Silencing of the Charcot–Marie–Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2 + homeostasis by reducing store-operated Ca2 + entry
Graphical abstract
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
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These authors contributed equally.