A CADM3 variant causes Charcot-Marie-Tooth disease with marked upper limb involvement

The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified—by whole exome sequencing—three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations. A correction has been published: Brain, Volume 144, Issue 7, July 2021, Page e64, https://doi.org/10.1093/brain/awab18

Biallelic mutations in SORD cause a common and potentially treatable hereditary neuropathy with implications for diabetes

Here we report biallelic mutations in the sorbitol dehydrogenase gene (SORD) as the most frequent recessive form of hereditary neuropathy. We identified 45 individuals from 38 families across multiple ancestries carrying the nonsense c.757delG (p.Ala253GlnfsTer27) variant in SORD, in either a homozygous or compound heterozygous state. SORD is an enzyme that converts sorbitol into fructose in the two-step polyol pathway previously implicated in diabetic neuropathy. In patient-derived fibroblasts, we found a complete loss of SORD protein and increased intracellular sorbitol. Furthermore, the serum fasting sorbitol levels in patients were dramatically increased. In Drosophila, loss of SORD orthologs caused synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient-derived fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a novel and potentially treatable cause of neuropathy and may contribute to a better understanding of the pathophysiology of diabetes

Function Over Form: Modeling Groups of Inherited Neurological Conditions in Zebrafish

Zebrafish are a unique cell to behavior model for studying the basic biology of human inherited neurological conditions. Conserved vertebrate genetics and optical transparency provide in vivo access to the developing nervous system as well as high-throughput approaches for drug screens. Here we review zebrafish modeling for two broad groups of inherited conditions that each share genetic and molecular pathways and overlap phenotypically: neurodevelopmental disorders such as Autism Spectrum Disorders (ASD), Intellectual Disability (ID) and Schizophrenia (SCZ), and neurodegenerative diseases, such as Cerebellar Ataxia (CATX), Hereditary Spastic Paraplegia (HSP) and Charcot-Marie Tooth Disease (CMT). We also conduct a small meta-analysis of zebrafish orthologs of high confidence neurodevelopmental disorder and neurodegenerative disease genes by looking at duplication rates and relative protein sizes. In the past zebrafish genetic models of these neurodevelopmental disorders and neurodegenerative diseases have provided insight into cellular, circuit and behavioral level mechanisms contributing to these conditions. Moving forward, advances in genetic manipulation, live imaging of neuronal activity and automated high-throughput molecular screening promise to help delineate the mechanistic relationships between different types of neurological conditions and accelerate discovery of therapeutic strategies

Genetic compensation in a stable slc25a46 mutant zebrafish: A case for using F0 CRISPR mutagenesis to study phenotypes caused by inherited disease.

A phenomenon of genetic compensation is commonly observed when an organism with a disease-bearing mutation shows incomplete penetrance of the disease phenotype. Such incomplete phenotypic penetrance, or genetic compensation, is more commonly found in stable knockout models, rather than transient knockdown models. As such, these incidents present a challenge for the disease modeling field, although a deeper understanding of genetic compensation may also hold the key for novel therapeutic interventions. In our study we created a knockout model of slc25a46 gene, which is a recently discovered important player in mitochondrial dynamics, and deleterious mutations in which are known to cause peripheral neuropathy, optic atrophy and cerebellar ataxia. We report a case of genetic compensation in a stable slc25a46 homozygous zebrafish mutant (hereafter referred as "mutant"), in contrast to a penetrant disease phenotype in the first generation (F0) slc25a46 mosaic mutant (hereafter referred as "crispant"), generated with CRISPR/Cas-9 technology. We show that the crispant phenotype is specific and rescuable. By performing mRNA sequencing, we define significant changes in slc25a46 mutant's gene expression profile, which are largely absent in crispants. We find that among the most significantly altered mRNAs, anxa6 gene stands out as a functionally relevant player in mitochondrial dynamics. We also find that our genetic compensation case does not arise from mechanisms driven by mutant mRNA decay. Our study contributes to the growing evidence of the genetic compensation phenomenon and presents novel insights about Slc25a46 function. Furthermore, our study provides the evidence for the efficiency of F0 CRISPR screens for disease candidate genes, which may be used to advance the field of functional genetics

Zebrafish: A Pharmacogenetic Model for Anesthesia

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery

Erratum: A CADM3 variant causes Charcot-Marie-Tooth disease with marked upper limb involvement (Brain (2021) 144:4 (1197-1213) DOI: 10.1093/brain/awab019)

The authors and publishers apologize for errors in the author affiliations and the References section. These have been corrected

A CADM3 variant causes Charcot-Marie-Tooth disease with marked upper limb involvement

The CADM family of proteins consists of four neuronal specific adhesion molecules (CADM1, CADM2, CADM3 and CADM4) that mediate the direct contact and interaction between axons and glia. In the peripheral nerve, axon-Schwann cell interaction is essential for the structural organization of myelinated fibres and is primarily mediated by the binding of CADM3, expressed in axons, to CADM4, expressed by myelinating Schwann cells. We have identified - by whole exome sequencing - three unrelated families, including one de novo patient, with axonal Charcot-Marie-Tooth disease (CMT2) sharing the same private variant in CADM3, Tyr172Cys. This variant is absent in 230 000 control chromosomes from gnomAD and predicted to be pathogenic. Most CADM3 patients share a similar phenotype consisting of autosomal dominant CMT2 with marked upper limb involvement. High resolution mass spectrometry analysis detected a newly created disulphide bond in the mutant CADM3 potentially modifying the native protein conformation. Our data support a retention of the mutant protein in the endoplasmic reticulum and reduced cell surface expression in vitro. Stochastic optical reconstruction microscopy imaging revealed decreased co-localization of the mutant with CADM4 at intercellular contact sites. Mice carrying the corresponding human mutation (Cadm3Y170C) showed reduced expression of the mutant protein in axons. Cadm3Y170C mice showed normal nerve conduction and myelin morphology, but exhibited abnormal axonal organization, including abnormal distribution of Kv1.2 channels and Caspr along myelinated axons. Our findings indicate the involvement of abnormal axon-glia interaction as a disease-causing mechanism in CMT patients with CADM3 mutations

Elevated preoptic brain activity in zebrafish glial glycine transporter mutants is linked to lethargy-like behaviors and delayed emergence from anesthesia

Abstract Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 − / −) mutant model. We compared locomotor behaviors; dose–response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1−/− mutants and their siblings. To identify differentially active brain regions in glyt1−/− mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1−/− mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose–response curves, glyt1−/− mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1−/− mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1−/− mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1−/− mutants

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants