4 regulates sleep through modulating GABA transmission.

Sleep is an essential and evolutionarily conserved behavior that is closely related to synaptic function. However, whether neuroligins (Nlgs), which are cell adhesion molecules involved in synapse formation and synaptic transmission, are involved in sleep is not clear. Here, we show that Drosophila Nlg4 (DNlg4) is highly expressed in large ventral lateral clock neurons (l-LNvs) and that l-LNv-derived DNlg4 is essential for sleep regulation. GABA transmission is impaired in mutant l-LNv, and sleep defects in dnlg4 mutant flies can be rescued by genetic manipulation of GABA transmission. Furthermore, dnlg4 mutant flies exhibit a severe reduction in GABAA receptor RDL clustering, and DNlg4 associates with RDLs in vivo. These results demonstrate that DNlg4 regulates sleep through modulating GABA transmission in l-LNvs, which provides the first known link between a synaptic adhesion molecule and sleep in Drosophila.

Prolonged Gq activity triggers fly rhodopsin endocytosis and degradation, and reduces photoreceptor sensitivity

Rapid deactivation of the Drosophila light receptor rhodopsin, through a visual arrestin Arr2 and a pathway that involves a transcription factor dCAMTA, is required for timely termination of light responses in the photoreceptor neuron. Here we report that this process is also critical for maintenance of the photoreceptor sensitivity. In both dCAMTA‐ and arr2‐mutant flies, the endocytosis of the major rhodopsin Rh1 was dramatically increased, which was mediated by a Gq protein that signals downstream of rhodopsin in the visual transduction pathway. Consequently, the Rh1 level was downregulated and the photoreceptor became less sensitive to light. Remarkably, the Gq‐stimulated Rh1 endocytosis does not require phospholipase C, a known effector of Gq, but depends on a tetraspanin protein. Our work has identified an arrestin‐independent endocytic pathway of G protein‐coupled receptor in the fly. This pathway may also function in mammals and mediate an early feedback regulation of receptor signaling.

A Drosophila metallophosphoesterase mediates deglycosylation of rhodopsin

Oligosaccharide chains of newly synthesized membrane receptors are trimmed and modified to optimize their trafficking and/or signalling before delivery to the cell surface. For most membrane receptors, the functional significance of oligosaccharide chain modification is unknown. During the maturation of Rh1 rhodopsin, a Drosophila light receptor, the oligosaccharide chain is trimmed extensively. Neither the functional significance of this modification nor the enzymes mediating this process are known. Here, we identify a dmppe (Drosophila metallophosphoesterase) mutant with incomplete deglycosylation of Rh1, and show that the retained oligosaccharide chain does not affect Rh1 localization or signalling. The incomplete deglycosylation, however, renders Rh1 more sensitive to endocytic degradation, and causes morphological and functional defects in photoreceptors of aged dmppe flies. We further demonstrate that the dMPPE protein functions as an Mn2+/Zn2+‐dependent phosphoesterase and mediates in vivo dephosphorylation of α‐Man‐II. Most importantly, the dephosphorylated α‐Man‐II is required for the removal of the Rh1 oligosaccharide chain. These observations suggest that the glycosylation status of membrane proteins is controlled through phosphorylation/dephosphorylation, and that MPPE acts as the phosphoesterase in this regulation.

Drosophila Neuroligin3 Regulates Neuromuscular Junction Development and Synaptic Differentiation

Background: Postsynaptic neuroligins play essential roles in synaptic maturation and function. Results: Drosophila Neuroligin3 regulates neuromuscular junction growth, GluRIIA recruitment, synaptic vesicle recycling, and postsynaptic density maturation. Conclusion: This elucidates the in vivo roles of Drosophila Neuroligin3 in development and synaptic differentiation of NMJs. Significance: This highlights that Drosophila can be used to understand and uncover functional diversity in neuroligins. Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Mammalian studies have shown that different Nlgs have different roles in synaptic maturation and function. In Drosophila melanogaster, the roles of Drosophila neuroligin1 (DNlg1), neuroligin2, and neuroligin4 have been examined. However, the roles of neuroligin3 (dnlg3) in synaptic development and function have not been determined. In this study, we used the Drosophila neuromuscular junctions (NMJs) as a model system to investigate the in vivo role of dnlg3. We showed that DNlg3 was expressed in both the CNS and NMJs where it was largely restricted to the postsynaptic site. We generated dnlg3 mutants and showed that these mutants exhibited an increased bouton number and reduced bouton size compared with the wild-type (WT) controls. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were affected in dnlg3 mutants. This included abnormal synaptic vesicle endocytosis, increased postsynaptic density length, and reduced GluRIIA recruitment. In addition to impaired synaptic development and differentiation, we found that synaptic transmission was reduced in dnlg3 mutants. Altogether, our data showed that DNlg3 was required for NMJ development, synaptic differentiation, and function.

Neurexin Regulates Visual Function via Mediating Retinoid Transport to Promote Rhodopsin Maturation

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. Here, we show that the Drosophila homolog of α-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. We show that the decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, we identify that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, we demonstrate that Neurexin is essential for the apolipoproteins level. Our results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function.

Ih channels control feedback regulation from amacrine cells to photoreceptors.

In both vertebrates and invertebrates, photoreceptors’ output is regulated by feedback signals from interneurons that contribute to several important visual functions. Although synaptic feedback regulation of photoreceptors is known to occur in Drosophila, many questions about the underlying molecular mechanisms and physiological implementation remain unclear. Here, we systematically investigated these questions using a broad range of experimental methods. We isolated two Ih mutant fly lines that exhibit rhythmic photoreceptor depolarization without light stimulation. We discovered that Ih channels regulate glutamate release from amacrine cells by modulating calcium channel activity. Moreover, we showed that the eye-enriched kainate receptor (EKAR) is expressed in photoreceptors and receives the glutamate signal released from amacrine cells. Finally, we presented evidence that amacrine cell feedback regulation helps maintain light sensitivity in ambient light. Our findings suggest plausible molecular underpinnings and physiological effects of feedback regulation from amacrine cells to photoreceptors. These results provide new mechanistic insight into how synaptic feedback regulation can participate in network processing by modulating neural information transfer and circuit excitability.

dAcsl, the Drosophila Ortholog of Acyl-CoA Synthetase Long-Chain Family Member 3 and 4, Inhibits Synapse Growth by Attenuating Bone Morphogenetic Protein Signaling via Endocytic Recycling

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. We report here that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, our results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation.