Human Rab7 mutation mimics features of Charcot–Marie–Tooth neuropathy type 2B in Drosophila Katrien Janssens a,b , Sofie Goethals a,c , Derek Atkinson a,c , Biljana Ermanoska a,c , Erik Fransen d , Albena Jordanova a,c , Michaela Auer-Grumbach e , Bob Asselbergh a,c , Vincent Timmerman a,c, ⁎ a Molecular Genetics Department, VIB, University of Antwerp, B-2610 Antwerpen, Belgium b Department of Medical Genetics, University of Antwerp, B-2610 Antwerpen, Belgium c Neurogenetics Laboratory, Institute Born Bunge, University of Antwerp, B-2610 Antwerpen, Belgium d StatUa Center for Statistics, University of Antwerp, B-2000 Antwerpen, Belgium e Department of Orthopaedics, Medical University Vienna, A-1090 Vienna, Austria abstract article info Article history: Received 6 September 2013 Revised 23 December 2013 Accepted 30 January 2014 Available online 9 February 2014 Keywords: Rab7 Drosophila melanogaster Charcot–Marie–Tooth Behavioural assay Charcot–Marie–Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor in- volvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B. © 2014 Elsevier Inc. All rights reserved. Introduction Inherited peripheral neuropathies (IPNs) are a clinically and geneti- cally heterogeneous group of diseases that cause a length-dependent degeneration of the peripheral nerves. The IPNs are classified into three major disease categories, based on the degree of involvement of the motor, sensory and autonomic neurons. In the Hereditary Motor and Sensory Neuropathies (also collectively known as Charcot–Marie– Tooth Disease (CMT)), both afferent and efferent peripheral neurons can be affected. The resulting phenotype is muscular wasting and weakness in combination with impairment of some or all of the somato- sensory modalities (pain, temperature, touch, vibration and proprio- ception) (Barisic et al., 2008). A distinction is further made between axonal IPNs, in which axonal degeneration is the prime hallmark, and demyelinating IPNs, which are caused by defects of the myelin sheet surrounding the axons. CMT2B, an axonal form of IPN, is characterised by prominent sensory loss of all somatosensory modalities, ulcero-mutilations and variable motor abnormalities (Auer-Grumbach et al., 2000). So far, four missense mutations (L129F, K157N, N161T and V162M) in the RAB7A gene were reported as the cause for CMT2B (Houlden et al., 2004; Meggouh et al., 2006; Verhoeven et al., 2003). Genotype/phenotype correlations in CMT2B patients have been described in detail elsewhere (Rotthier et al., 2009). Rab7 is a member of the family of small GTPases and is im- portant in the intracellular transport and maturation of late endosomes, lysosomes, phagosomes and autophagosomes, vesicles whose contents are destined for lysosomal degradation (Bucci et al., 2000; Gutierrez et al., 2004; Harrison et al., 2004). Like all Rab GTPases, Rab7 switches between a GDP-bound inactive cytosolic state and a GTP-bound active membrane-associated state; in the latter, Rab7 will recruit downstream effector molecules through which it exerts its function. The four CMT2B- associated mutations affect amino acid clustering near the highly con- served G-loops that participate in the formation of the nucleotide- binding site. Structural analysis of the L129F mutant showed that this mutation alters the nucleotide-binding pocket, thereby decreasing the nucleotide affinity and deregulating nucleotide exchange. Consequent- ly, the GDP–GTP exchange occurs independently of the guanine ex- change factor and is accelerated, but this is partially counterbalanced Neurobiology of Disease 65 (2014) 211–219 ⁎ Corresponding author at: Peripheral Neuropathy Group, VIB - Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium. Fax: +32 3 265 11 12. E-mail address: vincent.timmerman@molgen.vib-ua.be (V. Timmerman). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nbd.2014.01.021 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi