Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Lack of effect from genetic deletion of Hdac6 in a humanized mouse model of CMT2D.

BACKGROUND: Inhibition of HDAC6 has been proposed as a broadly applicable therapeutic strategy for Charcot-Marie-Tooth disease (CMT). Inhibition of HDAC6 increases the acetylation of proteins important in axonal trafficking, such as α-tubulin and Miro, and has been shown to be efficacious in several preclinical studies using mouse models of CMT. AIMS: Here, we sought to expand on previous preclinical studies by testing the effect of genetic deletion of Hdac6 on mice carrying a humanized knockin allele of Gars1, a model of CMT-type 2D. METHODS: Gars1ΔETAQ mice were bred to an Hdac6 knockout strain, and the resulting offspring were evaluated for clinically relevant outcomes. RESULTS: The genetic deletion of Hdac6 increased α-tubulin acetylation in the sciatic nerves of both wild-type and Gars1ΔETAQ mice. However, when tested at 5 weeks of age, the Gars1ΔETAQ mice lacking Hdac6 showed no changes in body weight, muscle atrophy, grip strength or endurance, sciatic motor nerve conduction velocity, compound muscle action potential amplitude, or peripheral nerve histopathology compared to Gars1ΔETAQ mice with intact Hdac6. INTERPRETATION: Our results differ from those of two previous studies that demonstrated the benefit of the HDAC6 inhibitor tubastatin A in mouse models of CMT2D. While we cannot fully explain the different outcomes, our results offer a counterexample to the benefit of inhibiting HDAC6 in CMT2D, suggesting additional research is necessary.

Testing SIPA1L2 as a modifier of CMT1A using mouse models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), leading to muscle weakness and loss of sensation in the hands and feet. A recent case-only genome-wide association study of CMT1A patients conducted by the Inherited Neuropathy Consortium identified a strong association between strength of foot dorsiflexion and variants in signal induced proliferation associated 1 like 2 (SIPA1L2), indicating that it may be a genetic modifier of disease. To validate SIPA1L2 as a candidate modifier and to assess its potential as a therapeutic target, we engineered mice with deletion of exon 1 (including the start codon) of the Sipa1l2 gene and crossed them to the C3-PMP22 mouse model of CMT1A. Neuromuscular phenotyping showed that Sipa1l2 deletion in C3-PMP22 mice preserved muscular endurance assayed by inverted wire hang duration and changed femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggest involvement of Sipa1l2 in cholesterol biosynthesis, a pathway that is also implicated in C3-PMP22 mice. Although Sipa1l2 deletion did impact CMT1A-associated phenotypes, thereby validating a genetic interaction, the overall effect on neuropathy was mild.

Dominant NARS1 mutations causing axonal Charcot-Marie-Tooth disease expand NARS1-associated diseases.

Pathogenic variants in six aminoacyl-tRNA synthetase (ARS) genes are implicated in neurological disorders, most notably inherited peripheral neuropathies. ARSs are enzymes that charge tRNA molecules with cognate amino acids. Pathogenic variants in asparaginyl-tRNA synthetase (NARS1) cause a neurological phenotype combining developmental delay, ataxia and demyelinating peripheral neuropathy. NARS1 has not yet been linked to axonal Charcot-Marie-Tooth disease. Exome sequencing of patients with inherited peripheral neuropathies revealed three previously unreported heterozygous NARS1 variants in three families. Clinical and electrophysiological details were assessed. We further characterized all three variants in a yeast complementation model and used a knock-in mouse model to study variant p.Ser461Phe. All three variants (p.Met236del, p.Cys342Tyr and p.Ser461Phe) co-segregate with the sensorimotor axonal neuropathy phenotype. Yeast complementation assays show that none of the three NARS1 variants support wild-type yeast growth when tested in isolation (i.e. in the absence of a wild-type copy of NARS1), consistent with a loss-of-function effect. Similarly, the homozygous knock-in mouse model (p.Ser461Phe/Ser472Phe in mouse) also demonstrated loss-of-function characteristics. We present three previously unreported NARS1 variants segregating with a sensorimotor neuropathy phenotype in three families. Functional studies in yeast and mouse support variant pathogenicity. Thus, NARS1 is the seventh ARS implicated in dominant axonal Charcot-Marie-Tooth disease, further stressing that all dimeric ARSs should be evaluated for Charcot-Marie-Tooth disease.

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo.

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically-interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific vs. shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified PKC phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via MARCKS is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remains unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point, and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

Testing SIPA1L2 as a modifier of CMT1A using mouse models.

