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.

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.

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.

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.

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.

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.

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.

T Cells from NOD-PerIg Mice Target Both Pancreatic and Neuronal Tissue.

It has become increasingly appreciated that autoimmune responses against neuronal components play an important role in type 1 diabetes (T1D) pathogenesis. In fact, a large proportion of islet-infiltrating B lymphocytes in the NOD mouse model of T1D produce Abs directed against the neuronal type III intermediate filament protein peripherin. NOD-PerIg mice are a previously developed BCR-transgenic model in which virtually all B lymphocytes express the H and L chain Ig molecules from the intra-islet-derived anti-peripherin-reactive hybridoma H280. NOD-PerIg mice have accelerated T1D development, and PerIg B lymphocytes actively proliferate within islets and expand cognitively interactive pathogenic T cells from a pool of naive precursors. We now report adoptively transferred T cells or whole splenocytes from NOD-PerIg mice expectedly induce T1D in NOD.scid recipients but, depending on the kinetics of disease development, can also elicit a peripheral neuritis (with secondary myositis). This neuritis was predominantly composed of CD4+ and CD8+ T cells. Ab depletion studies showed neuritis still developed in the absence of NOD-PerIg CD8+ T cells but required CD4+ T cells. Surprisingly, sciatic nerve-infiltrating CD4+ cells had an expansion of IFN-γ- and TNF-α- double-negative cells compared with those within both islets and spleen. Nerve and islet-infiltrating CD4+ T cells also differed by expression patterns of CD95, PD-1, and Tim-3. Further studies found transitory early B lymphocyte depletion delayed T1D onset in a portion of NOD-PerIg mice, allowing them to survive long enough to develop neuritis outside of the transfer setting. Together, this study presents a new model of peripherin-reactive B lymphocyte-dependent autoimmune neuritis.

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4.

The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

Structural Variant in Mitochondrial-Associated Gene (MRPL3) Induces Adult-Onset Neurodegeneration with Memory Impairment in the Mouse.

An impediment to the development of effective therapies for neurodegenerative disease is that available animal models do not reproduce important clinical features such as adult-onset and stereotypical patterns of progression. Using in vivo magnetic resonance imaging and behavioral testing to study male and female decrepit mice, we found a stereotypical neuroanatomical pattern of progression of the lesion along the limbic system network and an associated memory impairment. Using structural variant analysis, we identified an intronic mutation in a mitochondrial-associated gene (Mrpl3) that is responsible for the decrepit phenotype. While the function of this gene is unknown, embryonic lethality in Mrpl3 knock-out mice suggests it is critical for early development. The observation that a mutation linked to energy metabolism precipitates a pattern of neurodegeneration via cell death across disparate but linked brain regions may explain how stereotyped patterns of neurodegeneration arise in humans or define a not yet identified human disease.SIGNIFICANCE STATEMENT The development of novel therapies for adult-onset neurodegenerative disease has been impeded by the limitations of available animal models in reproducing many of the clinical features. Here, we present a novel spontaneous mutation in a mitochondrial-associated gene in a mouse (termed decrepit) that results in adult-onset neurodegeneration with a stereotypical neuroanatomical pattern of progression and an associated memory impairment. The decrepit mouse model may represent a heretofore undiagnosed human disease and could serve as a new animal model to study neurodegenerative disease.

CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase.

Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes. Charcot-Marie-Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies. A subtype of these diseases--CMT type 2D (CMT2D)--is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function. How mutations in GlyRS (GlyRS(CMT2D)) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRS(CMT2D) acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRS(CMT2D) to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRS(CMT2D) that antagonizes the VEGF-Nrp1 interaction, and indicate that the VEGF-Nrp1 signalling axis is an actionable target for treating CMT2D.

DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members.

During neural development, self-avoidance ensures that a neuron's processes arborize to evenly fill a particular spatial domain. At the individual cell level, self-avoidance is promoted by genes encoding cell-surface molecules capable of generating thousands of diverse isoforms, such as Dscam1 (Down syndrome cell adhesion molecule 1) in Drosophila Isoform choice differs between neighboring cells, allowing neurons to distinguish "self" from "nonself". In the mouse retina, Dscam promotes self-avoidance at the level of cell types, but without extreme isoform diversity. Therefore, we hypothesize that DSCAM is a general self-avoidance cue that "masks" other cell type-specific adhesion systems to prevent overly exuberant adhesion. Here, we provide in vivo and in vitro evidence that DSCAM masks the functions of members of the cadherin superfamily, supporting this hypothesis. Thus, unlike the isoform-rich molecules tasked with self-avoidance at the individual cell level, here the diversity resides on the adhesive side, positioning DSCAM as a generalized modulator of cell adhesion during neural development.

Trk receptor signaling and sensory neuron fate are perturbed in human neuropathy caused by Gars mutations.

Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.

Model validity for preclinical studies in precision medicine: precisely how precise do we need to be?

The promise of personalized medicine is that each patient's treatment can be optimally tailored to their disease. In turn, their disease, as well as their response to the treatment, is determined by their genetic makeup and the "environment," which relates to their general health, medical history, personal habits, and surroundings. Developing such optimized treatment strategies is an admirable goal and success stories include examples such as switching chemotherapy agents based on a patient's tumor genotype. However, it remains a challenge to apply precision medicine to diseases for which there is no known effective treatment. Such diseases require additional research, often using experimentally tractable models. Presumably, models that recapitulate as much of the human pathophysiology as possible will be the most predictive. Here we will discuss the considerations behind such "precision models." What sort of precision is required and under what circumstances? How can the predictive validity of such models be improved? Ultimately, there is no perfect model, but our continually improving ability to genetically engineer a variety of systems allows the generation of more and more precise models. Furthermore, our steadily increasing awareness of risk alleles, genetic background effects, multifactorial disease processes, and gene by environment interactions also allows increasingly sophisticated models that better reproduce patients' conditions. In those cases where the research has progressed sufficiently far, results from these models appear to often be translating to effective treatments for patients.

Synaptic Deficits at Neuromuscular Junctions in Two Mouse Models of Charcot-Marie-Tooth Type 2d.

Patients with Charcot-Marie-Tooth Type 2D (CMT2D), caused by dominant mutations in Glycl tRNA synthetase (GARS), present with progressive weakness, consistently in the hands, but often in the feet also. Electromyography shows denervation, and patients often report that early symptoms include cramps brought on by cold or exertion. Based on reported clinical observations, and studies of mouse models of CMT2D, we sought to determine whether weakened synaptic transmission at the neuromuscular junction (NMJ) is an aspect of CMT2D. Quantal analysis of NMJs in two different mouse models of CMT2D (Gars(P278KY), Gars(C201R)), found synaptic deficits that correlated with disease severity and progressed with age. Results of voltage-clamp studies revealed presynaptic defects characterized by: (1) decreased frequency of spontaneous release without any change in quantal amplitude (miniature endplate current), (2) reduced amplitude of evoked release (endplate current) and quantal content, (3) age-dependent changes in the extent of depression in response to repetitive stimulation, and (4) release failures at some NMJs with high-frequency, long-duration stimulation. Drugs that modify synaptic efficacy were tested to see whether neuromuscular performance improved. The presynaptic action of 3,4 diaminopyridine was not beneficial, whereas postsynaptic-acting physostigmine did improve performance. Smaller mutant NMJs with correspondingly fewer vesicles and partial denervation that eliminates some release sites also contribute to the reduction of release at a proportion of mutant NMJs. Together, these voltage-clamp data suggest that a number of release processes, while essentially intact, likely operate suboptimally at most NMJs of CMT2D mice. SIGNIFICANCE STATEMENT: We have uncovered a previously unrecognized aspect of axonal Charcot-Marie-Tooth disease in mouse models of CMT2D. Synaptic dysfunction contributes to impaired neuromuscular performance and disease progression. This suggests that drugs which improve synaptic efficacy at the NMJ could be considered in treating the pathophysiology of CMT2D patients.

Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology.

Charcot-Marie-Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Here, using novel Drosophila models, we show a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.