Elsevier

Neurobiology of Disease

Volume 55, July 2013, Pages 140-151
Neurobiology of Disease

Silencing of the Charcot–Marie–Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2 + homeostasis by reducing store-operated Ca2 + entry

https://doi.org/10.1016/j.nbd.2013.03.010Get rights and content

Highlights

  • GDAP1 participates in the cell trafficking and mobilization of mitochondria.

  • Silencing of GDAP1 gene affects the mitochondrial network distribution.

  • GDAP1 depletion reduces of Ca2+ entry through store-operated Ca2+ channels (SOCE).

  • Abnormal mitochondrial distribution and altered SOCE could be the basis of the GDAP1-associated CMT disease pathophysiology.

Abstract

GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca2 + stores. GDAP1 silencing does not affect mitochondrial Ca2 + uptake, ER-Ca2 +, or Ca2 + flow from ER to mitochondria, but reduces Ca2 + inflow through store-operated Ca2 + entry (SOCE) following mobilization of ER-Ca2 + and SOCE-driven Ca2 + entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca2 + uptake in mitochondria.

Introduction

Charcot–Marie–Tooth (CMT) disease is the most common inherited neurological disorder affecting 1–4 of 10,000 inhabitants (Braathen et al., 2011, Combarros et al., 1987, Skre, 1974). GDAP1 (ganglioside-induced differentiation associated protein 1) gene causes either demyelinating autosomal recessive CMT4A (Baxter et al., 2002), axonal recessive AR-CMT2K (Cuesta et al., 2002) or dominant CMT2K (Claramunt et al., 2005) peripheral neuropathies. GDAP1 belongs to a glutathione S-transferase enzyme subfamily (Marco et al., 2004) that is mainly expressed not only in neurons (Pedrola et al., 2005, Pedrola et al., 2008) but also in Schwann cells (Niemann et al., 2005). GDAP1 is located in the mitochondrial outer membrane (MOM) (Niemann et al., 2005, Pedrola et al., 2005) and a role as a regulator of mitochondrial dynamics has been proposed (Niemann et al., 2005, Pedrola et al., 2008). Overexpression of GDAP1 in COS7 or HeLa cells causes mitochondrial fission and a substantial accumulation of mitochondria around the nucleus. The effect of GDAP1 mutations in mitochondrial dynamics seems to depend on the inheritance pattern (Niemann et al., 2009).

The aim of this work was to study the disease cell pathogenesis by investigating protein interactions of GDAP1 along with the role of the protein in mitochondrial dynamics and calcium homeostasis. In the last years, disruption of calcium homeostasis with involvement of mitochondria has been shown to play a role in a number of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases (Mattson, 2007), and may also be involved in Charcot–Marie–Tooth disease. Mitochondria are well known players in calcium homeostasis due to their capacity to take up Ca2 + which results in the shaping of cytosolic calcium signals and to their capacity to move towards and away of Ca2 + sources (Rizzuto et al., 2012) and any of these properties may be affected by mutations in GDAP1.

Here we demonstrate that GDAP1 is located at MAM fraction interacting with RAB6B, a protein involved in retrograde vesicle trafficking (Matanis et al., 2002, Stenmark, 2009, Wanschers et al., 2007) and caytaxin, a protein participating in the anterograde movement of mitochondria (Aoyama et al., 2009), which suggest that GDAP1 may be important for movement of mitochondria within the cell towards the endoplasmic reticulum (ER). GDAP1 silencing alters mitochondrial interconnectivity and motility of the mitochondrial network within the cell and calcium homeostasis. We propose that GDAP1-related CMT neuropathies may be the consequence of abnormal mitochondrial distribution and movement throughout cytoskeleton towards the ER and plasma membrane. This results in a failure to sustain store-operated Ca2 + entry (SOCE) upon mobilization of ER-Ca2 + due to an impaired SOCE-driven Ca2 + uptake in mitochondria.