Charcot-Marie-Tooth 1A is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), which produces muscle weakness and loss of sensation in the hands and feet. A recent case-only genome wide association study by the Inherited Neuropathy Consortium identified a strong association between variants in signal induced proliferation associated 1 like 2 (SIPA1L2) and strength of foot dorsiflexion. To validate SIPA1L2 as a candidate modifier, and to assess its potential as a therapeutic target, we engineered mice with a deletion in SIPA1L2 and crossed them to the C3-PMP22 mouse model of CMT1A. We performed neuromuscular phenotyping and identified an interaction between Sipa1l2 deletion and muscular endurance decrements assayed by wire-hang duration in C3-PMP22 mice, as well as several interactions in femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggested an involvement of Sipa1l2 in cholesterol biosynthesis, which was also implicated in C3-PMP22 mice. Though several interactions between Sipa1l2 deletion and CMT1A-associated phenotypes were identified, validating a genetic interaction, the overall effect on neuropathy was small.

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

Two new mouse models of Gjb1-associated Charcot-Marie-Tooth disease type 1X.

BACKGROUND: Charcot-Marie-Tooth disease type 1X is caused by mutations in GJB1, which is the second most common gene associated with inherited peripheral neuropathy. The GJB1 gene encodes connexin 32 (CX32), a gap junction protein expressed in myelinating glial cells. The gene is X-linked, and the mutations cause a loss of function. AIMS: A large number of disease-associated variants have been identified, and many result in mistrafficking and mislocalization of the protein. An existing knockout mouse lacking Gjb1 expression provides a valid animal model of CMT1X, but the complete lack of protein may not fully recapitulate the disease mechanisms caused by aberrant CX32 proteins. To better represent the spectrum of human CMT1X-associated mutations, we have generated a new Gjb1 knockin mouse model. METHODS: CRISPR/Cas9 genome editing was used to produce mice carrying the R15Q mutation in Gjb1. In addition, we identified a second allele with an early frame shift mutation in codon 7 (del2). Mice were analyzed using clinically relevant molecular, histological, neurophysiological, and behavioral assays. RESULTS: Both alleles produce protein detectable by immunofluorescence in Schwann cells, with some protein properly localizing to nodes of Ranvier. However, both alleles also result in peripheral neuropathy with thinly myelinated and demyelinated axons, as well as degenerating and regenerating axons, predominantly in distal motor nerves. Nerve conduction velocities were only mildly reduced at later ages and compound muscle action potential amplitudes were not reduced. Levels of neurofilament light chain in plasma were elevated in both alleles. The del2 mice have an onset at ~3 months of age, whereas the R15Q mice had a later onset at 5-6 months of age, suggesting a milder loss of function. Both alleles performed comparably to wild type littermates in accelerating rotarod and grip strength tests of neuromuscular performance. INTERPRETATION: We have generated and characterized two new mouse models of CMT1X that will be useful for future mechanistic and preclinical studies.

A Novel ENU-Induced Mfn2 Mutation Causes Motor Deficits in Mice without Causing Peripheral Neuropathy.

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication. In humans, over 100 MFN2 mutations are associated with a form of inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Here we describe an ENU-induced mutant mouse line with a recessive neuromuscular phenotype. Behavioral screening showed progressive weight loss and rapid deterioration of motor function beginning at 8 weeks. Mapping and sequencing revealed a missense mutation in exon 18 of Mfn2 (T1928C; Leu643Pro), within the transmembrane domain. Compared to wild-type and heterozygous littermates, Mfn2L643P/L643P mice exhibited diminished rotarod performance and decreases in activity in the open field test, muscular endurance, mean mitochondrial diameter, sensory tests, mitochondrial DNA content, and MFN2 protein levels. However, tests of peripheral nerve physiology and histology were largely normal. Mutant leg bones had reduced cortical bone thickness and bone area fraction. Together, our data indicate that Mfn2L643P causes a recessive motor phenotype with mild bone and mitochondrial defects in mice. Lack of apparent nerve pathology notwithstanding, this is the first reported mouse model with a mutation in the transmembrane domain of the protein, which may be valuable for researchers studying MFN2 biology.

An allelic series of spontaneous Rorb mutant mice exhibit a gait phenotype, changes in retina morphology and behavior, and gene expression signatures associated with the unfolded protein response.

The Retinoid-related orphan receptor beta (RORß) gene encodes a developmental transcription factor and has 2 predominant isoforms created through alternative first exon usage; one specific to the retina and another present more broadly in the central nervous system, particularly regions involved in sensory processing. RORß belongs to the nuclear receptor family and plays important roles in cell fate specification in the retina and cortical layer formation. In mice, loss of RORß causes disorganized retina layers, postnatal degeneration, and production of immature cone photoreceptors. Hyperflexion or "high-stepping" of rear limbs caused by reduced presynaptic inhibition by Rorb-expressing inhibitory interneurons of the spinal cord is evident in RORß-deficient mice. RORß variants in patients are associated with susceptibility to various neurodevelopmental conditions, primarily generalized epilepsies, but including intellectual disability, bipolar, and autism spectrum disorders. The mechanisms by which RORß variants confer susceptibility to these neurodevelopmental disorders are unknown but may involve aberrant neural circuit formation and hyperexcitability during development. Here we report an allelic series in 5 strains of spontaneous Rorb mutant mice with a high-stepping gait phenotype. We show retinal abnormalities in a subset of these mutants and demonstrate significant differences in various behavioral phenotypes related to cognition. Gene expression analyses in all 5 mutants reveal a shared over-representation of the unfolded protein response and pathways related to endoplasmic reticulum stress, suggesting a possible mechanism of susceptibility relevant to patients.

