International Journal of Molecular Sciences Review Recent Advances in Drosophila Models of Charcot-Marie-Tooth Disease Fukiko Kitani-Morii 1,2,* and Yu-ichi Noto 2 1 Department of Molecular Pathobiology of Brain Disease, Kyoto Prefectural University of Medicine, Kyoto 6028566, Japan 2 Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto 6028566, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-75-251-5793 Received: 31 August 2020; Accepted: 6 October 2020; Published: 8 October 2020 Abstract: Charcot-Marie-Tooth disease (CMT) is one of the most common inherited peripheral neuropathies. CMT patients typically show slowly progressive muscle weakness and sensory loss in a distal dominant pattern in childhood. The diagnosis of CMT is based on clinical symptoms, electrophysiological examinations, and genetic testing. Advances in genetic testing technology have revealed the genetic heterogeneity of CMT; more than 100 genes containing the disease causative mutations have been identified. Because a single genetic alteration in CMT leads to progressive neurodegeneration, studies of CMT patients and their respective models revealed the genotype-phenotype relationships of targeted genes. Conventionally, rodents and cell lines have often been used to study the pathogenesis of CMT. Recently, Drosophila has also attracted attention as a CMT model. In this review, we outline the clinical characteristics of CMT, describe the advantages and disadvantages of using Drosophila in CMT studies, and introduce recent advances in CMT research that successfully applied the use of Drosophila, in areas such as molecules associated with mitochondria, endosomes/lysosomes, transfer RNA, axonal transport, and glucose metabolism. Keywords: Charcot-Marie-Tooth disease (CMT); Drosophila melanogaster; human disease model; neurodegeneration; peripheral neuropathy 1. Introduction 1.1. Clinical Features of CMT Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. The average prevalence of CMT is reported to be about 1 in 2,500 people [1]; however, the CMT prevalence rate varies markedly among epidemiological studies due to a large variety of CMT symptoms [2]. At present, the reported prevalence of CMT among Europeans is about 10–30 per 100,000 people, while that in the East Asia is 5.3–10.8 per 100,000 people [3–6]. The diagnosis of CMT is based on clinical symptoms, electrophysiological studies, genetic testing, and nerve biopsy [7]. CMT is usually juvenile-onset, and typical symptoms are slow, progressive muscle weakness and sensory disturbance in a distal dominant pattern. Patients show clumsiness, foot deformity (such as pes cavus), and gait disturbance. Some patients show additional symptoms such as hearing loss and scoliosis [8]. Interestingly, there are accumulating case reports of cerebral white matter abnormalities mainly in X-linked CMT type 1, and recently, abnormal diffusion-tensor imaging on brain MRI was shown to correlate with clinical disability in various CMT subgroups, suggesting subclinical central nervous system involvement in addition to clinical peripheral neuropathy [9–11]. Most patients develop symptoms in childhood, while there are marked individual differences in the severity and Int. J. Mol. Sci. 2020, 21, 7419; doi:10.3390/ijms21197419 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7419 2 of 19 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 17 progression rate even in those with the same genetic alteration [12]. A nerve conduction study is performedperformed forfor patientspatients toto estimate the background pathology of CMT. AA decreased nerve conduction velocityvelocity (NCV) (< 3838 m/s) m/s) indicates indicates CMT1. CMT1. The The main main pathological pathological feature feature of of CMT1 CMT1 is isa destruction a destruction of ofthe the myelin myelin sheath, sheath, which which is produced is produced by Schwann by Schwann cells. On cells. the other On the hand, other the hand, presence the of presence decreased of decreasedcompound compound muscle and muscle sensory and action sensory potentials action with potentials normal withNCV normal(> 38 m/s) NCV indicates (>38m CMT2,/s) indicates whose CMT2,pathological whose feature pathological is primary feature axonal is primary damage axonal. The intermediate damage. The NCV intermediate (30-45 m/s) NCV is associated (30-45 m /withs) is associatedthe mixed pathology with the mixed of damaged pathology myelin of damaged (demyelination myelin) (demyelination) and axon (axonopathy and axon) [13] (axonopathy). Genetic testing [13]. Genetichas made testing marked has progress made marked in recent progress years, and in more recent than years, 100and genes more containing than 100 causative genes containing mutations causativehave been mutationsidentified havewith the been widespread identified use with ofthe next widespread-generation usesequencing of next-generation technologies sequencing, revealing technologies,significant genetic revealing heterogeneity significant of genetic CMT [2,14,15 heterogeneity] (Figure of 1 CMT). Previous [2,14, 15studies] (Figure showed1). Previous that about studies 60% showedof CMT thatpatients about received 60% of a CMT definitive patients diagnosis received by a genetic definitive testing, diagnosis and over by genetic 90% of testing, genetically and overdiagnosed 90% of patients genetically show diagnosed an alteration patients in one showof the anfollowing alteration four in genes: one of Peripheral the following Myelin four Protein genes: 22- PeripheralkDa (PMP22 Myelin), Gap Protein Junction 22-kDa Beta 1( PMP22(GJB1), ),MyelinGap Junction Protein BetaZero 1(MPZ(GJB1), ),andMyelin Mitofusin Protein 2 ( ZeroMFN2(MPZ) [2,16,17), and]. MitofusinHowever, 2in(MFN2 the Mediterranean)[2,16,17]. However, area (e.g., in southern the Mediterranean Italy and eastern area (e.g.,Spain southern), mutation Italys in andGanglioside eastern- Spain),induced mutationsdifferentiation in Ganglioside-induced-associated protein 1 di(GDAP1fferentiation-associated) are the thirdprotein most common 1 (GDAP1 genetic) are the diagnosis third most of commonCMT after genetic PMP22 diagnosis duplication of CMT and after mutationsPMP22 induplication GJB1 [18,19 and]. No mutations GDAP1 inmutationGJB1 [18 was,19]. identified No GDAP1 in mutationa German was cohort, identified suggesting in a German that the cohort, geographical suggesting area that affects the geographicalthe genetic distribution area affects [20] the. genetic Nerve distributionbiopsy, which [20 was]. Nerve previously biopsy, the which key diagnostic was previously step, is thebeing key replaced diagnostic by genetic step, is testing, being replaced but it is bystill genetic important testing, in atypical but it is cases still [7] important. Typical in findings atypical of cases nerve [ 7biopsy]. Typical in demyelinating findings of nerve-type biopsy CMT are in demyelinating-typeonion bulb formation CMT and are a onion thinned bulb myelin formation sheath, and awhich thinned are myelin the resul sheath,t of which repeated arethe de result- and ofremyelination, repeated de- andand remyelination,these findings and are these uniformly findings observed are uniformly throughout observed the throughout nerve. In thecontrast, nerve. Inaxonopathy contrast, axonopathy leads to a decreased leads to a decreasednumber of number axons ofand axons the anddisappearance the disappearance of Schwann of Schwann cells in cells the inabsence the absence of demyelination of demyelination [21]. [21]. Figure 1. Schematic summary showing various Charcot-Marie-Tooth disease (CMT)-related genes and Figure 1. Schematic summary showing various Charcot-Marie-Tooth disease (CMT)-related genes pathways in the peripheral nerve. If each gene has multiple functions, the most representative one is and pathways in the peripheral nerve. If each gene has multiple functions, the most representative described. The enlarged box shows a cross-sectional view of the peripheral nerve. one is described. The enlarged box shows a cross-sectional view of the peripheral nerve. 1.2. Classification of CMT CMT is clinically divided into subgroups according to the combination of the inheritance pattern and NCV, which helps to estimate the underlying pathology (demyelination or axonopathy) Int. J. Mol. Sci. 2020, 21, 7419 3 of 19 1.2. Classification of CMT CMT is clinically divided into subgroups according to the combination of the inheritance pattern and NCV, which helps to estimate the underlying pathology (demyelination or axonopathy) (https://neuromuscular.wustl.edu/)[8] (Table1). At present, CMT1 is further classified into seven subgroups, from CMT1A to 1G. CMT1A (MIM #118220) is the most frequent subtype of CMT caused by a 1.5-Mb duplication on chromosome 17p11.2 containing the PMP22 gene, present in about half of all CMT patients [16,17,22]. Mutations within MPZ gene are causative for CMT1B (MIM #118200), accounting for around 10% of gene-mutation-identified CMT. CMT1 is the most common subtype of CMT, but CMT1 studies in Drosophila are not practical, because Drosophila does not have a mature myelin sheath like that of humans. CMT2, which accounts for 20% of genetically diagnosed CMT patients, shows an autosomal dominant inheritance pattern and decreased nerve action potential amplitudes with normal NCV (which indicates axonopathy) [16,17]. Among many genes in which mutations are reported to cause CMT2, research on the following important genes and their functions