Section snippets

Microorganism strains, plasmids and Y2H assay

GDAP11–318 cDNA was cloned in pBTM116 fused in 5′ with LexA and then used for yeast two-hybrid screening (Fields and Song, 1989) against a commercial human brain cDNA library, pACT2-GAL4 (Clontech Laboratories Inc., San Jose, CA) as described by Moreno et al. (2009). RAB6B ORF (OriGene, Rockville, MD) and ATCAY ORF (Geneservice Ltd., Cambridge) were amplified and cloned in pCMV-HA or pCMV-myc for coimmunoprecipitation and immunofluorescence assays. GDAP1 and GDAP1 missense mutations were cloned

GDAP1 silencing induces changes in mitochondrial network distribution and motility

GDAP1 overexpression induces fragmentation of the mitochondrial network suggesting that GDAP1 is related to the fission pathway of mitochondrial dynamics (Niemann et al., 2005, Pedrola et al., 2005). Thus, we expected that GDAP1 depletion should balance the mitochondrial reticulum dynamics towards the fusion pathway, causing mitochondrial tubulation as observed in Niemann et al. (2005). To investigate such hypothesis, we generated five GDAP1 knock-down clones of the human neuroblastoma SH-SY5Y

Discussion

GDAP1, a mitochondrial outer membrane protein, has been related to mitochondrial dynamics pathways because overexpression of wild type protein produces drastic fragmentation of mitochondrion (Niemann et al., 2005, Pedrola et al., 2005). Our data confirm previous results (Pedrola et al., 2008) that suggest that overexpression of mutant alleles does not produce additional changes in mitochondrial morphology different to that of overexpression of wild type protein, suggesting that GDAP1 may have

Conflict of interest

The authors declare no competing financial interests.

Acknowledgments

We thank Prof. P. Sanz, Institute of Biomedicine of Valencia, for advising on the 2-hybrid screening and gift TAT7 strain and pBTM cloning vector. This work has been funded by grants from the Spanish Ministry of Science and Innovation SAF2009-07063 (to F.P.) and BFU2011-30456-C02-01/BMC (to J.S.), the Generalitat Valenciana Prometeo Programme 2009/059 (to F.P.), the Comunidad de Madrid S2010/BMD-2402 MITOLAB-CM (to J.S.), by an institutional grant from the Fundación Ramón Areces to the Centro

References (56)

  • R. Rizzuto

    Ca(2 +) transfer from the ER to mitochondria: when, how and why

    Biochim. Biophys. Acta

    (2009)
  • K. Singaravelu

    Mitofusin 2 regulates STIM1 migration from the Ca2 + store to the plasma membrane in cells with depolarized mitochondria

    J. Biol. Chem.

    (2011)
  • K. Van Acker

    IP(3)-mediated Ca(2 +) signals in human neuroblastoma SH-SY5Y cells with exogenous overexpression of type 3 IP(3) receptor

    Cell Calcium

    (2002)
  • B.F. Wanschers

    A role for the Rab6B Bicaudal–D1 interaction in retrograde transport in neuronal cells

    Exp. Cell Res.

    (2007)
  • M.D. Abramoff et al.

    Computation and visualization of three-dimensional soft tissue motion in the orbit

    IEEE Trans. Med. Imaging

    (2002)
  • T. Aoyama

    Cayman ataxia protein caytaxin is transported by kinesin along neurites through binding to kinesin light chains

    J. Cell Sci.

    (2009)
  • R.V. Baxter

    Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot–Marie–Tooth disease type 4A/8q21

    Nat. Genet.

    (2002)
  • S. Bolte et al.

    A guided tour into subcellular colocalization analysis in light microscopy

    J. Microsc.

    (2006)
  • G.J. Braathen

    Genetic epidemiology of Charcot–Marie–Tooth in the general population

    Eur. J. Neurol.

    (2011)
  • R. Bravo

    Increased ER–mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress

    J. Cell Sci.

    (2011)
  • R. Claramunt

    Genetics of Charcot–Marie–Tooth disease type 4A: mutations, inheritance, phenotypic variability, and founder effect

    J. Med. Genet.

    (2005)
  • O. Combarros

    Prevalence of hereditary motor and sensory neuropathy in Cantabria

    Acta Neurol. Scand.

    (1987)
  • A. Cuesta

    The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot–Marie–Tooth type 4A disease

    Nat. Genet.

    (2002)
  • O.M. de Brito et al.

    Mitofusin 2 tethers endoplasmic reticulum to mitochondria

    Nature

    (2008)
  • O.M. de Brito et al.

    An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

    EMBO J.

    (2010)
  • S. Fields et al.

    A novel genetic system to detect protein–protein interactions

    Nature

    (1989)
  • G. Gemes

    Store-operated Ca2 + entry in sensory neurons: functional role and the effect of painful nerve injury

    J. Neurosci.

    (2011)
  • V. Lupo

    Missense mutations in the SH3TC2 protein causing Charcot–Marie–Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway

    Hum. Mol. Genet.

    (2009)
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