tRNA Dysregulation in Neurodevelopmental and Neurodegenerative Diseases.

Transfer RNAs (tRNAs) decode messenger RNA codons to peptides at the ribosome. The nuclear genome contains many tRNA genes for each amino acid and even each anticodon. Recent evidence indicates that expression of these tRNAs in neurons is regulated, and they are not functionally redundant. When specific tRNA genes are nonfunctional, this results in an imbalance between codon demand and tRNA availability. Furthermore, tRNAs are spliced, processed, and posttranscriptionally modified. Defects in these processes lead to neurological disorders. Finally, mutations in the aminoacyl tRNA synthetases (aaRSs) also lead to disease. Recessive mutations in several aaRSs cause syndromic disorders, while dominant mutations in a subset of aaRSs lead to peripheral neuropathy, again due to an imbalance between tRNA supply and codon demand. While it is clear that disrupting tRNA biology often leads to neurological disease, additional research is needed to understand the sensitivity of neurons to these changes.

A Unique Role for Protocadherin γC3 in Promoting Dendrite Arborization through an Axin1-Dependent Mechanism.

The establishment of a functional cerebral cortex depends on the proper execution of multiple developmental steps, culminating in dendritic and axonal outgrowth and the formation and maturation of synaptic connections. Dysregulation of these processes can result in improper neuronal connectivity, including that associated with various neurodevelopmental disorders. The γ-Protocadherins (γ-Pcdhs), a family of 22 distinct cell adhesion molecules that share a C-terminal cytoplasmic domain, are involved in multiple aspects of neurodevelopment including neuronal survival, dendrite arborization, and synapse development. The extent to which individual γ-Pcdh family members play unique versus common roles remains unclear. We demonstrated previously that the γ-Pcdh-C3 isoform (γC3), via its unique "variable" cytoplasmic domain (VCD), interacts in cultured cells with Axin1, a Wnt-pathway scaffold protein that regulates the differentiation and morphology of neurons. Here, we confirm that γC3 and Axin1 interact in the cortex in vivo and show that both male and female mice specifically lacking γC3 exhibit disrupted Axin1 localization to synaptic fractions, without obvious changes in dendritic spine density or morphology. However, both male and female γC3 knock-out mice exhibit severely decreased dendritic complexity of cortical pyramidal neurons that is not observed in mouse lines lacking several other γ-Pcdh isoforms. Combining knock-out with rescue constructs in cultured cortical neurons pooled from both male and female mice, we show that γC3 promotes dendritic arborization through an Axin1-dependent mechanism mediated through its VCD. Together, these data identify a novel mechanism through which γC3 uniquely regulates the formation of cortical circuitry.SIGNIFICANCE STATEMENT The complexity of a neuron's dendritic arbor is critical for its function. We showed previously that the γ-Protocadherin (γ-Pcdh) family of 22 cell adhesion molecules promotes arborization during development; it remained unclear whether individual family members played unique roles. Here, we show that one γ-Pcdh isoform, γC3, interacts in the brain with Axin1, a scaffolding protein known to influence dendrite development. A CRISPR/Cas9-generated mutant mouse line lacking γC3 (but not lines lacking other γ-Pcdhs) exhibits severely reduced dendritic complexity of cerebral cortex neurons. Using cultured γC3 knock-out neurons and a variety of rescue constructs, we confirm that the γC3 cytoplasmic domain promotes arborization through an Axin1-dependent mechanism. Thus, γ-Pcdh isoforms are not interchangeable, but rather can play unique neurodevelopmental roles.

Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

p53-mediated neurodegeneration in the absence of the nuclear protein Akirin2.

Proper gene regulation is critical for both neuronal development and maintenance as the brain matures. We previously demonstrated that Akirin2, an essential nuclear protein that interacts with transcription factors and chromatin remodeling complexes, is required for the embryonic formation of the cerebral cortex. Here we show that Akirin2 plays a mechanistically distinct role in maintaining healthy neurons during cortical maturation. Restricting Akirin2 loss to excitatory cortical neurons resulted in progressive neurodegeneration via necroptosis and severe cortical atrophy with age. Comparing transcriptomes from Akirin2-null postnatal neurons and cortical progenitors revealed that targets of the tumor suppressor p53, a regulator of both proliferation and cell death encoded by Trp53, were consistently upregulated. Reduction of Trp53 rescued neurodegeneration in Akirin2-null neurons. These data: (1) implicate Akirin2 as a critical neuronal maintenance protein, (2) identify p53 pathways as mediators of Akirin2 functions, and (3) suggest Akirin2 dysfunction may be relevant to neurodegenerative diseases.

NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

Precision mouse models of Yars/dominant intermediate Charcot-Marie-Tooth disease type C and Sptlc1/hereditary sensory and autonomic neuropathy type 1.

Animal models of neurodegenerative diseases such as inherited peripheral neuropathies sometimes accurately recreate the pathophysiology of the human disease, and sometimes accurately recreate the genetic perturbations found in patients. Ideally, models achieve both, but this is not always possible; nonetheless, such models are informative. Here we describe two animal models of inherited peripheral neuropathy: mice with a mutation in tyrosyl tRNA-synthetase, YarsE196K , modeling dominant intermediate Charcot-Marie-Tooth disease type C (diCMTC), and mice with a mutation in serine palmitoyltransferase long chain 1, Sptlc1C133W , modeling hereditary sensory and autonomic neuropathy type 1 (HSAN1). YarsE196K mice develop disease-relevant phenotypes including reduced motor performance and reduced nerve conduction velocities by 4 months of age. Peripheral motor axons are reduced in size, but there is no reduction in axon number and plasma neurofilament light chain levels are not increased. Unlike the dominant human mutations, the YarsE196K mice only show these phenotypes as homozygotes, or as compound heterozygotes with a null allele, and no phenotype is observed in E196K or null heterozygotes. The Sptlc1C133W mice carry a knockin allele and show the anticipated increase in 1-deoxysphingolipids in circulation and in a variety of tissues. They also have mild behavioral defects consistent with HSAN1, but do not show neurophysiological defects or axon loss in peripheral nerves or in the epidermis of the hind paw or tail. Thus, despite the biochemical phenotype, the Sptlc1C133W mice do not show a strong neuropathy phenotype. Surprisingly, these mice were lethal as homozygotes, but the heterozygous genotype studied corresponds to the dominant genetics seen in humans. Thus, YarsE196K homozygous mice have a relevant phenotype, but imprecisely reproduce the human genetics, whereas the Sptlc1C133W mice precisely reproduce the human genetics, but do not recreate the disease phenotype. Despite these shortcomings, both models are informative and will be useful for future research.

A longitudinal and cross-sectional study of plasma neurofilament light chain concentration in Charcot-Marie-Tooth disease.

Advances in genetic technology and small molecule drug development have paved the way for clinical trials in Charcot-Marie-Tooth disease (CMT); however, the current FDA-approved clinical trial outcome measures are insensitive to detect a meaningful clinical response. There is, therefore, a need to identify sensitive outcome measures or clinically relevant biomarkers. The aim of this study was to further evaluate plasma neurofilament light chain (NFL) as a disease biomarker in CMT. Plasma NFL was measured using SIMOA technology in both a cross-sectional study of a US cohort of CMT patients and longitudinally over 6 years in a UK CMT cohort. In addition, plasma NFL was measured longitudinally in two mouse models of CMT2D. Plasma concentrations of NFL were increased in a US cohort of patients with CMT1B, CMT1X and CMT2A but not CMT2E compared with controls. In a separate UK cohort, over a 6-year interval, there was no significant change in plasma NFL concentration in CMT1A or HSN1, but a small but significant reduction in patients with CMT1X. Plasma NFL was increased in wild type compared to GARSC201R mice. There was no significant difference in plasma NFL in GARSP278KY compared to wild type mice. In patients with CMT1A, the small difference in cross-sectional NFL concentration vs healthy controls and the lack of change over time suggests that plasma NFL may lack sufficient sensitivity to detect a clinically meaningful treatment response in adulthood.

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase.

Heterozygous mutations in six transfer RNA (tRNA) synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT mutant tRNA synthetases inhibit protein synthesis by an unknown mechanism. We found that CMT mutant glycyl-tRNA synthetases bound tRNAGly but failed to release it, resulting in tRNAGly sequestration. This sequestration potentially depleted the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome. Accordingly, we found ribosome stalling at glycine codons and activation of the integrated stress response (ISR) in affected motor neurons. Moreover, transgenic overexpression of tRNAGly rescued protein synthesis, peripheral neuropathy, and ISR activation in Drosophila and mouse CMT disease type 2D (CMT2D) models. Conversely, inactivation of the ribosome rescue factor GTPBP2 exacerbated peripheral neuropathy. Our findings suggest a molecular mechanism for CMT2D, and elevating tRNAGly levels may thus have therapeutic potential.