Advertisement

Neurodegenerative Charcot–Marie–Tooth disease as a case study to decipher novel functions of aminoacyl-tRNA synthetases

  • Na Wei
    Affiliations
    From the Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037
    Search for articles by this author
  • Qian Zhang
    Affiliations
    From the Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037
    Search for articles by this author
  • Xiang-Lei Yang
    Correspondence
    To whom correspondence should be addressed. Tel.:858-784-8976; Fax:858-784-7250
    Affiliations
    From the Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037
    Search for articles by this author
  • Author Footnotes
    3 M. Shy, IUBMB Focused Meeting on the Aminoacyl-tRNA Synthetases, Oct 29-Nov 2, 2017. p. 13, Clearwater, FL.
    4 S. Bervoets, N. Wei, M.-L. Erfurth, S. Yusein-Myashkova, B. Ermanoska, L. Mateiu, B. Asselbergh, D. Blocquel, F. P. Thomas, V. Guergueltcheva, I. Tournev, A. Jordanova, and X.-L. Yang, unpublished data.
    2 The abbreviations used are: aaRSaminoacyl-tRNA synthetaseCMTCharcot–Marie–Tooth diseasePDBProtein Data BankRMSDroot mean square deviationSAXSsmall angle X-ray scatteringHDXhydrogen–deuterium exchangeVEGFvascular endothelial growth factorNCVnerve conduction velocityMSCmultisynthetase complexTrktropomyosin receptor kinase.
Open AccessPublished:January 14, 2019DOI:https://doi.org/10.1074/jbc.REV118.002955
      Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that catalyze the first reaction in protein biosynthesis, namely the charging of transfer RNAs (tRNAs) with their cognate amino acids. aaRSs have been increasingly implicated in dominantly and recessively inherited human diseases. The most common aaRS-associated monogenic disorder is the incurable neurodegenerative disease Charcot–Marie–Tooth neuropathy (CMT), caused by dominant mono-allelic mutations in aaRSs. With six currently known members (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, and MetRS), aaRSs represent the largest protein family implicated in CMT etiology. After the initial discovery linking aaRSs to CMT, the field has progressed from understanding whether impaired tRNA charging is a critical component of this disease to elucidating the specific pathways affected by CMT-causing mutations in aaRSs. Although many aaRS CMT mutants result in loss of tRNA aminoacylation function, animal genetics studies demonstrated that dominant mutations in GlyRS cause CMT through toxic gain-of-function effects, which also may apply to other aaRS-linked CMT subtypes. The CMT-causing mechanism is likely to be multifactorial and involves multiple cellular compartments, including the nucleus and the extracellular space, where the normal WT enzymes also appear. Thus, the association of aaRSs with neuropathy is relevant to discoveries indicating that aaRSs also have nonenzymatic regulatory functions that coordinate protein synthesis with other biological processes. Through genetic, functional, and structural analyses, commonalities among different mutations and different aaRS-linked CMT subtypes have begun to emerge, providing insights into the nonenzymatic functions of aaRSs and the pathogenesis of aaRS-linked CMT to guide therapeutic development to treat this disease.

      Introduction

      As an essential component of the translation machinery executing in the central dogma of molecular biology, aminoacyl-tRNA synthetases (aaRSs)
      The abbreviations used are: aaRS
      aminoacyl-tRNA synthetase
      CMT
      Charcot–Marie–Tooth disease
      PDB
      Protein Data Bank
      RMSD
      root mean square deviation
      SAXS
      small angle X-ray scattering
      HDX
      hydrogen–deuterium exchange
      VEGF
      vascular endothelial growth factor
      NCV
      nerve conduction velocity
      MSC
      multisynthetase complex
      Trk
      tropomyosin receptor kinase.
      in all three domains of life have been studied for decades (
      • Woese C.R.
      • Olsen G.J.
      • Ibba M.
      • Söll D.
      Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.
      ). Regulatory functions of aaRSs beyond their classic enzymatic role in protein synthesis have also been widely discovered in recent years (
      • Guo M.
      • Schimmel P.
      Essential nontranslational functions of tRNA synthetases.
      ,
      • Yao P.
      • Fox P.L.
      Aminoacyl-tRNA synthetases in medicine and disease.
      • Kim S.
      • You S.
      • Hwang D.
      Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping.
      ). With the rise of genomics in health care, aaRSs are increasingly implicated in human diseases. The focus of the review is on the neurodegenerative Charcot–Marie–Tooth disease (CMT), the first and the most common monogenic disorder associated with aaRSs. Before we go into the studies and considerations on the etiology, our review starts with a brief introduction of the enzymatic and the nonenzymatic roles of aaRSs, a synopsis on the disease association of aaRSs to highlight the uniqueness of the CMT-associated aaRS subset, and a list of the relevant mutations and established animal models. Despite some heterogeneities, clinical presentations of CMT patients with aaRS mutations are overall similar, implying shared disease mechanisms. The field is now at the point where loss of tRNA aminoacylation is thought unlikely to serve as the mechanism for aaRS-linked CMT and where new insights regarding commonality in pathogenesis among different aaRSs start to emerge.

      Introduction of aminoacyl-tRNA synthetase

      Aminoacyl-tRNA synthetases in the human genome

      Protein synthesis requires aminoacylated transfer RNAs (tRNAs) to decode the mRNA with proper amino acids so that the genetic information can be translated into proteins. The job of creating the aminoacylated, or the “charged,” tRNAs is carried out by a family of enzymes called aminoacyl-tRNA synthetases (aaRSs). To charge the commonly used 20 proteinogenic amino acids onto their corresponding tRNAs, 20 members are included in the aaRS family.
      In human cells, two sets of aaRSs exist for their respective use in cytosolic and mitochondrial protein synthesis. A total of 37 aaRS genes are encoded by the human nuclear genome, including 18 for cytoplasm only, 17 for mitochondria only, and 2 for both sites. GlyRS and LysRS are the two dual-localized aaRSs, with their mitochondria targeting sequences included or excluded by alternative mRNA splicing or alternative sites of translation initiation (
      • Alexandrova J.
      • Paulus C.
      • Rudinger-Thirion J.
      • Jossinet F.
      • Frugier M.
      Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase.
      ,
      • Tolkunova E.
      • Park H.
      • Xia J.
      • King M.P.
      • Davidson E.
      The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual: alternative splicing of the primary transcript.
      ). Mitochondrial GlnRS is missing from the human genome, and the aminoacylation of mitochondrial tRNAGln is achieved through an indirect pathway, where tRNAGln is first mischarged by mitochondrial GluRS with Glu, which is then modified to Gln by an aminoacyl-tRNA aminotransferase (GatCAB) (
      • Nagao A.
      • Suzuki T.
      • Katoh T.
      • Sakaguchi Y.
      • Suzuki T.
      Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.
      ). The 18 genes encoding for cytoplasmic-only aaRSs contain two separate genes for the two subunits of PheRS (PheRS-a and PheRS-b) and one gene for the fused GluRS and ProRS (GluProRS). We should note that a single-letter amino acid code for aaRS genes (e.g. YARS for cytoplasmic TyrRS and YARS2 for mitochondrial TyrRS) was recently adopted by the HUGO Gene Nomenclature Committee (HGNC) database. In parallel, the 3-letter amino acid code, as the convention in the field, has been continuously used as a prefix to refer to the gene product for its easier and more explicit recognition as an amino acid. (For example, the name “AARS” can be confused between acronyms for “aminoacyl-tRNA synthetase” and for “alanyl-tRNA synthetase,” whereas “AlaRS” explicitly refers to “alanyl-tRNA synthetase”.) For this review, both naming systems are used; they are essentially interchangeable.

      Two-step aminoacylation reaction, two classes of aaRSs and MSC

      The evolutionarily conserved aminoacylation reaction is carried out in two steps. In the first step, aaRSs bind to amino acid and ATP to catalyze the formation of an enzyme-bound aminoacyl-adenylate, while liberating pyrophosphate (PPi). In the second step, the activated amino acid subsequently reacts with a tRNA to yield the aminoacyl-tRNA.
      Based on their unique active-site architecture and the corresponding conserved sequence motifs for ATP binding, aaRSs are evenly divided into two classes, with 10 members in each class of the human enzymes (
      • Eriani G.
      • Delarue M.
      • Poch O.
      • Gangloff J.
      • Moras D.
      Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.
      ,
      • Ribas de Pouplana L.
      • Schimmel P.
      Two classes of tRNA synthetases.
      ). The catalytic domains of class I aaRSs are based on the Rossmann nucleotide binding fold composed of parallel β-sheets and connecting α-helices, whereas the catalytic domains of class II aaRSs are made of anti-parallel β-sheets and flanking α-helices. Class I aaRSs are monomeric, except for TyrRS and TrpRS, which are dimers. The dimerization is mediated through the catalytic domain and is required for their catalytic activity. Because of their capacity to function as homodimers, the genes encoding TyrRS and TrpRS are significantly smaller than other class I aaRS genes. The requirement of a dimer form (or tetramer [dimer of dimer] for PheRS) for catalytic function applies to most class II aaRSs, except for AlaRS (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ).
      An interesting feature of cytoplasmic aaRSs in higher eukaryotes (from flies to humans) is that nine aaRSs (from both class I and class II), together with three nonenzymatic factors, form a multisynthetase complex (MSC). The functional significance of the MSC is unclear, but two hypotheses have been proposed. One suggested that the complex could improve translation efficiency by channeling the charged tRNAs to the ribosome (
      • Kyriacou S.V.
      • Deutscher M.P.
      An important role for the multienzyme aminoacyl-tRNA synthetase complex in mammalian translation and cell growth.
      ); the other proposed that the complex could serve as a “depot” to sequestrate the MSC components with regard to their diverse nonenzymatic, regulatory functions, only to be released in response to certain stimulations (
      • Ray P.S.
      • Arif A.
      • Fox P.L.
      Macromolecular complexes as depots for releasable regulatory proteins.
      ). Of course, these two hypotheses are not mutually exclusive.

      Pleiotropic regulatory functions of cytoplasmic aaRSs

      Coinciding with the emergence of the MSC, widespread regulatory functions of cytoplasmic aaRSs beyond their classic role in protein synthesis have been increasingly reported for both MSC and non-MSC components (
      • Guo M.
      • Schimmel P.
      Essential nontranslational functions of tRNA synthetases.
      ,
      • Yao P.
      • Fox P.L.
      Aminoacyl-tRNA synthetases in medicine and disease.
      • Kim S.
      • You S.
      • Hwang D.
      Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping.
      ). The cytoplasmic aaRSs are not only found in the cytosol where protein synthesis occurs but are also frequently detected in the nucleus and in the extracellular space, such as in cell culture media and in the systemic circulations of humans and mice. The nonenzymatic functions of aaRSs regulate many physiological processes, such as angiogenesis, hematopoiesis, immune response, and stress responses, and are thought to provide a mechanism to coordinate protein synthesis with other biological processes (
      • Fu G.
      • Xu T.
      • Shi Y.
      • Wei N.
      • Yang X.-L.
      tRNA-controlled nuclear import of a human tRNA synthetase.
      ). The functional expansion during evolution concurs with the incorporation of new domains and motifs. Almost all human cytoplasmic aaRSs have at least one new domain or sequence extension, added to the N or C terminus of the evolutionarily conserved catalytic core (
      • Guo M.
      • Yang X.-L.
      • Schimmel P.
      New functions of aminoacyl-tRNA synthetases beyond translation.
      ,
      • Guo M.
      • Schimmel P.
      • Yang X.-L.
      Functional expansion of human tRNA synthetases achieved by structural inventions.
      • Yang X.-L.
      Structural disorder in expanding the functionome of aminoacyl-tRNA synthetases.
      ). Most of the extensions and new domains are dispensable for the aminoacylation activity, suggesting that they function to mediate or regulate the nonenzymatic roles of aaRSs, including facilitating the cellular translocation of human aaRSs from cytoplasm to other cellular compartments (
      • Xu X.
      • Shi Y.
      • Zhang H.-M.
      • Swindell E.C.
      • Marshall A.G.
      • Guo M.
      • Kishi S.
      • Yang X.-L.
      Unique domain appended to vertebrate tRNA synthetase is essential for vascular development.
      ).

      Disease association of aminoacyl-tRNA synthetases

      aaRSs constitute the largest family of proteins implicated in CMT

      The first human disease linked to mutations in aaRSs was a neurodegenerative disease that specifically affects the peripheral nervous system (Fig. 1) (
      • Antonellis A.
      • Ellsworth R.E.
      • Sambuughin N.
      • Puls I.
      • Abel A.
      • Lee-Lin S.-Q.
      • Jordanova A.
      • Kremensky I.
      • Christodoulou K.
      • Middleton L.T.
      • Sivakumar K.
      • Ionasescu V.
      • Funalot B.
      • Vance J.M.
      • Goldfarb L.G.
      • et al.
      Glycyl tRNA synthetase mutations in Charcot–Marie–Tooth disease type 2D and distal spinal muscular atrophy type V.
      ). It was named Charcot–Marie–Tooth disease (CMT), after the three physicians who first described the disease in 1886. The disease, also known as hereditary motor and sensory neuropathy, affects peripheral nerves in a length-dependent manner and is characterized by weakness and wasting of the distal limb muscles leading to progressive motor impairment, sensory loss, and skeletal deformities (
      • Rossor A.M.
      • Polke J.M.
      • Houlden H.
      • Reilly M.M.
      Clinical implications of genetic advances in Charcot–Marie–Tooth disease.
      ,
      • Rossor A.M.
      • Tomaselli P.J.
      • Reilly M.M.
      Recent advances in the genetic neuropathies.
      • Gutmann L.
      • Shy M.
      Update on Charcot–Marie–Tooth disease.
      ). Based on the predominant pathological features, CMT is divided into two major types: the demyelinating type 1, where abnormalities occur in the myelin sheath surrounding peripheral axons, and the axonal type 2, where the damage is within the axon itself, whereas intermediate forms also exist (
      • Rossor A.M.
      • Polke J.M.
      • Houlden H.
      • Reilly M.M.
      Clinical implications of genetic advances in Charcot–Marie–Tooth disease.
      ,
      • Rossor A.M.
      • Tomaselli P.J.
      • Reilly M.M.
      Recent advances in the genetic neuropathies.
      • Gutmann L.
      • Shy M.
      Update on Charcot–Marie–Tooth disease.
      ).
      Figure thumbnail gr1
      Figure 1Human disease association of aminoacyl-tRNA synthetases. CMT is the first human disease linked to aaRSs through dominant mono-allelic mutations. So far, only a selective set of aaRSs, including the dual-localized GARS and the cytosolic YARS, AARS, MARS, HARS, and WARS, have been linked to CMT (green). In contrast, recessive bi-allelic mutations in all mitochondrial aaRSs (orange) and the vast majority of cytosolic aaRSs (purple) have been linked to severe multisystem disorders. The mitochondrial aaRSs are designated with a “2” suffix to the symbol of the genes.
      GlyRS (or GARS) was the first aaRS causally linked to CMT through dominant mono-allelic mutations (Fig. 1) (
      • Antonellis A.
      • Ellsworth R.E.
      • Sambuughin N.
      • Puls I.
      • Abel A.
      • Lee-Lin S.-Q.
      • Jordanova A.
      • Kremensky I.
      • Christodoulou K.
      • Middleton L.T.
      • Sivakumar K.
      • Ionasescu V.
      • Funalot B.
      • Vance J.M.
      • Goldfarb L.G.
      • et al.
      Glycyl tRNA synthetase mutations in Charcot–Marie–Tooth disease type 2D and distal spinal muscular atrophy type V.
      ,
      • Motley W.W.
      • Talbot K.
      • Fischbeck K.H.
      GARS axonopathy: not every neuron's cup of tRNA.
      ). The initial finding was a surprise to the field. How can a protein broadly required for protein synthesis in all cells be linked to a disease of extreme tissue specificity? However, the connection between aaRS and CMT was quickly reinforced by reports of additionally linked family members and by increasing numbers of aaRS mutations identified in CMT patients (
      • Boczonadi V.
      • Jennings M.J.
      • Horvath R.
      The role of tRNA synthetases in neurological and neuromuscular disorders.
      ,
      • Oprescu S.N.
      • Griffin L.B.
      • Beg A.A.
      • Antonellis A.
      Predicting the pathogenicity of aminoacyl-tRNA synthetase mutations.
      • Storkebaum E.
      Peripheral neuropathy via mutant tRNA synthetases: inhibition of protein translation provides a possible explanation.
      ). Currently, with five members (i.e. GARS, YARS, AARS, HARS, and WARS) firmly linked and another one (MARS) possibly associated, aaRSs represent the largest family of proteins implicated in the etiology of CMT (Fig. 1). The various aaRS-linked CMT subtypes usually present as axonal peripheral neuropathies, and sometimes the nerve conduction velocity (NCV) can be in the range associated with demyelination (
      • Jordanova A.
      • Irobi J.
      • Thomas F.P.
      • Van Dijck P.
      • Meerschaert K.
      • Dewil M.
      • Dierick I.
      • Jacobs A.
      • De Vriendt E.
      • Guergueltcheva V.
      • Rao C.V.
      • Tournev I.
      • Gondim F.A.
      • D’Hooghe M.
      • Van Gerwen V.
      • et al.
      Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot–Marie–Tooth neuropathy.
      ,
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      ). The patients have predominantly motor deficits, with highly variable symptoms in terms of severity (
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      ). Although the lower limbs are mainly affected by CMT, most patients with GlyRS mutations have upper limb predominance (
      • Motley W.W.
      • Talbot K.
      • Fischbeck K.H.
      GARS axonopathy: not every neuron's cup of tRNA.
      ,
      • Christodoulou K.
      • Kyriakides T.
      • Hristova A.H.
      • Georgiou D.-M.
      • Kalaydjieva L.
      • Yshpekova B.
      • Ivanova T.
      • Weber J.L.
      • Middleton L.T.
      Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p.
      ). Despite these heterogeneities, clinical presentations of CMT patients with aaRS mutations are similar,
      M. Shy, IUBMB Focused Meeting on the Aminoacyl-tRNA Synthetases, Oct 29-Nov 2, 2017. p. 13, Clearwater, FL.
      implying shared disease mechanisms.

      Mono-allelic versus bi-allelic mutations in aaRSs for disease association

      Except for GlyRS being a dual-localized aaRS used for both cytoplasmic and mitochondrial protein synthesis (
      • Alexandrova J.
      • Paulus C.
      • Rudinger-Thirion J.
      • Jossinet F.
      • Frugier M.
      Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase.
      ), all CMT-linked aaRSs belong to the cytoplasmic set. Although dominant mono-allelic mutations in aaRSs exclusively affect the peripheral nerves system, recessive bi-allelic mutations in aaRSs–both cytoplasmic and mitochondrial–have been linked to a variety of syndromes, which affect multiple organ systems and are sometimes accompanied with developmental delays. These findings were reported in rapid succession in recent years. (Patients with bi-allelic mutations have a mutation (not necessarily the same mutation) in both alleles of a single gene (paternal and maternal), whereas patients with a mono-allelic mutation only have one of the two alleles mutated yet still exhibit a disease phenotype. Therefore, the mono-allelic mutation is considered “dominant” in disease presentation.) So far, bi-allelic, but not mono-allelic (except for the dual-localized GlyRS), mutations in every mitochondrial aaRSs have been linked to human diseases, which mostly affected organs with high metabolic demand, such as the central nervous system and the heart (Fig. 1) (
      • Sissler M.
      • González-Serrano L.E.
      • Westhof E.
      Recent advances in mitochondrial aminoacyl-tRNA synthetases and disease.
      ). Trailing not too far behind, bi-allelic mutations in 16 cytoplasmic aaRSs have emerged as causative to various disease conditions, with an even broader range of organ systems affected than those by mutations in the mitochondrial aaRSs (Fig. 1) (
      • Boczonadi V.
      • Jennings M.J.
      • Horvath R.
      The role of tRNA synthetases in neurological and neuromuscular disorders.
      ,
      • Meyer-Schuman R.
      • Antonellis A.
      Emerging mechanisms of aminoacyl-tRNA synthetase mutations in recessive and dominant human disease.
      ). Because of the importance of protein synthesis in almost all cell types and other essential nonenzymatic roles of aaRSs, we would not be surprised if soon all cytoplasmic aaRSs are implicated in human diseases through bi-allelic mutations as well.

      CMT is specifically linked to mono-allelic mutations in a selective set of cytoplasmic aaRSs

      Although the broad disease phenotypes associated with bi-allelic aaRS mutations may sometimes include neuropathies (
      • McLaughlin H.M.
      • Sakaguchi R.
      • Liu C.
      • Igarashi T.
      • Pehlivan D.
      • Chu K.
      • Iyer R.
      • Cruz P.
      • Cherukuri P.F.
      • Hansen N.F.
      • Mullikin J.C.
      • NISC Comparative Sequencing Program
      • Biesecker L.G.
      • Wilson T.E.
      • Ionasescu V.
      • et al.
      Compound heterozygosity for loss-of-function lysyl-tRNA synthetase mutations in a patient with peripheral neuropathy.
      ), pure CMT and the related neuropathies are specifically associated with cytoplasmic aaRSs through mono-allelic mutations, indicating a special sensitivity and vulnerability of the peripheral nervous system toward mono-allelic aaRS mutations. It is possible that additional aaRSs may be linked; however, some characteristics that emerged from the existing list suggest a potential selectivity of certain aaRSs to be associated with CMT (Fig. 2).
      Figure thumbnail gr2
      Figure 2Prevalent characteristics of CMT-linked aaRSs. Prevalence rate is calculated as a ratio between the number of CMT-linked aaRSs and the total number of aaRSs within a certain category. Class I aaRSs include VARS, IARS, LARS, MARS, CARS, RARS, EARS, QARS, YARS, and WARS, and class II includes SARS, TARS, PARS, GARS, HARS, AARS, DARS, NARS, KARS, and FARS. The monomeric aaRSs are class I aaRSs except for YARS and WARS, which are dimers. Class II aaRSs are dimeric. FARS are tetrameric, which can be considered as a dimer of dimer. The MSC contains IARS, LARS, MARS, RARS, QARS KARS, DARS, EPRS/GluProRS, and three nonenzymatic factors. The WHEP domain–containing aaRSs include GARS, HARS, WARS, MARS, and EPRS.
      The six aaRSs associated with CMT are evenly distributed into the two classes of aaRSs (TyrRS, TrpRS, and MetRS in class I; GlyRS, HisRS, and AlaRS in class II) (Fig. 2). However, CMT-associated aaRSs are mostly dimeric (5 out of 6, except for MetRS), are not associated with the MSC (5 out of 6, except for MetRS), and have a specific appended domain called the WHEP domain (4 out of 6, except for TyrRS and AlaRS) (Fig. 2). In fact, all single WHEP domain–containing aaRSs (GlyRS, HisRS, TrpRS, and MetRS) are CMT-associated (
      • Guo M.
      • Schimmel P.
      • Yang X.-L.
      Functional expansion of human tRNA synthetases achieved by structural inventions.
      ).

      CMT-linked mutations in aaRS genes

      A large number of dominant mutations in the six aforementioned aaRSs has been linked to CMT.

      GlyRS (GARS)

      Human GlyRS protein is composed of three domains: a metazoan-specific helix-turn-helix WHEP domain, the evolutionarily conserved class II catalytic domain, and the anticodon binding domain (Fig. 3). The catalytic domain of GlyRS, and of three other CMT-linked aaRS (i.e. TyrRS, HisRS, and TrpRS), mediates the dimerization of the synthetase, which is necessary for the catalytic activity of these synthetases and for providing a complete set of binding sites for their tRNA substrates. So far, at least 19 mono-allelic mutations in GlyRS with various degrees of genetic evidence have been linked to CMT subtype 2D (CMT2D/AD-CMTax-GARS) in patients (
      • Storkebaum E.
      Peripheral neuropathy via mutant tRNA synthetases: inhibition of protein translation provides a possible explanation.
      ,
      • Liao Y.C.
      • Liu Y.T.
      • Tsai P.C.
      • Chang C.C.
      • Huang Y.H.
      • Soong B.W.
      • Lee Y.C.
      Two novel de novo GARS mutations cause early-onset axonal Charcot–Marie–Tooth disease.
      ). Most of the mutations, especially the ones with the strongest genetic evidence and extensive family histories (e.g. E71G, L129P, G240R, E279D, H418R, D500N, and G526R), are located in the catalytic domain of the enzyme. Two anticodon binding domain mutations (i.e. S581L and G598A) lack strong genetic evidence but are recurrently identified in patients with neuropathy phenotypes (
      • James P.A.
      • Cader M.Z.
      • Muntoni F.
      • Childs A.M.
      • Crow Y.J.
      • Talbot K.
      Severe childhood SMA and axonal CMT due to anticodon binding domain mutations in the GARS gene.
      • Griffin L.B.
      • Sakaguchi R.
      • McGuigan D.
      • Gonzalez M.A.
      • Searby C.
      • Züchner S.
      • Hou Y.M.
      • Antonellis A.
      Impaired function is a common feature of neuropathy-associated glycyl-tRNA synthetase mutations.
      ,
      • Boczonadi V.
      • Meyer K.
      • Gonczarowska-Jorge H.
      • Griffin H.
      • Roos A.
      • Bartsakoulia M.
      • Bansagi B.
      • Ricci G.
      • Palinkas F.
      • Zahedi R.P.
      • Bruni F.
      • Kaspar B.
      • Lochmüller H.
      • Boycott K.M.
      • Müller J.S.
      • Horvath R.
      Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.
      ,
      • Eskuri J.M.
      • Stanley C.M.
      • Moore S.A.
      • Mathews K.D.
      Infantile onset CMT2D/dSMA V in monozygotic twins due to a mutation in the anticodon-binding domain of GARS.
      • McMillan H.J.
      • Schwartzentruber J.
      • Smith A.
      • Lee S.
      • Chakraborty P.
      • Bulman D.E.
      • Beaulieu C.L.
      • Majewski J.
      • Boycott K.M.
      • Geraghty M.T.
      Compound heterozygous mutations in glycyl-tRNA synthetase are a proposed cause of systemic mitochondrial disease.
      ).
      Figure thumbnail gr3
      Figure 3Distribution of CMT-linked mutations in aaRSs. For GlyRS, the mutations are numbered according to the cytosolic form of the human protein and with the 54-amino acid mitochondrial targeting sequence omitted. The net increase of positive or negative charge(s) due to a CMT-associated mutation is indicated by “+” or “−” signs.
      Because GlyRS is a dual-localized aaRS and because two mutations in the Gars gene in mice were found to cause a CMT-like neuropathy (see below), it is important to note that the mutations are numbered according to the cytosolic form of the human protein and with the mitochondrial targeting sequence omitted in our review. In contrast, many reports numbered the same mutation sites with the mitochondrial targeting sequence included, and thereby resulted in an increase of 54 in amino acid residue numbers. (The mitochondrial targeting sequence is supposedly deleted after protein importation to the mitochondria.)

      TyrRS (YARS)

      Human TyrRS protein is composed of three domains: the evolutionarily conserved catalytic (class I) and anticodon binding domains and a C-terminal EMAP-II–like domain (from insects to humans) (Fig. 3). Five dominant mutations with different degrees of genetic association have been linked to an intermediate form of CMT with both demyelinating and axonal features (DI-CMTC/AD-CMTin-YARS) (
      • Jordanova A.
      • Irobi J.
      • Thomas F.P.
      • Van Dijck P.
      • Meerschaert K.
      • Dewil M.
      • Dierick I.
      • Jacobs A.
      • De Vriendt E.
      • Guergueltcheva V.
      • Rao C.V.
      • Tournev I.
      • Gondim F.A.
      • D’Hooghe M.
      • Van Gerwen V.
      • et al.
      Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot–Marie–Tooth neuropathy.
      ,
      • Hyun Y.S.
      • Park H.J.
      • Heo S.H.
      • Yoon B.R.
      • Nam S.H.
      • Kim S.B.
      • Park C.I.
      • Choi B.O.
      • Chung K.W.
      Rare variants in methionyl- and tyrosyl-tRNA synthetase genes in late-onset autosomal dominant Charcot–Marie–Tooth neuropathy.
      ,
      • Gonzaga-Jauregui C.
      • Harel T.
      • Gambin T.
      • Kousi M.
      • Griffin L.B.
      • Francescatto L.
      • Ozes B.
      • Karaca E.
      • Jhangiani S.N.
      • Bainbridge M.N.
      • Lawson K.S.
      • Pehlivan D.
      • Okamoto Y.
      • Withers M.
      • Mancias P.
      • et al.
      Exome sequence analysis suggests that genetic burden contributes to phenotypic variability and complex neuropathy.
      ). All mutations are located in the catalytic domain. Three (G41R, E196K, and E196Q) segregated with the disease in large families, whereas D81I and Δ153–156(VKQV) are de novo mutations, and each was found in a single patient. Nevertheless, through transgenic overexpression, the pathogenicity of Δ153–156, as well as that of G41R and E196K, has been recapitulated in Drosophila models (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ,
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ).

      HisRS (HARS)

      HisRS has an identical domain structure as that of GlyRS, including the WHEP domain, the class II catalytic domain, and the C-terminal anticodon binding domain (Fig. 3). So far, eight mutations have been linked to CMT subtype 2W (CMT2W/AD-CMTax-HARS), and five of them (T132I, P134H, V155G, D175E, and D364Y) exhibit clear segregation with disease in large families (
      • Safka Brozkova D.
      • Deconinck T.
      • Griffin L.B.
      • Ferbert A.
      • Haberlova J.
      • Mazanec R.
      • Lassuthova P.
      • Roth C.
      • Pilunthanakul T.
      • Rautenstrauss B.
      • Janecke A.R.
      • Zavadakova P.
      • Chrast R.
      • Rivolta C.
      • Zuchner S.
      • et al.
      Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.
      ,
      • Abbott J.A.
      • Meyer-Schuman R.
      • Lupo V.
      • Feely S.
      • Mademan I.
      • Oprescu S.N.
      • Griffin L.B.
      • Alberti M.A.
      • Casasnovas C.
      • Aharoni S.
      • Basel-Vanagaite L.
      • Züchner S.
      • De Jonghe P.
      • Baets J.
      • Shy M.E.
      • et al.
      Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.
      ). All mutations are located in the catalytic domain. Through transgenic overexpression, the toxicity of D364Y and R137Q mutations has been recapitulated in Caenorhabditis elegans neurons (
      • Safka Brozkova D.
      • Deconinck T.
      • Griffin L.B.
      • Ferbert A.
      • Haberlova J.
      • Mazanec R.
      • Lassuthova P.
      • Roth C.
      • Pilunthanakul T.
      • Rautenstrauss B.
      • Janecke A.R.
      • Zavadakova P.
      • Chrast R.
      • Rivolta C.
      • Zuchner S.
      • et al.
      Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.
      ,
      • Vester A.
      • Velez-Ruiz G.
      • McLaughlin H.M.
      • NISC Comparative Sequencing Program
      • Lupski J.R.
      • Talbot K.
      • Vance J.M.
      • Züchner S.
      • Roda R.H.
      • Fischbeck K.H.
      • Biesecker L.G.
      • Nicholson G.
      • Beg A.A.
      • Antonellis A.
      A loss-of-function variant in the human histidyl-tRNA synthetase (HARS) gene is neurotoxic in vivo.
      ).

      TrpRS (WARS)

      TrpRS also has a N-terminal WHEP domain, followed by a class I catalytic domain and the anticodon binding domain (Fig. 3). Although only one mutation (H257Q, located in the catalytic domain) in WARS has been linked to CMT, it is recurrently identified in multiple families with a clear disease segregation (
      • Tsai P.C.
      • Soong B.W.
      • Mademan I.
      • Huang Y.H.
      • Liu C.R.
      • Hsiao C.T.
      • Wu H.T.
      • Liu T.T.
      • Liu Y.T.
      • Tseng Y.T.
      • Lin K.P.
      • Yang U.C.
      • Chung K.W.
      • Choi B.O.
      • Nicholson G.A.
      • et al.
      A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy.
      ).

      AlaRS (AARS)

      Human AlaRS is the only cytoplasmic tRNA synthetase that has not acquired any new domain during evolution (
      • Guo M.
      • Schimmel P.
      • Yang X.-L.
      Functional expansion of human tRNA synthetases achieved by structural inventions.
      ). AlaRS is also unique among the CMT-linked aaRSs in that it does not have an anticodon binding domain, but has an evolutionarily conserved editing domain and a C-terminal domain designated as C-Ala (Fig. 3). The tRNA recognition of AlaRS does not involve the anticodon and is entirely based on a G3:U70 bp in the acceptor stem of the tRNA (
      • Hou Y.-M.
      • Schimmel P.
      A simple structure feature is a major determinant of the identity of a transfer RNA.
      ). At the same time, due to structural similarities between alanine and some other amino acids (e.g. serine), AlaRS can misactivate or mischarge noncognate amino acids and therefore requires a hydrolytic editing function to ensure the accuracy of the tRNA aminoacylation reaction (
      • Beebe K.
      • Ribas De Pouplana L.
      • Schimmel P.
      Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability.
      ). Although the C-Ala domain is conserved from prokaryotes to humans, its sequence and function have evolved from enhancing tRNA binding in prokaryotes (
      • Guo M.
      • Chong Y.E.
      • Beebe K.
      • Shapiro R.
      • Yang X.-L.
      • Schimmel P.
      The C-Ala domain brings together editing and aminoacylation functions on one tRNA.
      ) to engendering new roles outside of aminoacylation in humans (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ). So far, nine mutations, five of which are located in the catalytic domain (N71Y, G102R, R326W, R329R, and E337K), two in the editing domain (S627L and E688G), and two others in the C-Ala domain (E778A and D893N), have been linked to CMT subtype 2N (CMT2N/AD-CMTax-AARS) (Fig. 3) (
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      ,
      • Lin K.-P.
      • Soong B.-W.
      • Yang C.-C.
      • Huang L.-W.
      • Chang M.-H.
      • Lee I.-H.
      • Antonellis A.
      • Antonellis A.
      • Lee Y.-C.
      The mutational spectrum in a cohort of Charcot–Marie–Tooth Disease type 2 among the Han Chinese in Taiwan.
      • Motley W.W.
      • Griffin L.B.
      • Scherer S.S.
      A novel AARS mutation in a family with dominant myeloneuropathy.
      ,
      • Latour P.
      • Thauvin-Robinet C.
      • Baudelet-Méry C.
      • Soichot P.
      • Cusin V.
      • Faivre L.
      • Locatelli M.C.
      • Mayençon M.
      • Sarcey A.
      • Broussolle E.
      • Camu W.
      • David A.
      • Rousson R.
      A major determinant for binding and aminoacylation of tRNAAla in cytoplasmic alanyl-tRNA synthetase is mutated in dominant axonal Charcot–Marie–Tooth Disease.
      ,
      • McLaughlin H.M.
      • Sakaguchi R.
      • Giblin W.
      • NISC Comparative Sequencing Program
      • Wilson T.E.
      • Biesecker L.
      • Lupski J.R.
      • Talbot K.
      • Vance J.M.
      • Züchner S.
      • Lee Y.C.
      • Kennerson M.
      • Hou Y.M.
      • Nicholson G.
      • Antonellis A.
      A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot–Marie–Tooth disease type 2N (CMT2N).
      ,
      • Bansagi B.
      • Antoniadi T.
      • Burton-Jones S.
      • Murphy S.M.
      • McHugh J.
      • Alexander M.
      • Wells R.
      • Davies J.
      • Hilton-Jones D.
      • Lochmüller H.
      • Chinnery P.
      • Horvath R.
      Genotype/phenotype correlations in AARS-related neuropathy in a cohort of patients from the United Kingdom and Ireland.
      • Zhao Z.
      • Hashiguchi A.
      • Hu J.
      • Sakiyama Y.
      • Okamoto Y.
      • Tokunaga S.
      • Zhu L.
      • Shen H.
      • Takashima H.
      Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy.
      ). All mutations segregate with disease. In particular, the R329H mutation in the catalytic domain has been recurrently identified in multiple families (
      • Latour P.
      • Thauvin-Robinet C.
      • Baudelet-Méry C.
      • Soichot P.
      • Cusin V.
      • Faivre L.
      • Locatelli M.C.
      • Mayençon M.
      • Sarcey A.
      • Broussolle E.
      • Camu W.
      • David A.
      • Rousson R.
      A major determinant for binding and aminoacylation of tRNAAla in cytoplasmic alanyl-tRNA synthetase is mutated in dominant axonal Charcot–Marie–Tooth Disease.
      ,
      • McLaughlin H.M.
      • Sakaguchi R.
      • Giblin W.
      • NISC Comparative Sequencing Program
      • Wilson T.E.
      • Biesecker L.
      • Lupski J.R.
      • Talbot K.
      • Vance J.M.
      • Züchner S.
      • Lee Y.C.
      • Kennerson M.
      • Hou Y.M.
      • Nicholson G.
      • Antonellis A.
      A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot–Marie–Tooth disease type 2N (CMT2N).
      • Bansagi B.
      • Antoniadi T.
      • Burton-Jones S.
      • Murphy S.M.
      • McHugh J.
      • Alexander M.
      • Wells R.
      • Davies J.
      • Hilton-Jones D.
      • Lochmüller H.
      • Chinnery P.
      • Horvath R.
      Genotype/phenotype correlations in AARS-related neuropathy in a cohort of patients from the United Kingdom and Ireland.
      ). Although AlaRS-linked CMT is designated as type 2, some CMT2N patients (i.e. some patients carrying the E337K mutation) also exhibit demyelinating features (
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      ).

      MetRS (MARS)

      The conserved class I catalytic domain and anticodon binding domain of human MetRS are sandwiched between an N-terminal appended GST domain to anchor it to the MSC and a C-terminal WHEP domain with unknown function (Fig. 3). MetRS is the only CMT-linked aaRS that does not form a dimer (Fig. 2). Three mutations, all in the anticodon binding domain, have been linked to CMT type 2U (CMT2U/AD-CMTax-MARS). The R618C mutation was identified in two CMT patients within a family, with incomplete penetrance (
      • Gonzalez M.
      • McLaughlin H.
      • Houlden H.
      • Guo M.
      • Yo-Tsen L.
      • Hadjivassilious M.
      • Speziani F.
      • Yang X.-L.
      • Antonellis A.
      • Reilly M.M.
      • Züchner S.
      Inherited Neuropathy Consortium
      Exome sequencing identifies a significant variant in methionyl-tRNA synthetase (MARS) in a family with late-onset CMT2.
      ). The same mutation was found in the seemingly unaffected father of a patient with the R618C allele compounded with another mutant allele in MARS, causing recessive interstitial lung and liver disease (
      • Rips J.
      • Meyer-Schuman R.
      • Breuer O.
      • Tsabari R.
      • Shaag A.
      • Revel-Vilk S.
      • Reif S.
      • Elpeleg O.
      • Antonellis A.
      • Harel T.
      MARS variant associated with both recessive interstitial lung and liver disease and dominant Charcot–Marie–Tooth disease.
      ). The P800T mutation has been recurrently identified in multiple CMT patients (
      • Hyun Y.S.
      • Park H.J.
      • Heo S.H.
      • Yoon B.R.
      • Nam S.H.
      • Kim S.B.
      • Park C.I.
      • Choi B.O.
      • Chung K.W.
      Rare variants in methionyl- and tyrosyl-tRNA synthetase genes in late-onset autosomal dominant Charcot–Marie–Tooth neuropathy.
      ,
      • Hirano M.
      • Oka N.
      • Hashiguchi A.
      • Ueno S.
      • Sakamoto H.
      • Takashima H.
      • Higuchi Y.
      • Kusunoki S.
      • Nakamura Y.
      Histopathological features of a patient with Charcot–Marie–Tooth disease type 2U/AD-CMTax-MARS.
      ,
      • Nam S.H.
      • Hong Y.B.
      • Hyun Y.S.
      • Nam da E.
      • Kwak G.
      • Hwang S.H.
      • Choi B.O.
      • Chung K.W.
      Identification of genetic causes of inherited peripheral neuropathies by targeted gene panel sequencing.
      ), and the R737W mutation was reported to be a likely pathogenic variant (
      • Sagi-Dain L.
      • Shemer L.
      • Zelnik N.
      • Zoabi Y.
      • Orit S.
      • Adir V.
      • Schif A.
      • Peleg A.
      Whole-exome sequencing reveals a novel missense mutation in the MARS gene related to a rare Charcot–Marie–Tooth neuropathy type 2U.
      ). However, at this point in time, none of the cases provided unequivocal genetic evidence for the mutations to be CMT-causing.

      Animal models of aaRS-linked CMT

      To study the aaRS-linked CMT diseases in vivo, animal models have been generated and characterized.

