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

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

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 induced enhancement of synaptic release is suppressed by heterozygosity in BMP receptor <i>wit</i>.

<p>(A) Representative traces for EJCs and mEJCs in control (<i>OK371-Gal4</i>/+; <i>UAS-luciferase</i>/+), Khc-73 OE (<i>OK371-Gal4</i>/<i>UAS-Khc-73</i>) and Khc-73 OE; <i>wit</i>^<i>HA4</i>/+ (<i>OK371-Gal4</i>/<i>UAS-Khc-73</i>; <i>wit</i>^<i>HA4</i>/+). (B) Quantification of mEJC, EJC and QC for genotypes in control (<i>OK371-Gal4</i>/+; <i>UAS-luciferase</i>/+), <i>wit</i>^<i>HA4</i>/+ (<i>OK371-Gal4</i>/+; <i>wit</i>^<i>HA4</i>/+), Khc-73 OE (<i>OK371-Gal4</i>/<i>UAS-Khc-73</i>) and Khc-73 OE; <i>wit</i>^<i>HA4</i>/+ (<i>OK371-Gal4</i>/<i>UAS-Khc-73</i>; <i>wit</i>^<i>HA4</i>/+). N = 10, 10, 10 and 10. Error Bars are SEM. Student’s t-test. *P<0.05, **P<0.01, ***P<0.001. ns-no statistical significance.</p

Activation of BMP signaling is suppressed in <i>Khc-73</i> mutant larvae.

<p>(A) pMad staining at the NMJ for Control (<i>BG380-GAL4</i>/+), Motoneuron>WIT (<i>BG380-GAL4</i>/+; <i>UAS-Wit</i>/+) and Motoneuron>Wit, <i>Khc-73</i> (<i>BG380-GAL4</i>/+; <i>UAS-Wit</i>, <i>Khc-73</i>^<i>149</i>/+, <i>Khc-73</i>^<i>149</i>). (B) Quantification of mean pMad fluorescence intensity for genotypes in (A). N = 22, 44 and 10 NMJs. (C) pMad staining at the NMJ for 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>/+) (D) Quantification of mean pMad fluorescence intensity for genotypes in (C). N = 12, 12, and 15 NMJs. Error bars are S.E.M. Student’s t-test. *P<0.05, ***P<0.001. Scale bar is 10μm.</p

Enhanced BMP signaling suppresses loss of Brp puncta in <i>Khc-73</i> mutants.

<p>(A) Brp puncta in terminal boutons of muscle 4 NMJs in Control (<i>BG380-Gal4</i>/+; <i>UAS-luciferase</i>/+), TKV^ACT (<i>BG380-Gal4</i>/+; <i>UAS-TKV</i>^<i>ACT</i>/+), <i>Khc-73</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>/<i>Khc-73</i>^<i>149</i>), <i>Khc-73</i>; <i>TKV</i>^<i>ACT</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>; <i>UAS-TKV</i>^<i>ACT</i>/+) larvae. (B) Quantification of BRP puncta per NMJ in genotypes in (A). N = 18, 17, 13, 12 NMJs. (C) Overexpression of activated form of TKV in motoneurons does not enhance BRP intensity at the NMJ. Control (<i>BG380-Gal4</i>/+; <i>UAS-luciferase</i>/+); TKV^ACT (<i>BG380-Gal4</i>/+; UAS-<i>TKV</i>^<i>ACT</i>). N = 19, 17 NMJs. Error bars are SEM. Student’s t-test. ***<0.001. ns-no significance. Scale bar is 5μm.</p

Synaptic proteins Brp and synaptotagmin accumulate in axons of <i>Khc-73</i> and <i>mad</i> mutants.

<p>(A) Brp puncta in axons of Control (<i>Khc-73</i>^<i>100</i><i>/+</i>), <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>) and <i>mad</i> (<i>mad</i>^{<i>K00237</i>}). (B) Quantification of Brp density (Number of Brp puncta/μm³) expressed as a percentage of control. N = 19, 10, 10. (C) SYT puncta in axons of Control (<i>Khc-73</i>^<i>100</i><i>/+</i>), <i>Khc-73</i> (<i>Khc-73</i>^<i>149</i>) and <i>mad</i> (<i>mad</i>^{<i>K00237</i>}). (D) Quantification of SYT density (Number of Brp puncta/μm³) expressed as a percentage of control. N = 19, 10, 10. (E) Brp puncta in axons of Control (<i>BG380-Gal4</i>/+), TKV^ACT (<i>BG380-Gal4</i>/+; <i>UAS-TKV</i>^<i>ACT</i>/+), <i>Khc-73</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>), <i>Khc-73</i>; <i>TKV</i>^<i>ACT</i> (<i>BG380-Gal4</i>/+; <i>Khc-73</i>^<i>149</i>; <i>UAS-TKV</i>^<i>ACT</i>/+). (F) Quantification of Brp puncta axon density for genotypes in (E). N = 10, 5, 10 and 9 larvae. Error Bars are SEM. Student’s t-test. **P<0.01, ***P<0.001. ns-no statistical significance. Scale bar is 5μm.</p