Pathologic classification of a late-onset peripheral neuropathy in a spontaneous Labrador retriever dog model.

Late-onset peripheral neuropathy (LPN) is a heritable canine neuropathy commonly found in Labrador retrievers and is characterized by laryngeal paralysis and pelvic limb paresis. Our objective was to establish canine LPN as a model for human hereditary peripheral neuropathy by classifying it as either an axonopathy or myelinopathy and evaluating length-dependent degeneration. We conducted a motor nerve conduction study of the sciatic and ulnar nerves, electromyography (EMG) of appendicular and epaxial musculature, and histologic analysis of sciatic and recurrent laryngeal nerves in LPN-affected and control dogs. LPN-affected dogs exhibited significant decreases in compound muscle action potential (CMAP) amplitude, CMAP area, and pelvic limb latencies. However, no differences were found in motor nerve conduction velocity, residual latencies, or CMAP duration. Distal limb musculature showed greater EMG changes in LPN-affected dogs. Histologically, LPN-affected dogs exhibited a reduction in the number of large-diameter axons, especially in distal nerve regions. In conclusion, LPN in Labrador retrievers is a common, spontaneous, length-dependent peripheral axonopathy that is a novel animal model of age-related peripheral neuropathy that could be used for fundamental research and clinical trials.

Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.

Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

Neurofilament light chain concentration does not correlate with disease status in Labrador Retrievers affected with idiopathic laryngeal paralysis.

OBJECTIVE: The aim of this study was to investigate whether plasma neurofilament light chain (pNfL) concentration was altered in Labrador Retrievers with idiopathic laryngeal paralysis (ILP) compared to a control population. A secondary aim was to investigate relationships between age, height, weight, and body mass index in the populations studied. ANIMALS: 123 dogs: 62 purebred Labrador Retrievers with ILP (ILP Cases) and 61 age-matched healthy medium- to large-breed dogs (Controls). METHODS: Dogs, recruited from August 1, 2016, to March 1, 2022, were categorized as case or control based on a combination of physical exam, neurologic exam, and history. Blood plasma was collected, and pNfL concentration was measured. pNfL concentrations were compared between ILP Cases and Controls. Covariables including age, height, and weight were collected. Relationships between pNfL and covariables were analyzed within and between groups. In dogs where 2 plasma samples were available from differing time points, pNfL concentrations were measured to evaluate alterations over time. RESULTS: No significant difference in pNfL concentration was found between ILP Cases and Control (P = .36). pNfL concentrations had moderate negative correlations with weight and height in the Control group; other variables did not correlate with pNfL concentrations in ILP Case or Control groups. pNfL concentrations do not correlate with ILP disease status or duration in Labrador Retrievers. CLINICAL RELEVANCE: There is no evidence that pNfL levels are altered due to ILP disease duration or progression when compared with healthy controls. When evaluating pNfL concentrations in the dog, weight and height should be considered.

Peripheral Nervous System (PNS) Myelin Diseases.

This is a review of inherited and acquired causes of human demyelinating neuropathies and a subset of disorders that affect axon-Schwann cell interactions. Nearly all inherited demyelinating neuropathies are caused by mutations in genes that are expressed by myelinating Schwann cells, affecting diverse functions in a cell-autonomous manner. The most common acquired demyelinating neuropathies are Guillain-Barré syndrome and chronic, inflammatory demyelinating polyneuropathy, both of which are immune-mediated. An additional group of inherited and acquired disorders affect axon-Schwann cell interactions in the nodal region. Overall, these disorders affect the formation of myelin and its maintenance, with superimposed axonal loss that is clinically important.

NetREm: Network Regression Embeddings reveal cell-type transcription factor coordination for gene regulation.

Transcription factor (TF) coordination plays a key role in gene regulation such as protein-protein interactions (PPIs) and DNA co-bindings. Single-cell technologies facilitate gene expression measurement for individual cells and cell-type identification, yet the connection between TF coordination and gene regulation of various cell types remains unclear. To address this, we have developed a novel computational approach, Network Regression Embedding (NetREm), to reveal cell-type TF-TF coordination activities for gene regulation. NetREm leverages network-constrained regularization using prior interaction knowledge (e.g., protein, chromatin, TF binding) to analyze single-cell gene expression data. We test NetREm by simulation data and apply it to analyze various cell types in both central and peripheral nerve systems (PNS) such as neuronal, glial and Schwann cells as well as in Alzheimer's disease (AD). Our findings uncover cell-type coordinating TFs and identify new TF-target gene candidate links. We also validate our top predictions using Cut&Run and knockout loss-of-function expression data in rat and mouse models and compare our results with additional functional genomic data including expression quantitative trait loci (eQTL) and Genome-Wide Association Studies (GWAS) to link genetic variants to TF coordination. NetREm is open-source available at https://github.com/SaniyaKhullar/NetREm .

Post-transcriptional microRNA repression of PMP22 dose in severe Charcot-Marie-Tooth disease type 1.

Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3' untranslated region (3'-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3'-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wild-type and mutant 3'-UTR showed significantly increased reporter assay activity in the latter, which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development.

Modulation of Schwann cell homeostasis by the BAP1 deubiquitinase.

Schwann cell programming during myelination involves transcriptional networks that activate gene expression but also repress genes that are active in neural crest/embryonic differentiation of Schwann cells. We previously found that a Schwann cell-specific deletion of the EED subunit of the Polycomb Repressive Complex (PRC2) led to inappropriate activation of many such genes. Moreover, some of these genes become re-activated in the pro-regenerative response of Schwann cells to nerve injury, and we found premature activation of the nerve injury program in a Schwann cell-specific knockout of Eed. Polycomb-associated histone modifications include H3K27 trimethylation formed by PRC2 and H2AK119 monoubiquitination (H2AK119ub1), deposited by Polycomb repressive complex 1 (PRC1). We recently found dynamic regulation of H2AK119ub1 in Schwann cell genes after injury. Therefore, we hypothesized that H2AK119 deubiquitination modulates the dynamic polycomb repression of genes involved in Schwann cell maturation. To determine the role of H2AK119 deubiquitination, we generated a Schwann cell-specific knockout of the H2AK119 deubiquitinase Bap1 (BRCA1-associated protein). We found that loss of Bap1 causes tomacula formation, decreased axon diameters and eventual loss of myelinated axons. The gene expression changes are accompanied by redistribution of H2AK119ub1 and H3K27me3 modifications to extragenic sites throughout the genome. BAP1 interacts with OGT in the PR-DUB complex, and our data suggest that the PR-DUB complex plays a multifunctional role in repression of the injury program. Overall, our results indicate Bap1 is required to restrict the spread of polycomb-associated histone modifications in Schwann cells and to promote myelin homeostasis in peripheral nerve.

BACH2 in TRegs Limits the Number of Adipose Tissue Regulatory T Cells and Restrains Type 2 Immunity to Fungal Allergens.

FoxP3+ regulatory T cells (Tregs) are essential for self-tolerance and moderating tissue-damaging inflammation. Tregs that develop and mature in the thymus are classified as central Tregs or effector Tregs based on whether Tregs predominately inhabit secondary lymphoid organs (central Tregs) or tissues (effector Tregs). By generating mice that are conditionally deficient for Bach2 in peripheral Tregs, we have examined the role of Bach2 in regulating Treg homeostasis and effector functions. Unlike global and T cell-specific Bach2-deficient mice, Treg-specific Bach2 ablation did not result in unprovoked TH2 inflammation in the lungs. However, Bach2 deficiency in Tregs led to augmented expressions of IRF4, BATF, and GATA3 and a significant increase in the accumulation of ST2 (IL-33R)+ve effector Tregs in the spleen and visceral adipose tissue (VAT) but not in the lungs. Enhanced Bach2-deficient Treg numbers in VAT was not linked to hyperresponsiveness to exogenous IL-33 in vivo. Most strikingly, Treg-specific Bach2 deficiency resulted in enhanced fungal protease-induced Type 2 allergic inflammation in the lungs, with no detectable effects on Type 1 responses to systemic or respiratory viral infections. In summary, we ascribe vital roles for Bach2 in peripheral Tregs: as a transcriptional checkpoint to limit precocious differentiation into effector Tregs in lymphoid tissues and as a regulator of the functional program that restrains Type 2 but not Type 1 inflammation in lungs. Results presented in this manuscript implicate dysregulated Tregs in the pathogenesis of airway hypersensitivities, asthma, and other allergic disorders.