      Mouse models

      So far, mammalian disease models are only reported for GlyRS-linked CMT2D (Table 1). A spontaneous (P234KY) and a mutagen-induced (C157R) mutation were each found in the GlyRS gene in mice to give rise to CMT-like phenotypes (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ,
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      ). Both GarsP234KY/+ (also known as GarsNmf249/+) and GarsC157R/+ (also known as GarsC201R/+) mice display pathological features of distal motor and sensory neuropathy as seen in the human disease, recapitulating the dominant trait of the human disease. GarsP234KY/+ mice have more severe deficits than GarsC157R/+ mice (Table 1). GarsP234KY/+ mice show a genetic background-dependent mortality at 6–8 weeks (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ), whereas GarsC157R/+ mice have a normal life span (
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      ), resembling the situation for most CMT2D patients. Both models show diminished body weight, reduced distal muscle force, and decreased NCV. The decreased NCV is likely caused by loss of large-parameter peripheral axons. No myelination defect was found in either model, consistent with type 2 CMT. In contrast to axon loss in distal nerves, ventral root axons and spinal cord cell bodies remain unperturbed, indicating that the axonopathy progresses in a distal to proximal manner (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ). Obvious morphological defects of distal neuromuscular junctions, including denervation of distal muscles (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ,
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      ), which are accompanied by synaptic dysfunction (
      • Spaulding E.L.
      • Sleigh J.N.
      • Morelli K.H.
      • Pinter M.J.
      • Burgess R.W.
      • Seburn K.L.
      Synaptic deficits at neuromuscular junctions in two mouse models of Charcot–Marie–Tooth type 2d.
      ), have been observed in both models. Moreover, developmental defects of distal neuromuscular junction were observed prior to synaptic degeneration, highlighting the neuromuscular junction as an important site of early, selective pathology in CMT2D mice (
      • Sleigh J.N.
      • Grice S.J.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Neuromuscular junction maturation defects precede impaired lower motor neuron connectivity in Charcot–Marie–Tooth type 2D mice.
      ).
      Table 1Existing animal models for aaRS-induced CMT through dominant mutations
      Gene/CMT subtypeAnimalMutationGenetic manipulationPhenotype severityRefs.
      GARS/CMT2DMouseC157RKnockinP234KY > C157R
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      P234KYKnockin
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      DrosophilaE71GTransgenic overexpressionP234KY > G240R > G526R > E71G
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      P234KYTransgenic overexpression
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      G240RTransgenic overexpression
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      G526RTransgenic overexpression
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      YARS/DI-CMTCDrosophilaG41RTransgenic overexpressionE196K > G41R > Δ153–156
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      Δ153–156Transgenic overexpression
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      E196KTransgenic overexpression
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      AARS/CMT2NZebrafishR326WmRNA injection of embryosVaries among experiments
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      E337KmRNA injection of embryos
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      S627LmRNA injection of embryos
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      HARS/CMT2WC. elegansR137QTransgenic overexpressionNot reported
      • Vester A.
      • Velez-Ruiz G.
      • McLaughlin H.M.
      • NISC Comparative Sequencing Program
      • Lupski J.R.
      • Talbot K.
      • Vance J.M.
      • Züchner S.
      • Roda R.H.
      • Fischbeck K.H.
      • Biesecker L.G.
      • Nicholson G.
      • Beg A.A.
      • Antonellis A.
      A loss-of-function variant in the human histidyl-tRNA synthetase (HARS) gene is neurotoxic in vivo.
      D364YTransgenic overexpression
      • Safka Brozkova D.
      • Deconinck T.
      • Griffin L.B.
      • Ferbert A.
      • Haberlova J.
      • Mazanec R.
      • Lassuthova P.
      • Roth C.
      • Pilunthanakul T.
      • Rautenstrauss B.
      • Janecke A.R.
      • Zavadakova P.
      • Chrast R.
      • Rivolta C.
      • Zuchner S.
      • et al.
      Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

      Fly model

      Expressing the cytosolic form of GlyRS mutants (GlyRSE71G, GlyRSP234KY, GlyRSG240R, and GlyRSG526R) (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ,
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ) and of TyrRS mutants (TyrRSG41R, TyrRSE196K, and TyrRSΔ153–156) (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ) induced neuronal phenotypes that successfully recapitulated some of the hallmarks of the human disease, including progressive motor performance deficits, electrophysiological evidence of neuronal dysfunction, and terminal axonal degeneration (Table 1). Expression of the Drosophila TyrRS gene containing CMT-associated mutations induced similar defects as the human TyrRS mutants, suggesting evolutionary conservation between the Drosophila and human orthologs (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ). The mouse mutation P234KY induced greater toxicity in fly than the human mutation G240R, consistent with the severity of the mouse model (
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ). Among the human GlyRS mutations tested, the order of phenotypic strength ranks as G240R > G526R > E71G, whereas the phenotypic severity rank for TyrRS mutations is E196K > G41R > Δ153–156 based on their ability to induce motor performance deficits (Table 1) (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ,
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ). Importantly, overexpression of a benign variant TyrRSK265N did not induce signs of toxicity, validating Drosophila as a readout platform to evaluate the pathogenicity of CMT mutations (
      • Leitão-Gonçalves R.
      • Ermanoska B.
      • Jacobs A.
      • De Vriendt E.
      • Timmerman V.
      • Lupski J.R.
      • Callaerts P.
      • Jordanova A.
      Drosophila as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT.
      ).

      Worm model

      C. elegans is another organism successfully used to recapitulate neurotoxicity of CMT mutations in a dominant manner. Transgenic overexpression of the C. elegans HisRS gene containing the mutation equivalent to R137Q or D364Y in human HisRS, but not of the WT gene, in GABAergic neurons (a subclass of motor neurons in nematodes) caused morphological neurotoxicity denoted by dorsal and ventral nerve gaps, axonal blebbing, and severely aberrant axonal processes (Table 1) (
      • Safka Brozkova D.
      • Deconinck T.
      • Griffin L.B.
      • Ferbert A.
      • Haberlova J.
      • Mazanec R.
      • Lassuthova P.
      • Roth C.
      • Pilunthanakul T.
      • Rautenstrauss B.
      • Janecke A.R.
      • Zavadakova P.
      • Chrast R.
      • Rivolta C.
      • Zuchner S.
      • et al.
      Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.
      ,
      • Vester A.
      • Velez-Ruiz G.
      • McLaughlin H.M.
      • NISC Comparative Sequencing Program
      • Lupski J.R.
      • Talbot K.
      • Vance J.M.
      • Züchner S.
      • Roda R.H.
      • Fischbeck K.H.
      • Biesecker L.G.
      • Nicholson G.
      • Beg A.A.
      • Antonellis A.
      A loss-of-function variant in the human histidyl-tRNA synthetase (HARS) gene is neurotoxic in vivo.
      ).

      Fish model

      Most recently, zebrafish have been used successfully to demonstrate the dominant toxicity of CMT mutants (Table 1). Injection of mRNAs of three different AlaRS CMT mutants (R326W, E337K, and S627L) produced neural developmental toxicity in the embryos, whereas the same amount of WT mRNA did not (
      • Weterman M.A.J.
      • Kuo M.
      • Kenter S.B.
      • Gordillo S.
      • Karjosukarso D.W.
      • Takase R.
      • Bronk M.
      • Oprescu S.
      • van Ruissen F.
      • Witteveen R.J.W.
      • Bienfait H.M.E.
      • Breuning M.
      • Verhamme C.
      • Hou Y.-M.
      • Visser M.
      • et al.
      Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.
      ).

      Studies on disease mechanism

      Gain-of-function rather than loss-of-function mechanism demonstrated for CMT2D

      The mono-allelic nature of the CMT-causing mutations in aaRSs and the fact that heterozygous null mice (Gars+/−) do not develop neuropathy (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ) suggest a gain-of-function rather than a loss-of-function disease mechanism. Nevertheless, genetic experiments have been carried out in mouse and fly models to specifically address this question (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Motley W.W.
      • Seburn K.L.
      • Nawaz M.H.
      • Miers K.E.
      • Cheng J.
      • Antonellis A.
      • Green E.D.
      • Talbot K.
      • Yang X.L.
      • Fischbeck K.H.
      • Burgess R.W.
      Charcot–Marie–Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels.
      ). The strategy was to overexpress the WT GlyRS in the CMT2D animal model (Fig. 4). If the disease is caused by a loss of function (either through haplo-insufficiency or a dominant-negative effect), then the overexpression would suppress the phenotypes; otherwise, a toxic gain of function by the mutation would be the cause of the disease phenotypes (Fig. 4). For both GarsP234KY/+ and GarsC157R/+ mice, despite a very high level (10-fold more than the endogenous level) of overexpression of the WT GlyRS, neuropathy phenotypes did not improve (
      • Motley W.W.
      • Seburn K.L.
      • Nawaz M.H.
      • Miers K.E.
      • Cheng J.
      • Antonellis A.
      • Green E.D.
      • Talbot K.
      • Yang X.L.
      • Fischbeck K.H.
      • Burgess R.W.
      Charcot–Marie–Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels.
      ), thus supporting strongly toxic gain-of-function effects for both mutations (Fig. 4). The same conclusion on the human CMT2D mutation (G240R) was reached by another study using the Drosophila CMT model (Fig. 4). The G240R mutation per se causes a loss of aminoacylation activity as shown by in vitro assays and induces strong phenotypes in the fly model (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Nangle L.A.
      • Zhang W.
      • Xie W.
      • Yang X.-L.
      • Schimmel P.
      Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect.
      ). However, overexpression of the WT gene in the mutant fly did not provide any phenotypic rescue (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ). Thus, genetic studies conclusively and consistently have demonstrated that dominant mutations in GlyRS cause CMT through toxic gain-of-function effects.
      Figure thumbnail gr4
      Figure 4Genetic experiments used to clarify whether CMT2D is caused by a gain- or loss-of-function mechanism in mouse and fly models. CMT-like neuropathy phenotypes cannot be rescued by overexpressing WT GlyRS, suggesting toxic gain-of-function effects by the mutations as the cause of CMT2D. In contrast, phenotypes beyond CMT2D and caused by recessive bi-allelic mutations can be rescued by the WT GlyRS overexpression, suggesting some mutations do have loss-of-function properties, although they are not the cause of CMT.

      CMT2D mutations may have loss-of-function properties but they are not the cause of CMT

      Although the genetic experiments in mice and flies have ruled out loss of function as the mechanism for CMT2D, the mutations per se do cause loss-of-function effects as manifested in phenotypes beyond neuropathy in the homozygous GlyRS mutant mice. For example, GarsP234KY/P234KY mice are not viable; GarsC157R/C157R mice can be born, but they show reduced viability and early death (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ,
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      ). Compared with heterozygous GarsC157R/+ mice, GarsC157R/C157R mice have more severe neuropathies and also present abnormalities in the central nervous system, possibly related to the central nervous system phenotypes observed in patients with bi-allelic GlyRS mutations (
      • Boczonadi V.
      • Meyer K.
      • Gonczarowska-Jorge H.
      • Griffin H.
      • Roos A.
      • Bartsakoulia M.
      • Bansagi B.
      • Ricci G.
      • Palinkas F.
      • Zahedi R.P.
      • Bruni F.
      • Kaspar B.
      • Lochmüller H.
      • Boycott K.M.
      • Müller J.S.
      • Horvath R.
      Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.
      ,
      • McMillan H.J.
      • Schwartzentruber J.
      • Smith A.
      • Lee S.
      • Chakraborty P.
      • Bulman D.E.
      • Beaulieu C.L.
      • Majewski J.
      • Boycott K.M.
      • Geraghty M.T.
      Compound heterozygous mutations in glycyl-tRNA synthetase are a proposed cause of systemic mitochondrial disease.
      ,
      • Nafisinia M.
      • Riley L.G.
      • Gold W.A.
      • Bhattacharya K.
      • Broderick C.R.
      • Thorburn D.R.
      • Simons C.
      • Christodoulou J.
      Compound heterozygous mutations in glycyl-tRNA synthetase (GARS) cause mitochondrial respiratory chain dysfunction.
      ). Importantly, overexpression of WT GARS was able to rescue the perinatal death, but not the neuropathy phenotypes of the homozygous GarsC157R/C157R mice, again supporting that the neuropathy phenotype is caused by a toxic gain-of-function effect, whereas the phenotypes beyond neuropathy are caused by a loss of function, which may include enzymatic and/or nonenzymatic activities (Fig. 4). Similarly, Chihara et al. (
      • Chihara T.
      • Luginbuhl D.
      • Luo L.
      Cytoplasmic and mitochondrial protein translation in axonal and dendritic terminal arborization.
      ) showed that a homozygous mutation in the GlyRS gene (P98L) causes preferential loss of dendritic and axonal terminal arborization in Drosophila olfactory projection neurons, and this defect can be fully rescued by transgenic expression of WT GlyRS, demonstrating that a loss of function (undefined) can cause developmental defects in the nervous system (Fig. 4) (
      • Chihara T.
      • Luginbuhl D.
      • Luo L.
      Cytoplasmic and mitochondrial protein translation in axonal and dendritic terminal arborization.
      ). However, the defect cannot be rescued or fully rescued by CMT2D mutants (L129P and E71G, respectively), suggesting that the mutants do have some undefined loss-of-function properties, albeit they are not the cause of the neuropathy.

      Loss of function in aminoacylation is not a shared property of CMT-causing mutations

      Because the aminoacylation function is an essential activity of aaRS, and because the CMT-causing mutations are predominantly located in the catalytic domains, the mutational effects on tRNA aminoacylation have been extensively characterized through in vitro aminoacylation assays and in vivo genetic complementation assays using yeast (
      • Storkebaum E.
      Peripheral neuropathy via mutant tRNA synthetases: inhibition of protein translation provides a possible explanation.
      ,
      • Wallen R.C.
      • Antonellis A.
      To charge or not to charge: mechanistic insights into neuropathy-associated tRNA synthetase mutations.
      ). Although many mutations per se do affect the aminoacylation function, it became clear that loss of aminoacylation is not a shared property of CMT-causing mutations. For example, GlyRSE71G (
      • Nangle L.A.
      • Zhang W.
      • Xie W.
      • Yang X.-L.
      • Schimmel P.
      Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect.
      ,
      • Antonellis A.
      • Lee-Lin S.-Q.
      • Wasterlain A.
      • Leo P.
      • Quezado M.
      • Goldfarb L.G.
      • Myung K.
      • Burgess S.
      • Fischbeck K.H.
      • Green E.D.
      Functional analyses of glycyl-tRNA synthetase mutations suggest a key role for tRNA-charging enzymes in peripheral axons.
      ) and TyrRSE196K (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ,
      • Froelich C.A.
      • First E.A.
      Dominant intermediate Charcot–Marie–Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase.
      ), both of which segregate with CMT in large families, exhibit no defect in these assays. In addition, the level of defect in aminoacylation does not correlate with the severity of the disease phenotype. For example, flies expressing the enzymatically intact TyrRSE196K mutant show a stronger defect in motor performance than flies expressing the aminoacylation-compromised mutants TyrRSG41R and TyrRSΔ153–156 (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ).

      CMT patients and animal models are unlikely to be deficient in tRNA aminoacylation

      With regard to the aminoacylation activity, it is important to consider the presence of the WT allele in the CMT patients. If a defect caused by a mono-allelic mutation at the molecular level can be suppressed by the presence of the WT protein, and therefore is not manifested at the level of cells and tissues, this defect is unlikely to be disease-causing. Studies using the two CMT2D mouse models discussed above provided results consistent with this scenario. No significant decrease in enzymatic activity was found in tissues of GarsP234KY/+ and GarsC157R/+ mice compared with the WT animals (
      • Seburn K.L.
      • Nangle L.A.
      • Cox G.A.
      • Schimmel P.
      • Burgess R.W.
      An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot–Marie–Tooth 2D mouse model.
      ,
      • Achilli F.
      • Bros-Facer V.
      • Williams H.P.
      • Banks G.T.
      • AlQatari M.
      • Chia R.
      • Tucci V.
      • Groves M.
      • Nickols C.D.
      • Seburn K.L.
      • Kendall R.
      • Cader M.Z.
      • Talbot K.
      • van Minnen J.
      • Burgess R.W.
      • et al.
      An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot–Marie–Tooth type 2D peripheral neuropathy.
      ). Consistently, Northern blot analysis revealed that the endogenous aminoacylation levels of tRNAGly in Drosophila were not altered by transgenic expression of WT or mutant GlyRS (i.e. E71G, G240R, and G526R), regardless of the effect of the mutations on the aminoacylation activity of GlyRS per se (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ).

      Mutant GlyRS may cause CMT through cell-autonomous and noncell-autonomous mechanisms

      The ubiquitously expressed mutant tRNA synthetases may give rise to CMT phenotypes through cell-autonomous and/or noncell-autonomous manners. In the case of a neurological disease, such as CMT, if the expression of a mutant protein in neuronal cells alone is able to induce a disease phenotype, the effect is cell-autonomous. Conversely, a noncell-autonomous effect is one in which the neuronal phenotype is caused the mutant protein expressed from non-neuronal cells. This question has been addressed in the Drosophila model using the GAL4 overexpression system with different cell-type–specific promoters. Both ubiquitous (actin-GAL4) and neuron-specific (Nsyb-GAL4) expression of TyrRS mutants, but not muscle-specific expression (MHC-GAL4), induced motor performance deficits, thereby indicating an intrinsic toxicity of TyrRS mutants to neurons (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ). However, for GlyRS mutants, both cell-autonomous and noncell-autonomous mechanisms were observed. By using Nsyb-GAL4 (pan-neuronal) to drive the expression of GlyRSP234KY or GlyRSG240R, and the OK371-GAL4 (motor neuron–specific) to drive the expression of GlyRSE71G, GlyRSG240R, or GlyRSG526R, the two studies each showed that neuronal expression of GlyRS mutants induce motor performance deficits that are cell-autonomous (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ). The GlyRSCMT mutations do not affect the localization of GlyRS within neuronal cells, including neuromuscular junctions (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ,
      • Stum M.
      • McLaughlin H.M.
      • Kleinbrink E.L.
      • Miers K.E.
      • Ackerman S.L.
      • Seburn K.L.
      • Antonellis A.
      • Burgess R.W.
      An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase mutations.
      ). In contrast, Grice et al. (
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ) suggested that the neuronal toxicity of GlyRSP234KY is, at least in part, noncell-autonomous, as expression of the mutant GlyRS in mesoderm or muscle alone results in motor deficits and progressive denervation at the neuromuscular junction.
      Supporting the noncell-autonomous mechanism, the secretion of GlyRS has been demonstrated in many cell types, including neuronal and muscle cells (
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ,
      • Park M.C.
      • Kang T.
      • Jin D.
      • Han J.M.
      • Kim S.B.
      • Park Y.J.
      • Cho K.
      • Park Y.W.
      • Guo M.
      • He W.
      • Yang X.-L.
      • Schimmel P.
      • Kim S.
      PNAS plus: secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis.
      ,
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ). In the fly model, Grice et al. (
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ) showed that muscle-expressed GlyRSP234KY, but not GlyRSWT, accumulates outside muscle cells and on the pre-synaptic membrane of axon terminals. Interestingly, at least in Cos-7 cells, GlyRSP234KY and GlyRSWT have similar levels of secretion (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ).

      Distribution of CMT-associated residues on aaRS structures

      Crystal structures have been solved for all the WT CMT-linked aaRSs (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ,
      • Xie W.
      • Nangle L.A.
      • Zhang W.
      • Schimmel P.
      • Yang X.-L.
      Long-range structural effects of a Charcot Marie Tooth disease-causing mutation in human glycyl-tRNA synthetase.
      • Yang X.-L.
      • Skene R.J.
      • McRee D.E.
      • Schimmel P.
      Crystal structure of a human aminoacyl-tRNA synthetase cytokine.
      ,
      • Yang X.-L.
      • Otero F.J.
      • Skene R.J.
      • McRee D.E.
      • Schimmel P.
      • Ribas de Pouplana L.
      Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains.
      ,
      • Sun L.
      • Gomes A.C.
      • He W.
      • Zhou H.
      • Wang X.
      • Pan D.W.
      • Schimmel P.
      • Pan T.
      • Yang X.L.
      Evolutionary gain of alanine mischarging to noncognate tRNAs with a G4:U69 base pair.
      • Kim Y.K.
      • Chang J.E.
      • Kim S.
      • Jeon Y.H.
      Structural characteristics of human histidyl-tRNA synthetase.
      ) and thus provide a structural platform to understand the CMT-associated mutations. There is an apparent concentration of CMT mutants near the dimer interface of GlyRS, TyrRS, TrpRS, and HisRS (Fig. 5). For example, at least nine CMT-associated residues in GlyRS are located in the immediate vicinity of the dimer interface; also, the only reported CMT-associated residue in TrpRS (His-257) is adjacent to the dimer interface. However, not all CMT mutations in GlyRS, TyrRS, and HisRS are located near the dimer interface, and the effect of different mutations on dimer formation varies: some mutations (e.g. G526R and S581L in GlyRS) strengthen, and others (e.g. L129P and G240R in GlyRS and E196K in TyrRS) significantly weaken the dimers (
      • Nangle L.A.
      • Zhang W.
      • Xie W.
      • Yang X.-L.
      • Schimmel P.
      Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect.
      ,
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ,
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ).
      Figure thumbnail gr5
      Figure 5Distribution of CMT-linked mutation sites in relationship to the dimer interface of aaRSs. A, GlyRS; B, TyrRS; C, HisRS; D, TrpRS; E, AlaRS. For clarity, aaRS dimers are shown in ribbon representation for one subunit and in space-filling model for the second subunit. CMT mutation sites directly located at the dimerization interface are colored in red; CMT mutation sites near but not immediately at the dimerization interface are labeled in green; CMT mutation sites far away from the dimerization interface are colored in dark blue. CMT-linked residues from the space-filling subunits are shown in italic type with ′. The crystal structures of human GlyRS (A), TyrRS (B) (without the C-terminal EMAP-II domain), and TrpRS (D) dimers are provided by PDB entries 2PME, 1N3L, and 1R6T, respectively. The structure of human HisRS dimer (C) is obtained by re-processing the deposited data of PDB 4X5O to reveal the WHEP domain (B. Kuhle, personal communication). The full-length human AlaRS dimer (E) is modeled based on PDB entries 3WQY, 5KNN, and 5T5S. The AlaRS editing domain from Archaeoglobus fulgidus (PDB 3WQY) was docked onto human AlaRS catalytic domain (PDB 5KNN) by superimposing the catalytic domains (RMSD 1.903 Å). Human C-Ala domain (PDB 5T5S) was further docked onto the model by following the domain arrangement of human AlaRS based on SAXS analysis in solution (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ). The crystal structure of human C-Ala dimer (PDB 5T5S) provided the dimer interface.
      It is worth noting again that most, if not all, CMT mutations in GlyRS, TyrRS, TrpRS, and HisRS are located in the catalytic domain (Fig. 3) and that the catalytic domain mediates dimer formation for these four enzymes. In contrast, when the catalytic domain is not involved in dimerization, as in the case of AlaRS, the CMT mutations are no longer concentrated in the catalytic domain (Fig. 3). Although AlaRS can dimerize through the C-Ala domain (Fig. 5), the dimerization, as well as the C-Ala domain itself, does not contribute to the catalytic activity (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ). The same idea applies to MetRS. MetRS does not form dimers, and all of the putative CMT-associated residues are located in the anticodon binding domain (Fig. 3). Which aspect, being in the catalytic domain or being at the dimer interface, is more fundamentally connected to disease, or is neither?

      Potential connection with tRNA

      In addition to the quasi-concentrations of CMT-associated residues at the dimer interface and the catalytic domain, the tRNA-binding surface is another possible consideration. The tRNAs are large molecules compared with the amino acid and ATP substrates, being about 76 nucleotides with a molecular mass of 25 kilodaltons. They fold into L-shaped structures and occupy large binding surfaces on aaRSs, which span from the catalytic domain to the anticodon domain of GlyRS, TyrRS, HisRS, TrpRS, and MetRS or, in the unique case of AlaRS, from the catalytic to the editing and the C-Ala domains (Fig. 6). The CMT-associated residues are mapped onto the co-crystal structures or structure models of the aaRS/tRNA complexes in Fig. 6. Although some CMT-linked residues are closer than others to the tRNA contact sites, the large areas of aaRSs involved in tRNA binding make it possible to imagine a tRNA connection.
      Figure thumbnail gr6
      Figure 6Distribution of CMT-linked mutation sites in relationship to tRNA-binding sites on aaRSs. A dimeric aaRS is shown for GlyRS (A), TyrRS (B), HisRS (C), and TrpRS (D) because the dimer form is required to provide the complete binding sites for a single tRNA and is necessary for catalysis. In contrast, monomeric AlaRS (E) and MetRS (F) are sufficient for tRNA aminoacylation. In the dimeric cases, one subunit of the dimer is in ribbon representation, and the other subunit is in a space-filling model. CMT mutation sites are indicated as orange-red spheres. Mutations from surface presentation subunits are labeled in italic type with ′. A, human GlyRS/tRNAGly complex (PDB 5E6M). Insertion III (residues 423–518) was modeled by superimposing PDB 5E6M with another human GlyRS/tRNAGly structure (PDB 4QEI) with an RMSD of 0.803 Å, which indicates high accuracy of the structure model. B, human TyrRS/tRNATyr complex model. The complex was modeled by superimposing archaeal TyrRS/tRNATyr complex (PDB 1J1U) with human TyrRS catalytic and anticodon domains (PDB 1N3L) with an RMSD of 1.833 Å. In this model, two 3′-nucleotides of the tRNA are missing. C, human HisRS/tRNAHis complex model. The complex was modeled by superimposing the Thermos thermophilus HisRS/tRNAHis complex (PDB 4RDX) with human HisRS structure (PDB 4X5O), with an RMSD of 3.815 Å. Although the large RMSD indicates potential inaccuracy of the model, the tRNA fits well on the structure of human HisRS. D, human TrpRS/tRNATrp complex (PDB 2DR2). The WHEP domain was docked in by superimposing the complex with human TrpRS structure (PDB 1R6T) with a small RMSD of 0.6 Å. E, human AlaRS/tRNAAla complex model. The AlaRS and tRNAAla complex from A. fulgidus (PDB ID: 3WQY) was superimposed with the human AlaRS catalytic domain (PDB 5KNN) through the catalytic domains (RMSD 1.903 Å). Human C-Ala domain (PDB 5T5S) was further docked onto the model by following the domain arrangement of human AlaRS based on SAXS analysis in solution (
      • Sun L.
      • Song Y.
      • Blocquel D.
      • Yang X.-L.
      • Schimmel P.
      Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
      ). The distances from the CMT mutation sites in C-Ala to tRNA may not be reliable, as the C-Ala domain may undergo structural re-arrangement upon tRNA binding. F, human MetRS/tRNAMet complex model. The Aquifex aeolicus MetRS/tRNAMet complex (PDB 2CSX) was superimposed with a truncated human MetRS structure (PDB 5GL7) with an RMSD of 1.67 Å.
      The binding between an aaRS and a tRNA is strongly contributed by electrostatic interactions between the positively-charged basic residues on the protein and the negatively-charged phosphate groups on the tRNA. Interestingly, CMT-associated mutations in aaRSs predominately result in a net increase of positive charges, suggesting a potential enhancement of the aaRS/tRNA interaction (Fig. 3). For example, although none of the CMT2D-associated mutations in GlyRS introduce a negatively charged residue, 12 mutations, including those with the strongest evidence for pathogenicity (e.g. E71G, C157R, P234KY, G240R, and G526R), cause a net increase in the positive charge. Except for the deletion mutation Δ153–156(VKQV), the same is true for DI-CMTC–linked TyrRS mutations; all mutations (i.e. G41R, D81I, E196K, and E196Q) lead to a net increase of positive charges (Fig. 3). The only reported CMT-associated residue in TrpRS (i.e. H257R) also introduces a positive charge, whereas six out of nine CMT2N mutations in AlaRS cause a net increase in positive charge (Fig. 3). In contrast, CMT-associated mutations in HisRS and MetRS do not show this tendency. It would be interesting to study the effect of CMT-causing mutations on tRNA binding. Any potential effects, however, are unlikely to be linked to CMT through affecting the status of tRNA aminoacylation. Insights into the potential tRNA connection to CMT may be inspired by recent findings on the roles of tRNA and tRNA fragments in multiple regulatory networks and that link tRNA to neurodegeneration (
      • Schimmel P.
      The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.
      ,
      • Ishimura R.
      • Nagy G.
      • Dotu I.
      • Zhou H.
      • Yang X.-L.
      • Schimmel P.
      • Senju S.
      • Nishimura Y.
      • Chuang J.H.
      • Ackerman S.L.
      Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration.
      • Shen Y.
      • Yu X.
      • Zhu L.
      • Li T.
      • Yan Z.
      • Guo J.
      Transfer RNA-derived fragments and tRNA halves: biogenesis, biological functions and their roles in diseases.
      ).

      Conformational opening induced by CMT mutations

      Although several CMT mutants have been successfully crystallized, their crystal structures revealed little conformational change compared with their corresponding WT enzymes. These include GlyRSG526R (
      • Xie W.
      • Nangle L.A.
      • Zhang W.
      • Schimmel P.
      • Yang X.-L.
      Long-range structural effects of a Charcot Marie Tooth disease-causing mutation in human glycyl-tRNA synthetase.
      ), GlyRSS581L (
      • Cader M.Z.
      • Ren J.
      • James P.A.
      • Bird L.E.
      • Talbot K.
      • Stammers D.K.
      Crystal structure of human wildtype and S581L-mutant glycyl-tRNA synthetase, an enzyme underlying distal spinal muscular atrophy.
      ), and GlyRSE71G (
      • Qin X.
      • Hao Z.
      • Tian Q.
      • Zhang Z.
      • Zhou C.
      • Xie W.
      Cocrystal structures of glycyl-tRNA synthetase in complex with tRNA suggest multiple conformational states in glycylation.
      ) and also TyrRSG41R and TyrRSE196K (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). In addition, we found that those mutants of GlyRS associated with a weakened dimer (e.g. GlyRSL129P and GlyRSG240R) (
      • Nangle L.A.
      • Zhang W.
      • Xie W.
      • Yang X.-L.
      • Schimmel P.
      Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect.
      ,
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ) were refractory to crystallization, suggesting a potential mutation-induced conformational change that might affect the ability of these mutants to be crystallized. Although conformational changes might also exist in the mutants that were successfully crystallized, crystal-packing interactions can suppress the conformational change and prevent it from being revealed.
      These considerations directed us to use solution-based methods, such as hydrogen–deuterium exchange (HDX) and small-angle X-ray scattering (SAXS), to study the structures and the dynamics of the CMT mutants. HDX monitored by MS determines which areas on the protein become more or less exposed to solvent due to a mutation. This information can be modeled in 3D using the structure template provided by the crystal structure of the WT protein. Although SAXS does not provide high-resolution information, it is useful for detecting a global conformational change. A combined use of these structural methods has so far provided significant insight into the conformational changes in GlyRS and TyrRS induced by the CMT mutations (Fig. 7) (
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ,
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ).
      Figure thumbnail gr7
      Figure 7Solution-based structural analyses reveal conformational opening induced by CMT-causing mutations to engender aberrant interactions. A, six different CMT2D mutations induce conformational change of GlyRS that opens consensus areas that overlap with the dimerization interface. B, three different DI-CMTC mutations open up a same area in TyrRS. The neomorphic surfaces are likely to be responsible for aberrant interactions made by the mutant aaRSs as illustrated.
      Five different human CMT2D mutants have been studied by HDX (
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ). These mutations were selected based on their distinct effects on dimerization: L129P and G240R significantly weaken the GlyRS dimer formation, whereas G526R, S581L, and G598A slightly strengthen the dimer. Despite having different effects on dimerization, all five mutations induce conformational openings of various degrees. Eight hot spots were consensus areas shared by each of the mutants, which partially overlap with the dimerization interface. The mouse mutation P234KY also induces a similar conformational opening in GlyRS (Fig. 7) (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ).
      A similar conclusion was reached for CMT mutations in TyrRS (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). We focused on the three mutations (G41R, E196K, and Δ153–156) that have been validated for their pathogenicity in the fly–CMT model (
      • Storkebaum E.
      • Leitão-Gonçalves R.
      • Godenschwege T.
      • Nangle L.
      • Mejia M.
      • Bosmans I.
      • Ooms T.
      • Jacobs A.
      • Van Dijck P.
      • Yang X.-L.
      • Schimmel P.
      • Norga K.
      • Timmerman V.
      • Callaerts P.
      • Jordanova A.
      Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot–Marie–Tooth neuropathy.
      ). The G41R and Δ153–156 mutations have no significant effect on dimerization, whereas E196K substantially weakens dimer formation (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). Despite the different effects of G41R and E196K on dimerization, they share a similar conformational opening effect on the catalytic domain in an area that is near the dimer interface (Fig. 7). In contrast, the deletion mutation (Δ153–156) does not induce a global conformational change. However, the area that is opened up by G41R and E196K sits right behind the loop containing 153VKQV156. Thus, the same area is also opened up by the deletion mutation (Fig. 7) (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ).

      CMT-causing mutations in aaRSs are unlikely to cause misfolding-induced aggregation

      The shared conformational impact among different CMT mutations in GlyRS and TyrRS provided a conceptually unifying molecular framework to consider the disease etiology. However, the structural change is different from the usual concept of protein misfolding, which is often associated with exposure of hydrophobic residues, reduction of protein stability, and formation of aggregates as an underlying mechanism for the development of many neurological diseases (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). The conformational change induced by the CMT mutations in GlyRS and TyrRS does not necessarily affect protein stability. In fact, many mutants, such as TyrRSG41R and TyrRSΔ153–156, as well as GlyRSG526R and GlyRSS581L, are more stable than their WT counterparts as purified proteins. Even for the mutants (e.g. GlyRSP234KY) that are less stable than the WT protein as purified proteins, the reduced in vitro stability does not translate into reduced stability in vivo. No aggregation or ubiquitin-positive inclusion was detected in neural tissues of the GarsP234KY/+ CMT2D mouse model (
      • Stum M.
      • McLaughlin H.M.
      • Kleinbrink E.L.
      • Miers K.E.
      • Ackerman S.L.
      • Seburn K.L.
      • Antonellis A.
      • Burgess R.W.
      An assessment of mechanisms underlying peripheral axonal degeneration caused by aminoacyl-tRNA synthetase mutations.
      ). These observations suggest protein misfolding and aggregation do not underlie the development of aaRS-linked CMT (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). Rather, we hypothesized that the conformational changes induced by CMT-linked aaRS mutations allow the mutant proteins to make specific aberrant interactions with other molecules (Fig. 7) (
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ,
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ).

      GlyRSCMT mutants interact with Nrp1/Plexin, Trk, and HDAC6

      The concept that CMT mutants make aberrant interactions through the neomorphic surfaces has been well validated. So far, at least three aberrant interaction partners have been identified for mutant GlyRS and a separate one for mutant TyrRS (Fig. 7). The first was Neuropilin 1 (Nrp1), identified to interact with GlyRSCMT mutants but not with GlyRSWT (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ). The aberrant GlyRS–Nrp1 interaction can also be detected in the neural tissue of the GarsP234KY/+ mouse and in the lymphocytes of CMT2D patients carrying the L129P mutation (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ).
      Nrp1 is a cell-surface receptor expressed in motor neurons, endothelial cells, and other cell types. Through binding to signaling proteins such as the semaphorins and vascular endothelial growth factor (VEGF), Nrp1 regulates both the nervous and vascular systems. The secreted mutant GlyRS interacts with the extracellular B1 domain of Nrp1, which is also the high-affinity binding site for both VEGF and semaphorins (
      • Kumanogoh A.
      • Kikutani H.
      Immunological functions of the neuropilins and plexins as receptors for semaphorins.
      ). Mutant GlyRS competes with VEGF for Nrp1 binding and thereby inhibits the neurotrophic VEGF–Nrp1 signaling, which in turn leads to the progressive motor neuron degeneration of CMT2D (Fig. 8) (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ). This model is supported by a genetic interaction between GarsP234KY/+ and Nrp1+/− mice, and by the rescue of motor performance deficits of the CMT2D mice through VEGF overexpression. Interestingly, CMT2D mice do not exhibit defects in the vasculature system (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ,
      • Sleigh J.N.
      • Gómez-Martín A.
      • Wei N.
      • Bai G.
      • Yang X.-L.
      • Schiavo G.
      Neuropilin 1 sequestration by neuropathogenic mutant glycyl-tRNA synthetase is permissive to vascular development and homeostasis.
      ).
      Figure thumbnail gr8
      Figure 8Multifactorial and multicompartmental pathogenic mechanisms proposed for aaRS-linked CMT. In the nucleus, TyrRSCMT aberrantly interacts with the TRIM28/HDAC1 complex to overactivate transcriptional factor E2F1, which is normally suppressed in neurons.4 In the cytosol, TyrRSCMT and GlyRSCMT repress protein translation in motor and sensory neurons through an unknown mechanism independent of tRNA aminoacylation (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ). The dual-localized GlyRSCMT also inhibits translation in mitochondria in mice and patient-induced neuronal progenitor cells (
      • Boczonadi V.
      • Meyer K.
      • Gonczarowska-Jorge H.
      • Griffin H.
      • Roos A.
      • Bartsakoulia M.
      • Bansagi B.
      • Ricci G.
      • Palinkas F.
      • Zahedi R.P.
      • Bruni F.
      • Kaspar B.
      • Lochmüller H.
      • Boycott K.M.
      • Müller J.S.
      • Horvath R.
      Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.
      ). GlyRSCMT aberrantly interacts with HDAC6, leading to hypo-acetylation of α-tubulin and axonal transport deficits (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ,
      • Benoy V.
      • Van Helleputte L.
      • Prior R.
      • d’Ydewalle C.
      • Haeck W.
      • Geens N.
      • Scheveneels W.
      • Schevenels B.
      • Cader M.Z.
      • Talbot K.
      • Kozikowski A.P.
      • Vanden Berghe P.
      • Van Damme P.
      • Robberecht W.
      • Van Den Bosch L.
      HDAC6 is a therapeutic target in mutant GARS-induced Charcot–Marie–Tooth disease.
      ). Extracellularly, secreted GlyRSCMT competes with VEGF to interact with the cell-surface receptor Nrp1 on motor neuron, interfering with the neurotrophic signaling of VEGF (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ). Secreted GlyRSCMT was also detected at the motor neuron terminal to compete with Sema2a for binding to the cell-surface receptor plexin B in a fly-CMT model (
      • Grice S.J.
      • Sleigh J.N.
      • Zameel Cader M.
      Plexin-semaphorin signaling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy.
      ). GlyRSCMT also aberrantly interacts with the cell-surface receptor TrkA/B/C in mice to cause developmental imbalance of sensory neurons (
      • Sleigh J.N.
      • Dawes J.M.
      • West S.J.
      • Wei N.
      • Spaulding E.L.
      • Gómez-Martín A.
      • Zhang Q.
      • Burgess R.W.
      • Cader M.Z.
      • Talbot K.
      • Yang X.-L.
      • Bennett D.L.
      • Schiavo G.
      Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.
      ).
      The impact of mutant GlyRS on semaphorin binding to Nrp1 has not yet been studied, because the neurological impact of semaphorin–Nrp1 signaling is thought to be mediated through the A1 domain of Nrp1 (
      • Janssen B.J.
      • Malinauskas T.
      • Weir G.A.
      • Cader M.Z.
      • Siebold C.
      • Jones E.Y.
      Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex.
      ,
      • Gu C.
      • Rodriguez E.R.
      • Reimert D.V.
      • Shu T.
      • Fritzsch B.
      • Richards L.J.
      • Kolodkin A.L.
      • Ginty D.D.
      Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.
      ), which is not the binding site of mutant GlyRS. However, it was recently reported that, in the fly–CMT model where GlyRSP234KY is expressed, the pre-synaptic localized mutant GlyRS interferes with plexin B signaling (
      • Grice S.J.
      • Sleigh J.N.
      • Zameel Cader M.
      Plexin-semaphorin signaling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy.
      ). Plexin B binds to Semaphorin-2A (Sema2a) and is a functional homolog of Nrp1 for semaphorin signaling in Drosophila. Plexin B co-localizes with mutant GlyRS at the neuromuscular junction, and plexin B levels modify association of mutant GlyRS with the presynaptic membrane. Furthermore, increasing availability of the Sema2a alleviates the pathology and the build up of mutant GlyRS, suggesting that mutant GlyRS competes with Sema2a for binding to plexin B, which contributes to the CMT phenotypes in the fly model (Fig. 8) (
      • Grice S.J.
      • Sleigh J.N.
      • Zameel Cader M.
      Plexin-semaphorin signaling modifies neuromuscular defects in a Drosophila model of peripheral neuropathy.
      ). It is interesting to note that in the SOD1G93A mouse model of ALS, semaphorin-3A signaling through Nrp1 was shown to be an early trigger for distal axonopathy (
      • Venkova K.
      • Christov A.
      • Kamaluddin Z.
      • Kobalka P.
      • Siddiqui S.
      • Hensley K.
      Semaphorin 3A signaling through neuropilin-1 is an early trigger for distal axonopathy in the SOD1G93A mouse model of amyotrophic lateral sclerosis.
      ). These studies highlight the importance of understanding the impact of GlyRSCMT on semaphorin-Nrp1 signaling.
      Tropomyosin receptor kinase (Trk) receptors were also shown to aberrantly interact with GlyRSCMT and have been suggested to explain the sensory involvement seen in CMT2D (Fig. 8) (
      • Sleigh J.N.
      • Dawes J.M.
      • West S.J.
      • Wei N.
      • Spaulding E.L.
      • Gómez-Martín A.
      • Zhang Q.
      • Burgess R.W.
      • Cader M.Z.
      • Talbot K.
      • Yang X.-L.
      • Bennett D.L.
      • Schiavo G.
      Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.
      ). Trk signaling is essential for sensory neuron differentiation and development. Mutant GlyRS binds and misactivates multiple Trk receptors, thereby subverting sensory neuron differentiation and/or survival during early stages of development. In contrast to the progressive motor neuron deficits, the sensory defect is developmental and nonprogressive in CMT2D mice. The developmental nature of the sensory defect might give rise to a binary presentation of sensory involvement in CMT2D patients and may explain the absence of sensory defect in some patients with GlyRS mutations (classified as dSMA-V or dHMN-V) (
      • Sleigh J.N.
      • Dawes J.M.
      • West S.J.
      • Wei N.
      • Spaulding E.L.
      • Gómez-Martín A.
      • Zhang Q.
      • Burgess R.W.
      • Cader M.Z.
      • Talbot K.
      • Yang X.-L.
      • Bennett D.L.
      • Schiavo G.
      Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.
      ).
      Recently, HDAC6 was also identified to aberrantly interact with all CMT2D mutants tested, including GlyRSE71G, GlyRSL129P, GlyRSS211F, GlyRSG240R, GlyRSE279D, GlyRSH418R, GlyRSG526R, GlyRSS581L, and GlyRSG598A (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ,
      • Benoy V.
      • Van Helleputte L.
      • Prior R.
      • d’Ydewalle C.
      • Haeck W.
      • Geens N.
      • Scheveneels W.
      • Schevenels B.
      • Cader M.Z.
      • Talbot K.
      • Kozikowski A.P.
      • Vanden Berghe P.
      • Van Damme P.
      • Robberecht W.
      • Van Den Bosch L.
      HDAC6 is a therapeutic target in mutant GARS-induced Charcot–Marie–Tooth disease.
      ). A main target of the HDAC6 deacetylase is α-tubulin, a critical component of the microtubule, which provides the tracks along which long-distance axonal transport occurs. Acetylation of α-tubulin facilitates axonal transport by promoting the binding of motor proteins to the microtubule. The aberrant GlyRS–HDAC6 interaction enhances the activity of the deacetylase, resulting in a decreased acetylation level of α-tubulin and leading to axonal transport defects (Fig. 8) (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ). Importantly, the decreased α-tubulin acetylation was only found in peripheral nerves but not in brain and spinal cord of the P234KY mouse model. Because the decrease in acetylation and the axonal transport deficits were found in advance of the onset of CMT symptoms in the mouse model, these defects appear not to be secondary to axonal degeneration (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ).
      It is worth noting that the two anticodon binding domain mutations S581L and G598A induce much stronger aberrant interactions with HDAC6 than other patient mutations in the catalytic domain of GlyRS (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ). The S581L and G598A patients have more severe distal weakness and wasting in the lower limbs (
      • James P.A.
      • Cader M.Z.
      • Muntoni F.
      • Childs A.M.
      • Crow Y.J.
      • Talbot K.
      Severe childhood SMA and axonal CMT due to anticodon binding domain mutations in the GARS gene.
      • Griffin L.B.
      • Sakaguchi R.
      • McGuigan D.
      • Gonzalez M.A.
      • Searby C.
      • Züchner S.
      • Hou Y.M.
      • Antonellis A.
      Impaired function is a common feature of neuropathy-associated glycyl-tRNA synthetase mutations.
      ,
      • Boczonadi V.
      • Meyer K.
      • Gonczarowska-Jorge H.
      • Griffin H.
      • Roos A.
      • Bartsakoulia M.
      • Bansagi B.
      • Ricci G.
      • Palinkas F.
      • Zahedi R.P.
      • Bruni F.
      • Kaspar B.
      • Lochmüller H.
      • Boycott K.M.
      • Müller J.S.
      • Horvath R.
      Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.
      ,
      • Eskuri J.M.
      • Stanley C.M.
      • Moore S.A.
      • Mathews K.D.
      Infantile onset CMT2D/dSMA V in monozygotic twins due to a mutation in the anticodon-binding domain of GARS.
      • McMillan H.J.
      • Schwartzentruber J.
      • Smith A.
      • Lee S.
      • Chakraborty P.
      • Bulman D.E.
      • Beaulieu C.L.
      • Majewski J.
      • Boycott K.M.
      • Geraghty M.T.
      Compound heterozygous mutations in glycyl-tRNA synthetase are a proposed cause of systemic mitochondrial disease.
      ), in contrast to the upper limb predominance found in other CMT2D patients (
      • Motley W.W.
      • Talbot K.
      • Fischbeck K.H.
      GARS axonopathy: not every neuron's cup of tRNA.
      ,
      • Christodoulou K.
      • Kyriakides T.
      • Hristova A.H.
      • Georgiou D.-M.
      • Kalaydjieva L.
      • Yshpekova B.
      • Ivanova T.
      • Weber J.L.
      • Middleton L.T.
      Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p.
      ). Thus, the aberrant GlyRS–HDAC6 interaction appears to correlate with the divergent clinical presentation among CMT2D patients. Moreover, the G598A mutation can induce strong aberrant interactions of GlyRS with both Nrp1 and HDAC6, potentially explaining the severe, early-onset clinical symptoms of patients carrying this mutation (
      • James P.A.
      • Cader M.Z.
      • Muntoni F.
      • Childs A.M.
      • Crow Y.J.
      • Talbot K.
      Severe childhood SMA and axonal CMT due to anticodon binding domain mutations in the GARS gene.
      ,
      • Eskuri J.M.
      • Stanley C.M.
      • Moore S.A.
      • Mathews K.D.
      Infantile onset CMT2D/dSMA V in monozygotic twins due to a mutation in the anticodon-binding domain of GARS.
      ). Although the aberrant Nrp1–Plexin and Trk interactions may be responsible for the motor and sensory neuron specificity of the disease, respectively, the aberrant HDAC6 interaction helps explain the length-dependent vulnerability of axons in CMT2D.

