Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology

Mechanism of inward rectification of neuronal nicotininc acetycholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are wide spread in the nervous system. Ample evidence indicates that many central nAChRs are located at the nerve terminals, where they act to facilitate neurotransmitter release; however, little is known about how these receptors carry out their function. The focus of my study is to understand the mechanism(s) that underlie the function of neuronal nAChRs. Neuronal nAChRs conduct inward current at negative membrane potentials, but conduct little or no outward current at positive membrane potentials, a process known as inward rectification. Inward rectification prevents the ACh-evoked conductance increase from short-circuiting the action potential at the nerve terminal, thereby ensuring optimal depolarization of the terminal and effective neurotransmitter release. Using the outside-out single channel patch-clamp technique, I demonstrate that intracellular polyamines block neuronal nAChRs with high affinity and in a voltage dependent manner; this is true for native nAChRs expressed by sympathetic neurons as well as recombinant alpha3beta4, alpha4beta2 nAChRs expressed in Xenopus oocytes. Given the physiological concentrations of polyamines inside cells, this block can fully account for the strong macroscopic inward rectification. Furthermore, using a combined approach of site-directed mutagenesis and electrophysiology, I show that the negatively charged residues at the cytoplasmic mouth of the pore (known as the intermediate ring) mediate the interaction of intracellular polyamines with the receptor; partial removal of these residues abolishes the strong inward rectification. Interestingly, I show that the intermediate ring influences the permeation of calcium through the receptor, indicating that a molecular link exists between calcium permeability and inward rectification of neuronal nAChRs. My experiments also show that extracellular polyamines and a polyamine-related toxin, Joro spider toxin, block neuronal

Removing 4E-BP Enables Synapses to Refine without Postsynaptic Activity

Summary: Throughout the developing nervous system, considerable synaptic re-organization takes place as postsynaptic neurons extend dendrites and incoming axons refine their synapses, strengthening some and eliminating others. It is well accepted that these processes rely on synaptic activity; however, the mechanisms that lead to this developmental reorganization are not fully understood. Here, we explore the regulation of cap-dependent translation, a mechanism known to play a role in synaptic growth and plasticity. Using sympathetic ganglia in α3 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, we establish that electrophysiologically silent synapses between preganglionic axons and postsynaptic sympathetic neurons do not refine, and the growth of dendrites and the targeting of synapses on postsynaptic neurons are impaired. Remarkably, genetically removing 4E-BP, a suppressor of cap-dependent translation, from these α3 nAChR-KO mice largely restores these features. We conclude that synaptic connections can re-organize and refine without postsynaptic activity during post-natal development when 4E-BP-regulated cap-dependent translation is enhanced. : Synaptic activity is required for synaptic refinement and reorganization during post-natal development. Chong et al. find that silent synapses in superior cervical ganglia (SCG) refine when 4E-BP is genetically removed, suggesting that enhanced cap-dependent translation promotes synaptic refinement in the absence of postsynaptic activity. Keywords: sympathetic neurons, cap-dependent translation, synaptic refinement, synaptic activity, nicotinic receptors, silent synapse

Retrograde BMP Signaling Controls Synaptic Growth at the NMJ by Regulating Trio Expression in Motor Neurons

SummaryRetrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the <i>Drosophila</i> neuromuscular junction

<div><p>Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in <i>Drosophila</i> larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the <i>Khc-73</i> mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in <i>Khc-73</i> mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of <i>Khc-73</i>. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.</p></div

BMP receptors Wit and TKV accumulate at <i>Khc-73</i> mutant NMJs.

<p>(A) Representative western blot of Wit levels in brain tissue from control (<i>w</i>^<i>1118</i>) and <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>) mutants. Actin loading control. (B) Quantification of Wit protein band intensity normalized to actin of (A) and shown as percentage of control. n = 3 blots. (C) Representative western blot of Wit levels in muscle tissue from control (<i>w</i>^<i>1118</i>) and <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>) mutants. Actin loading control. (D) Quantification of Wit protein band intensity normalized to actin for (C) and shown as percentage of control. n = 3 blots. (E) Live image of muscle 4 NMJs in unfixed larvae for Control (<i>BG380-Gal4</i>/+; <i>OK371-Gal4</i>/ <i>UAS-Wit-GFP</i>) and <i>Khc-73</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>, <i>OK371-Gal4</i> / <i>Khc-73</i>^<i>149</i>, <i>UAS-Wit-GFP</i>). Scale bar is 10μm. (F) Quantification of mean fluorescence intensity as percentage of control for genotypes in (E). N = 11, 6. (G) Live image of muscle 6/7 NMJs in unfixed larvae for Control (<i>BG380-Gal4</i>/+; <i>OK371-Gal4</i>/<i>UAS-Wit-GFP</i>) and <i>Khc-73</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>, <i>OK371-Gal4</i>/ <i>Khc-73</i>^<i>149</i>, <i>UAS-Wit-GFP</i>). Scale bar is 20μm. (H) Quantification of mean fluorescence intensity as percentage of control for genotypes in (G). N = 10, 9 NMJs. Error Bars are SEM. Student’s t-test. *P<0.05, **P<0.01. ns-no statistical significance.</p

<i>Khc-73</i> mutants have normal synaptic structure and function.

