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Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions

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Abstract

Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery–Dreifuss muscular dystrophy. Here, we describe a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. We show that larvae carrying a deletion of Msp-300 KASH domain (Msp-300 ∆KASH ) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. We show that Msp-300 ∆KASH mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. We could rescue Msp-300 ∆KASH locomotion phenotypes by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, we found that Msp-300 controls GluRIIA density at the neuromuscular junction. Our results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies.

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Abbreviations

ANC-1:

Nuclear anchorage protein-1

ARCA1:

Autosomal recessive cerebellar ataxia

Dlg:

Discs-large

EDMD:

Emery–Dreifuss muscular dystrophy

ER:

Endoplasmic reticulum

eEJC:

Evoked excitatory junctional current

eEJP:

Evoked excitatory junctional potential

GluRII:

Glutamate receptor type II

IkB:

Inhibitor of kappa B

KASH:

Klarsicht/ANC-1/Syne homology

LINC:

Linker of nucleoskeleton and cytoskeleton

MAGUK:

Membrane-associated guanylate kinase

mEJC:

Miniature excitatory junctional current

mEJP:

Miniature excitatory junctional potential

MHC:

Myosin heavy chain

Msp-300:

Muscle-specific protein-300

NF-kB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

NMJ:

Neuromuscular junction

RNAi:

RNA interference

ROI:

Region of interest

SR:

Spectrin repeats

SSR:

Subsynaptic reticulum

WT:

Wild type

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Acknowledgments

We would like to thank E. Goillot, E. Delaune, E. Girard, and N. Morel for critical comments on this manuscript and fruitful advice. We also thank C. Chamot, C. Lionnet, and O. Duc (Platim, ENS-Lyon) for their help with image acquisition and analysis, and V. Krakoviack for his help with locomotion analysis. We thank T. Volk, A. DiAntonio, S. Wassermann, and K. Giesler for generously sharing antibodies, A. DiAntonio, G. Davis, M. Baylies, Bloomington Stock Center and Vienna Drosophila RNAi Center for providing fly stocks and DSHB for providing antibodies. This work was supported by the AFM and the CNRS. The authors declare no competing financial interests.

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Authors and Affiliations

  1. Equipe Différenciation Neuromusculaire, CNRS, UMR5239, Ecole Normale Supérieure-Lyon, 46 allée d’Italie, 69364, Lyon Cedex 07, France

    Véronique Morel, Alexandre N. Rey & Laurent Schaeffer

  2. Institut de Génomique Fonctionnelle, CNRS, UMR5203, 141 rue de la cardonille, 34094, Montpellier Cedex 05, France

    Simon Lepicard & Marie-Laure Parmentier

  3. INSERM, U.661, Université Montpellier 1,2, Montpellier, France

    Simon Lepicard & Marie-Laure Parmentier

  4. Université Lyon 1, Villeurbanne, France

    Laurent Schaeffer

  5. Centre de biotechnologies cellulaires, Hospices civils de Lyon, Lyon, France

    Laurent Schaeffer

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  1. Véronique Morel
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  2. Simon Lepicard
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Correspondence to Véronique Morel.

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18_2014_1566_MOESM1_ESM.eps

Supplementary material 1 Suppl. Fig. v50192 muscle-specific RNAi results in nuclei clustering and Msp-300 disconnection from the nuclear envelope. KASH-containing Msp-300 isoforms were depleted specifically in muscles by the expression of v50192 RNAi amplicon with the 24B-Gal4 construct. (a-c) Confocal images of UAS-v50192/+ ; 24B-Gal4/+ third-instar wandering larvae showing Msp-300 subcellular localization (in grey) and nuclei (outlined by LaminA staining, in green) organization. Muscle-specific knock-down of Msp-300 KASH-containing isoforms results in nuclei clustering (a) and disconnection of Msp-300 from the nuclei (a, b) without perturbing Z-band Msp-300 localization (c), thus phenocopying the localization defects observed in Msp-300 ∆KASH mutants and validating the use of v50192 to knock-down Msp-300. Scale bar: 20 µm. (EPS 15529 kb)

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Morel, V., Lepicard, S., N. Rey, A. et al. Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions. Cell. Mol. Life Sci. 71, 3363–3379 (2014). https://doi.org/10.1007/s00018-014-1566-7

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