      TyrRSCMT mutants aberrantly interact with TRIM28

      TRIM28 was identified to interact with TyrRSWT through an interactome study of nuclear TyrRS (
      • Wei N.
      • Shi Y.
      • Truong L.N.
      • Fisch K.M.
      • Xu T.
      • Gardiner E.
      • Fu G.
      • Hsu Y.-S.O.S.O.
      • Kishi S.
      • Su A.I.
      • Wu X.
      • Yang X.-L.
      Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.
      ). TRIM28 is a scaffolding protein, which forms a complex with the deacetylase HDAC1 to suppress acetylation and activity of transcription factors such as E2F1. The TyrRS–TRIM28 interaction sequesters TRIM28 and HDAC1 and thereby activates E2F1. We found that all three validated DI-CMTC–causing mutations (G41R, E196K, and Δ153–156) caused an enhanced interaction with TRIM28 (
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ), presumably through the exposed neomorphic surface near the dimer interface (Fig. 7), leading to E2F1 hyperacetylation and overactivation
      S. Bervoets, N. Wei, M.-L. Erfurth, S. Yusein-Myashkova, B. Ermanoska, L. Mateiu, B. Asselbergh, D. Blocquel, F. P. Thomas, V. Guergueltcheva, I. Tournev, A. Jordanova, and X.-L. Yang, unpublished data.
      (Fig. 8). The aberrant interaction and E2F1 hyperactivation could be verified in patient-derived lymphocytes, suggesting transcriptional dysregulation is associated DI-CMTC. In fact, a broad transcriptional dysregulation network was identified with neuronal tissues of Drosophila expressing TyrRSE196K versus TyrRSWT, indicating additional transcription regulators that could be aberrantly interacted and dysregulated by mutant TyrRS. Remarkably, pharmacological inhibition of TyrRS nuclear entry reduced, whereas genetic nuclear exclusion of mutant TyrRS completely rescued, hallmark phenotypes of CMT in the Drosophila model, uncovering the importance of a nucleus-localized aaRS for CMT. The CMT-causing mutations do not appear to affect the nuclear localization of TyrRS.4

      WHEP domain

      Except for TyrRS and AlaRS, all CMT-associated aaRSs contain a WHEP domain, either at the N terminus (GlyRS, HisRS, and TrpRS) or at the C terminus (MetRS) (Fig. 3). The only other aaRSs that contain WHEP domains are the bi-functional EPRS, which is a component of the MSC and has three consecutive WHEP domains between the fused GluRS and ProRS. WHEP domains adopt a helix-turn-helix structure and have the capacities to bind to proteins and nucleic acids, including RNA and DNA (
      • Cahuzac B.
      • Berthonneau E.
      • Birlirakis N.
      • Guittet E.
      • Mirande M.
      A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases.
      ,
      • Rho S.B.
      • Lee J.S.
      • Jeong E.J.
      • Kim K.S.
      • Kim Y.G.
      • Kim S.
      A multifunctional repeated motif is present in human bifunctional tRNA synthetase.
      ). Although they do not significantly affect the tRNA-binding affinity and the aminoacylation activity of their host aaRSs (
      • Ko Y.G.
      • Kang Y.S.
      • Kim E.K.
      • Park S.G.
      • Kim S.
      Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis.
      ,
      • Wakasugi K.
      • Slike B.M.
      • Hood J.
      • Otani A.
      • Ewalt K.L.
      • Friedlander M.
      • Cheresh D.A.
      • Schimmel P.
      A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.
      • Qin X.
      • Deng X.
      • Chen L.
      • Xie W.
      Crystal structure of the wild-type human GlyRS bound with tRNA(Gly) in a productive conformation.
      ), the WHEP domains were found to regulate or mediate interactions of aaRSs with other proteins or nucleic acids for nonenzymatic functions. For example, the WHEP domains in EPRS are essential for the role of the bi-functional synthetase in translationally silencing specific mRNAs associated with the inflammatory response (
      • Mukhopadhyay R.
      • Jia J.
      • Arif A.
      • Ray P.S.
      • Fox P.L.
      The GAIT system: a gatekeeper of inflammatory gene expression.
      ,
      • Jia J.
      • Arif A.
      • Ray P.S.
      • Fox P.L.
      WHEP domains direct noncanonical function of glutamyl-prolyl tRNA synthetase in translational control of gene expression.
      • Sampath P.
      • Mazumder B.
      • Seshadri V.
      • Gerber C.A.
      • Chavatte L.
      • Kinter M.
      • Ting S.M.
      • Dignam J.D.
      • Kim S.
      • Driscoll D.M.
      • Fox P.L.
      Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation.
      ). Also, the WHEP domain of HisRS is the main epitope for the anti-Jo-1 antibodies in inflammatory myositis patients (
      • Howard O.M.
      • Dong H.F.
      • Yang D.
      • Raben N.
      • Nagaraju K.
      • Rosen A.
      • Casciola-Rosen L.
      • Härtlein M.
      • Kron M.
      • Yang D.
      • Yiadom K.
      • Dwivedi S.
      • Plotz P.H.
      • Oppenheim J.J.
      Histidyl–tRNA synthetase and asparaginyl–tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells.
      ,
      • Zhou J.J.
      • Wang F.
      • Xu Z.
      • Lo W.-S.
      • Lau C.-F.
      • Chiang K.P.
      • Nangle L.A.
      • Ashlock M.A.
      • Mendlein J.D.
      • Yang X.-L.
      • Zhang M.
      • Schimmel P.
      Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.
      ). The WHEP domain alone (HisRSWHEP) can be produced as a splice variant, and the expression of HisRSWHEP is up-regulated in the myositis patients (
      • Zhou J.J.
      • Wang F.
      • Xu Z.
      • Lo W.-S.
      • Lau C.-F.
      • Chiang K.P.
      • Nangle L.A.
      • Ashlock M.A.
      • Mendlein J.D.
      • Yang X.-L.
      • Zhang M.
      • Schimmel P.
      Secreted histidyl-tRNA synthetase splice variants elaborate major epitopes for autoantibodies in inflammatory myositis.
      ). Removal of the WHEP domain in TrpRS, either by proteolysis or alternative splicing, activates the anti-angiogenic activity of the synthetase by exposing its active site for interaction with the extracellular domain of the endothelial adhesion molecule VE-cadherin (
      • Wakasugi K.
      • Slike B.M.
      • Hood J.
      • Otani A.
      • Ewalt K.L.
      • Friedlander M.
      • Cheresh D.A.
      • Schimmel P.
      A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.
      ,
      • Zhou Q.
      • Kapoor M.
      • Guo M.
      • Belani R.
      • Xu X.
      • Kiosses W.B.
      • Hanan M.
      • Park C.
      • Armour E.
      • Do M.-H.
      • Nangle L.A.
      • Schimmel P.
      • Yang X.-L.
      Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.
      ). The WHEP domain of TrpRS also mediates direct interactions with DNA-PK and PARP-1 in the nucleus to activate p53 (
      • Sajish M.
      • Zhou Q.
      • Kishi S.
      • Valdez Jr., D.M.
      • Kapoor M.
      • Guo M.
      • Lee S.
      • Kim S.
      • Yang X.-L.
      • Schimmel P.
      Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-γ and p53 signaling.
      ). Interestingly, deletion of the WHEP domain from GlyRS creates a similar conformational opening as seen in the GlyRSCMT mutants (
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ).
      The exceptional prevalence of WHEP domains in CMT-associated aaRSs suggests a potential relevance of this appended domain for CMT. Indeed, Cader and co-workers (
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ) demonstrated that the toxicity of GlyRSP234KY in the fly model is WHEP domain-dependent. Deletion of the WHEP domain abrogated the toxicity of GlyRSP234KY through either ubiquitous or muscle-specific expression, although the mechanism underlying the rescue is not yet understood. Further investigations on the role of the WHEP domain in CMT will be of great interest.

      “Site of lesion” of aaRSs in CMT

      GlyRS is one of the two dual-localized aaRSs, functioning for both cytoplasmic and mitochondrial protein synthesis. Therefore, the initial link of GlyRS to CMT immediately triggered the question of which subcellular site–cytoplasm or mitochondria–is relevant to the disease. The subsequently identified aaRSs all encode enzymes specifically used in the cytoplasm but not in the mitochondria, suggesting that the mitochondrial site may not have a strong relevance for aaRS-linked CMT. Consistently, transgenic expression of the cytoplasmic version of the GlyRSCMT mutants successfully induced phenotypes that recapitulate the hallmarks of the human diseases (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ,
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ,
      • Grice S.J.
      • Sleigh J.N.
      • Motley W.W.
      • Liu J.L.
      • Burgess R.W.
      • Talbot K.
      • Cader M.Z.
      Dominant, toxic gain-of-function mutations in GARS lead to non-cell autonomous neuropathology.
      ). However, it became clear that these supposedly cytoplasmic-restricted enzymes also have the potential to be multilocalized in the mitochondria and nucleus and to be secreted (
      • Debard S.
      • Bader G.
      • De Craene J.O.
      • Enkler L.
      • Bär S.
      • Laporte D.
      • Hammann P.
      • Myslinski E.
      • Senger B.
      • Friant S.
      • Becker H.D.
      Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.
      ).
      A neuron-specific mitochondrial defect was detected in the induced neuronal progenitor cells of a CMT2D patient carrying a dominant mono-allelic mutation H162R GlyRS (reported as H216R due to the inclusion of the mitochondrial targeting sequence) (
      • Boczonadi V.
      • Meyer K.
      • Gonczarowska-Jorge H.
      • Griffin H.
      • Roos A.
      • Bartsakoulia M.
      • Bansagi B.
      • Ricci G.
      • Palinkas F.
      • Zahedi R.P.
      • Bruni F.
      • Kaspar B.
      • Lochmüller H.
      • Boycott K.M.
      • Müller J.S.
      • Horvath R.
      Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.
      ). The defects include reduced levels of both mtDNA and nucleus-encoded mitochondrial respiratory chain complexes and decreased mitochondrial respiration and a reduced level of vesicle-associated membrane protein-associated protein B (as part of the mitochondria-associated endoplasmic reticulum membrane complex) and its downstream signaling, including mitochondrial calcium uptake and autophagy. As calcium uptake regulates synaptic vesicles at the neuromuscular junction, this study may provide part of the explanation for the presynaptic defects of neuromuscular transmission in CMT2D mice (
      • Spaulding E.L.
      • Sleigh J.N.
      • Morelli K.H.
      • Pinter M.J.
      • Burgess R.W.
      • Seburn K.L.
      Synaptic deficits at neuromuscular junctions in two mouse models of Charcot–Marie–Tooth type 2d.
      ).
      As discussed above, GlyRSCMT could be secreted and interfere with proper signaling in motor and sensory neurons through aberrant interactions with ectodomains of membrane receptors (e.g. Nrp1 and Trk) (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ,
      • Sleigh J.N.
      • Dawes J.M.
      • West S.J.
      • Wei N.
      • Spaulding E.L.
      • Gómez-Martín A.
      • Zhang Q.
      • Burgess R.W.
      • Cader M.Z.
      • Talbot K.
      • Yang X.-L.
      • Bennett D.L.
      • Schiavo G.
      Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.
      ). Mutant GlyRSCMT also causes an axonal transport defect due to an aberrant interaction with intracellular protein HDAC6 (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ,
      • Benoy V.
      • Van Helleputte L.
      • Prior R.
      • d’Ydewalle C.
      • Haeck W.
      • Geens N.
      • Scheveneels W.
      • Schevenels B.
      • Cader M.Z.
      • Talbot K.
      • Kozikowski A.P.
      • Vanden Berghe P.
      • Van Damme P.
      • Robberecht W.
      • Van Den Bosch L.
      HDAC6 is a therapeutic target in mutant GARS-induced Charcot–Marie–Tooth disease.
      ), presumably in the cytoplasm. Moreover, the relevance of the nucleus in TyrRS-linked CMT has been demonstrated using the Drosophila model.4 Therefore, the “site of lesion” of CMT-linked aaRSs should not be restricted to the cytosol and/or the mitochondria and is likely to involve multiple compartments.

      Concluding remarks and future directions

      Peripheral neuropathy was the first human disease linked to aaRSs. The number of CMT-linked aaRSs has now expanded to a total of six family members (Figure 1, Figure 3). Although this number may be further increased, we speculate that not all aaRSs have the potential to be linked to CMT through dominant mutations. Certain molecular features, such as the capacity for dimerization, the lack of association to the large MSC, and the possession of a helix-turn-helix WHEP domain, are prevalent in CMT-linked aaRS members (Fig. 2), suggesting selectivity.
      Although CMT-causing mutations have loss-of-function properties, genetic studies have clearly demonstrated that dominant mutations in GlyRS cause CMT2D through toxic gain-of-function effects (Fig. 4), and this may apply to other aaRS-linked CMT subtypes. It is important to differentiate the disease-causing features of a mutation from its many other possible effects. This is particularly important for understanding dominantly transmitted diseases in which the functional effect of a mutation could be different in the absence or the presence of the WT protein.
      The most important future direction for the field is to understand the commonality in pathogenesis among different aaRS-linked CMT forms. The disease-causing mechanism of aaRS-linked CMT is likely to be multifactorial and to involve multiple cellular compartments, including the extracellular space. At the same time, some common pathogenic mechanisms among different aaRSs are expected to contribute to their overall similar clinical phenotypes. Commonalities have emerged from the genetic (
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ), functional (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ), and structural studies (Figure 5, Figure 67) (
      • He W.
      • Zhang H.-M.
      • Chong Y.E.
      • Guo M.
      • Marshall A.G.
      • Yang X.-L.
      Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening.
      ,
      • Blocquel D.
      • Li S.
      • Wei N.
      • Daub H.
      • Sajish M.
      • Erfurth M.-L.
      • Kooi G.
      • Zhou J.
      • Bai G.
      • Schimmel P.
      • Jordanova A.
      • Yang X.-L.
      Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy.
      ). For example, common genetic modifiers with nuclear localization were found in fly-based screens for the CMT-associated mutants GlyRS and TyrRS (
      • Ermanoska B.
      • Motley W.W.
      • Leitão-Gonçalves R.
      • Asselbergh B.
      • Lee L.H.
      • De Rijk P.
      • Sleegers K.
      • Ooms T.
      • Godenschwege T.A.
      • Timmerman V.
      • Fischbeck K.H.
      • Jordanova A.
      CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.
      ). Expression of either the GlyRS or TyrRS mutant in Drosophila impairs protein synthesis through an unknown mechanism that is independent of tRNA aminoacylation (
      • Niehues S.
      • Bussmann J.
      • Steffes G.
      • Erdmann I.
      • Köhrer C.
      • Sun L.
      • Wagner M.
      • Schäfer K.
      • Wang G.
      • Koerdt S.N.
      • Stum M.
      • Jaiswal S.
      • RajBhandary U.L.
      • Thomas U.
      • Aberle H.
      • Burgess R.W.
      • et al.
      Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases.
      ). Different CMT-linked mutations in different aaRSs (e.g. GlyRS and TyrRS) cause shared conformational openings that expose new protein surfaces for potential aberrant interactions with other proteins, nucleic acids, or small molecules (Fig. 7). This conceptual framework has guided the discovery of many aberrant interaction partners of CMT-linked aaRSs, including Nrp1–Plexin, Trk, HDAC6, and TRIM28. These interactions were identified through a combination of serendipity or hypothesis-based investigations. For future studies, unbiased, systematic approaches are necessary to further reveal the global aberrant interactome of aaRS mutants, from which commonality among different aaRS-linked CMT forms may emerge.
      Understanding the commonality in pathogenesis among different aaRS-linked CMT subtypes is key for therapeutic development. Although gene therapies have emerged strongly for monogenic disease such as CMT (
      • Zhao H.T.
      • Damle S.
      • Ikeda-Lee K.
      • Kuntz S.
      • Li J.
      • Mohan A.
      • Kim A.
      • Hung G.
      • Scheideler M.A.
      • Scherer S.S.
      • Svaren J.
      • Swayze E.E.
      • Kordasiewicz H.B.
      PMP22 antisense oligonucleotides reverse Charcot–Marie–Tooth disease type 1A features in rodent models.
      ), the large number of different mutations involved and the small number of patients affected by each mutation render classic gene therapy onerous for aaRS-linked CMT. Identifying a causal treatment strategy applicable to different mutations in several genes would therefore be the most attractive therapeutic approach.
      Several treatment strategies have been tested in animal models for proof-of-concept. For CMT2D, overexpressing VEGF in hindlimb muscles to overcome the competition of GlyRSP234KY for Nrp1 binding improved the motor performance of GarsP234KY/+ mice (
      • He W.
      • Bai G.
      • Zhou H.
      • Wei N.
      • White N.M.
      • Lauer J.
      • Liu H.
      • Shi Y.
      • Dumitru C.D.
      • Lettieri K.
      • Shubayev V.
      • Jordanova A.
      • Guergueltcheva V.
      • Griffin P.R.
      • Burgess R.W.
      • Pfaff S.L.
      • Yang X.-L.
      CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.
      ); administration of the HDAC6 inhibitor tubastatin A to block the aberrant HDAC6–GlyRS interaction and the resulting HDAC6 hyperactivation also improved motor performance of both GarsP234KY/+ and GarsC157R/+ mice (
      • Mo Z.
      • Zhao X.
      • Liu H.
      • Hu Q.
      • Chen X.Q.
      • Pham J.
      • Wei N.
      • Liu Z.
      • Zhou J.
      • Burgess R.W.
      • Pfaff S.L.
      • Caskey C.T.
      • Wu C.
      • Bai G.
      • Yang X.L.
      Aberrant GlyRS–HDAC6 interaction linked to axonal transport deficits in Charcot–Marie–Tooth neuropathy.
      ,
      • Benoy V.
      • Van Helleputte L.
      • Prior R.
      • d’Ydewalle C.
      • Haeck W.
      • Geens N.
      • Scheveneels W.
      • Schevenels B.
      • Cader M.Z.
      • Talbot K.
      • Kozikowski A.P.
      • Vanden Berghe P.
      • Van Damme P.
      • Robberecht W.
      • Van Den Bosch L.
      HDAC6 is a therapeutic target in mutant GARS-induced Charcot–Marie–Tooth disease.
      ). A symptomatic treatment using the postsynaptic-acting cholinesterase inhibitor physostigmine to overcome presynaptic defects at neuromuscular junctions also showed benefit (
      • Spaulding E.L.
      • Sleigh J.N.
      • Morelli K.H.
      • Pinter M.J.
      • Burgess R.W.
      • Seburn K.L.
      Synaptic deficits at neuromuscular junctions in two mouse models of Charcot–Marie–Tooth type 2d.
      ). For DI-CMTC, administration of the p300/CBP-associated factor inhibitor embelin to impede the nuclear entry of TyrRS improved the viability and motor performance of TyrRSE196K-expressing Drosophila .4 Although these function-based strategies are likely to benefit patients with different mutations, they may have limited efficacy as a monotherapy, because they target only one of many aspects of the pathology. If the mutation-induced, shared conformational opening is indeed a fundamental cause of aaRS-linked CMT forms, targeting the opening site on each aaRS to prevent aberrant interactions may be a more effective strategy.
      Another priority for the field is to develop additional mouse models based on human mutations and on aaRS-linked CMT subtypes beyond CMT2D. They are much needed to facilitate the study of common mechanisms and therapeutic development. Progress in recent years has benefited tremendously from the availability of animal models, especially the mouse models. These animal models also provide opportunities to understand the physiological significance of aaRSs with their enzymatic and nonenzymatic regulatory functions. In this regard, the neurodegenerative CMT disease phenotypes might represent a dysregulated state of the normal homeostatic, regulatory network made of a selective group of aaRSs.

      Acknowledgments

      We thank Paul Schimmel, James Sleigh, and Erik Storkebaum, Albena Jordanova, and Robert Burgess for advice on the manuscript and/or the figures.

      References

        • Woese C.R.
        • Olsen G.J.
        • Ibba M.
        • Söll D.
        Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.
        Microbiol. Mol. Biol. Rev. 2000; 64 (10704480): 202-236
        • Guo M.
        • Schimmel P.
        Essential nontranslational functions of tRNA synthetases.
        Nat. Chem. Biol. 2013; 9 (23416400): 145-153
        • Yao P.
        • Fox P.L.
        Aminoacyl-tRNA synthetases in medicine and disease.
        EMBO Mol. Med. 2013; 5 (23427196): 332-343
        • Kim S.
        • You S.
        • Hwang D.
        Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping.
        Nat. Rev. Cancer. 2011; 11 (21941282): 708-718
        • Alexandrova J.
        • Paulus C.
        • Rudinger-Thirion J.
        • Jossinet F.
        • Frugier M.
        Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase.
        RNA Biol. 2015; 12 (26327585): 1301-1313
        • Tolkunova E.
        • Park H.
        • Xia J.
        • King M.P.
        • Davidson E.
        The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual: alternative splicing of the primary transcript.
        J. Biol. Chem. 2000; 275 (10952987): 35063-35069
        • Nagao A.
        • Suzuki T.
        • Katoh T.
        • Sakaguchi Y.
        • Suzuki T.
        Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19805282): 16209-16214
        • Eriani G.
        • Delarue M.
        • Poch O.
        • Gangloff J.
        • Moras D.
        Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.
        Nature. 1990; 347 (2203971): 203-206
        • Ribas de Pouplana L.
        • Schimmel P.
        Two classes of tRNA synthetases.
        Cell. 2001; 104 (11269237): 191-193
        • Sun L.
        • Song Y.
        • Blocquel D.
        • Yang X.-L.
        • Schimmel P.
        Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS.
        Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27911835): 14300-14305
        • Kyriacou S.V.
        • Deutscher M.P.
        An important role for the multienzyme aminoacyl-tRNA synthetase complex in mammalian translation and cell growth.
        Mol. Cell. 2008; 29 (18313381): 419-427
        • Ray P.S.
        • Arif A.
        • Fox P.L.
        Macromolecular complexes as depots for releasable regulatory proteins.
        Trends Biochem. Sci. 2007; 32 (17321138): 158-164
        • Fu G.
        • Xu T.
        • Shi Y.
        • Wei N.
        • Yang X.-L.
        tRNA-controlled nuclear import of a human tRNA synthetase.
        J. Biol. Chem. 2012; 287 (22291016): 9330-9334
        • Guo M.
        • Yang X.-L.
        • Schimmel P.
        New functions of aminoacyl-tRNA synthetases beyond translation.
        Nat. Rev. Mol. Cell Biol. 2010; 11 (20700144): 668-674
        • Guo M.
        • Schimmel P.
        • Yang X.-L.
        Functional expansion of human tRNA synthetases achieved by structural inventions.
        FEBS Lett. 2010; 584 (19932696): 434-442
        • Yang X.-L.
        Structural disorder in expanding the functionome of aminoacyl-tRNA synthetases.
        Chem. Biol. 2013; 20 (24054183): 1093-1099
        • Xu X.
        • Shi Y.
        • Zhang H.-M.
        • Swindell E.C.
        • Marshall A.G.
        • Guo M.
        • Kishi S.
        • Yang X.-L.
        Unique domain appended to vertebrate tRNA synthetase is essential for vascular development.
        Nat. Commun. 2012; 3 (22353712): 681
        • Antonellis A.
        • Ellsworth R.E.
        • Sambuughin N.
        • Puls I.
        • Abel A.
        • Lee-Lin S.-Q.
        • Jordanova A.
        • Kremensky I.
        • Christodoulou K.
        • Middleton L.T.
        • Sivakumar K.
        • Ionasescu V.
        • Funalot B.
        • Vance J.M.
        • Goldfarb L.G.
        • et al.
        Glycyl tRNA synthetase mutations in Charcot–Marie–Tooth disease type 2D and distal spinal muscular atrophy type V.
        Am. J. Hum. Genet. 2003; 72 (12690580): 1293-1299
        • Rossor A.M.
        • Polke J.M.
        • Houlden H.
        • Reilly M.M.
        Clinical implications of genetic advances in Charcot–Marie–Tooth disease.
        Nat. Rev. Neurol. 2013; 9 (24018473): 562-571
        • Rossor A.M.
        • Tomaselli P.J.
        • Reilly M.M.
        Recent advances in the genetic neuropathies.
        Curr. Opin. Neurol. 2016; 29 (27584852): 537-548
        • Gutmann L.
        • Shy M.
        Update on Charcot–Marie–Tooth disease.
        Curr. Opin. Neurol. 2015; 28 (26263471): 462-467
        • Motley W.W.
        • Talbot K.
        • Fischbeck K.H.
        GARS axonopathy: not every neuron's cup of tRNA.
        Trends Neurosci. 2010; 33 (20152552): 59-66
        • Boczonadi V.
        • Jennings M.J.
        • Horvath R.
        The role of tRNA synthetases in neurological and neuromuscular disorders.
        FEBS Lett. 2018; 592 (29288497): 703-717
        • Oprescu S.N.
        • Griffin L.B.
        • Beg A.A.
        • Antonellis A.
        Predicting the pathogenicity of aminoacyl-tRNA synthetase mutations.
        Methods. 2017; 113 (27876679): 139-151
        • Storkebaum E.
        Peripheral neuropathy via mutant tRNA synthetases: inhibition of protein translation provides a possible explanation.
        BioEssays. 2016; 38 (27352040): 818-829
        • Jordanova A.
        • Irobi J.
        • Thomas F.P.
        • Van Dijck P.
        • Meerschaert K.
        • Dewil M.
        • Dierick I.
        • Jacobs A.
        • De Vriendt E.
        • Guergueltcheva V.
        • Rao C.V.
        • Tournev I.
        • Gondim F.A.
        • D’Hooghe M.
        • Van Gerwen V.
        • et al.
        Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot–Marie–Tooth neuropathy.
        Nat. Genet. 2006; 38 (16429158): 197-202