<p>(A) <i>Khc-73</i> synapse structure at muscle 4. Postsynaptic Dlg stain (green) and presynaptic neuron HRP stain (red). Control (<i>Khc-73</i>^<i>100</i>). Scale bar is 10μm. (B) Quantification of bouton number in Control (<i>Khc-73</i>^<i>100</i>) and <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>) mutants at muscle 4 n = 17, 18 NMJs. (C) Muscle surface area of muscle 4 in Control (<i>Khc-73</i>^<i>100</i>) and <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>). Muscle 4 n = 17, 18. (D) Representative traces of EJCs and mEJCs from third instar larval NMJ in precise excision <i>Khc-73</i>^<i>100</i> (top) and <i>Khc-73</i> mutant (<i>Khc-73</i>^<i>149</i>) (bottom). (E) Quantification of mEJC, EJC and QC for control <i>w</i>^<i>1118</i>, <i>Khc-73</i>^<i>100</i>, <i>Khc-73</i>^<i>193</i> and <i>Khc-73</i>^<i>149</i>. N = 9, 14, 7 and 11 NMJs. Error Bars are SEM. Student’s t-test. ns-no statistical significance.</p

Live imaging of Rab7:GFP at synaptic terminals.

<p>(A) Montage of RAB7:GFP retrograde movement at muscle 4 NMJ in control (<i>OK371-GAL4</i>/+; <i>UAS-RAB7</i>:<i>GFP</i>/+) and <i>Khc-73</i> (<i>OK371-GAL4</i>, <i>Khc-73</i>^<i>149</i>/+, <i>Khc-73</i>^<i>149</i>; <i>UAS-RAB7</i>:<i>GFP</i>/+) larvae. Scale bar is 5μm. Time is seconds. (B) Histogram of Anterograde velocities of RAB7:GFP puncta in (A). N = 18(42), 12(36) NMJs(puncta). (C) Histogram of Retrograde velocities of RAB7:GFP puncta in (A). N = 18(42), 12(36) NMJs(puncta). (D) Average anterograde velocity of RAB7:GFP puncta for genotypes in (A). N = 18(42), 12(36) NMJs(puncta). (E) Average retrograde velocity of RAB7:GFP puncta for genotypes in (A). N = 18(42), 12(36) NMJs(puncta). (F) Average time RAB7:GFP puncta spent in each pause event. For genotypes in (A). N = 18(42), 12(36) NMJs(puncta). (G) Average number of pauses per RAB7:GFP puncta.for genotypes in (A). N = 18(42), 12(36) NMJs(puncta). (H) Total time RAB7:GFP puncta remained paused within proximal axon for genotypes in (A). N = 18(42), 12(36) NMJs(puncta). (I) TKV-YFP expression in control (BG380-Gal4/+; UAS-<i>TKV-YFP</i>/+) and <i>Khc-73</i> (<i>BG380-GAL4</i>/+; <i>Khc-73</i>^<i>149</i>; <i>UAS-TKV-YFP</i>/+) larvae in the NMJ axon of muscle 4. (J) Quantification of the number of stationary puncta observed within the axon from time lapse movies of genotypes in (I). N = 6, 8 NMJs. Scale bar is 5 μm. Error Bars are SEM. Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. ns-no statistical significance.</p

Khc-73 is required for retrograde BMP signaling at the NMJ.

<p>(A) Representative traces of EJC and mEJCs of Control (<i>MHC-Gal4</i>/+), Muscle>Gbb (<i>UAS-Gbb</i>^<i>99</i>/+; <i>MHC-Gal4</i>/+) and Muscle>Gbb, <i>Khc-73</i> (<i>Khc-73</i>^<i>193</i>, <i>UAS-Gbb</i>^<i>99</i>/<i>Khc-73</i>^<i>149</i>, +; <i>MHC-Gal4</i>/+). (B) Quantification of mEJC, EJC and QC for genotypes shown in (A). n = 10, 10 and 7. (C) pMad staining in the motoneuron nuclei of ventral nerve cord in MHC Control (<i>MHC-GAL4</i>/+), MHC>Gbb (<i>UAS-Gbb</i>^<i>99</i>/+; <i>MHC-GAL4</i>/+) and MHC>Gbb, <i>Khc-73</i> (<i>Khc-73</i>^<i>193</i>, <i>UAS-Gbb</i>^<i>99</i>/<i>Khc-73</i>^<i>149</i>, +; <i>MHC-GAL4</i>/+) larvae. (D) Quantification of the mean fluorescence intensity of nuclei for genotypes in (C). n = 302(7), 372(9) and 5(210), Nuclei (larvae) respectively. Error Bars are SEM. One way ANOVA. Student’s t-test. *P<0.05, **P<0.01, ***P<0.001.</p