A novel mouse model of CMT1B identifies hyperglycosylation as a new pathogenetic mechanism.

Mutations in the Myelin Protein Zero gene (MPZ), encoding P0, the major structural glycoprotein of peripheral nerve myelin, are the cause of Charcot-Marie-Tooth (CMT) type 1B neuropathy, and most P0 mutations appear to act through gain-of-function mechanisms. Here, we investigated how misglycosylation, a pathomechanism encompassing several genetic disorders, may affect P0 function. Using in vitro assays, we showed that gain of glycosylation is more damaging for P0 trafficking and functionality as compared with a loss of glycosylation. Hence, we generated, via CRISPR/Cas9, a mouse model carrying the MPZD61N mutation, predicted to generate a new N-glycosylation site in P0. In humans, MPZD61N causes a severe early-onset form of CMT1B, suggesting that hyperglycosylation may interfere with myelin formation, leading to pathology. We show here that MPZD61N/+ mice develop a tremor as early as P15 which worsens with age and correlates with a significant motor impairment, reduced muscular strength and substantial alterations in neurophysiology. The pathological analysis confirmed a dysmyelinating phenotype characterized by diffuse hypomyelination and focal hypermyelination. We find that the mutant P0D61N does not cause significant endoplasmic reticulum stress, a common pathomechanism in CMT1B, but is properly trafficked to myelin where it causes myelin uncompaction. Finally, we show that myelinating dorsal root ganglia cultures from MPZD61N mice replicate some of the abnormalities seen in vivo, suggesting that they may represent a valuable tool to investigate therapeutic approaches. Collectively, our data indicate that the MPZD61N/+ mouse represents an authentic model of severe CMT1B affirming gain-of-glycosylation in P0 as a novel pathomechanism of disease.

JUN Regulation of Injury-Induced Enhancers in Schwann Cells.

Schwann cells play a critical role after peripheral nerve injury by clearing myelin debris, forming axon-guiding bands of Büngner, and remyelinating regenerating axons. Schwann cells undergo epigenomic remodeling to differentiate into a repair state that expresses unique genes, some of which are not expressed at other stages of Schwann cell development. We previously identified a set of enhancers that are activated in Schwann cells after nerve injury, and we determined whether these enhancers are preprogrammed into the Schwann cell epigenome as poised enhancers before injury. Poised enhancers share many attributes of active enhancers, such as open chromatin, but are marked by repressive histone H3 lysine 27 (H3K27) trimethylation rather than H3K27 acetylation. We find that most injury-induced enhancers are not marked as poised enhancers before injury indicating that injury-induced enhancers are not preprogrammed in the Schwann cell epigenome. Injury-induced enhancers are enriched with AP-1 binding motifs, and the c-JUN subunit of AP-1 had been shown to be critical to drive the transcriptional response of Schwann cells after injury. Using in vivo chromatin immunoprecipitation sequencing analysis in rat, we find that c-JUN binds to a subset of injury-induced enhancers. To test the role of specific injury-induced enhancers, we focused on c-JUN-binding enhancers upstream of the Sonic hedgehog (Shh) gene, which is only upregulated in repair Schwann cells compared with other stages of Schwann cell development. We used targeted deletions in male/female mice to show that the enhancers are required for robust induction of the Shh gene after injury.SIGNIFICANCE STATEMENT The proregenerative actions of Schwann cells after nerve injury depends on profound reprogramming of the epigenome. The repair state is directed by injury-induced transcription factors, like JUN, which is uniquely required after nerve injury. In this study, we test whether the injury program is preprogrammed into the epigenome as poised enhancers and define which enhancers bind JUN. Finally, we test the roles of these enhancers by performing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated deletion of JUN-bound injury enhancers in the Sonic hedgehog gene. Although many long-range enhancers drive expression of Sonic hedgehog at different developmental stages of specific tissues, these studies identify an entirely new set of enhancers that are required for Sonic hedgehog induction in Schwann cells after injury.

ACTL6a coordinates axonal caliber recognition and myelination in the peripheral nerve.

Cells elaborate transcriptional programs in response to external signals. In the peripheral nerves, Schwann cells (SC) sort axons of given caliber and start the process of wrapping their membrane around them. We identify Actin-like protein 6a (ACTL6a), part of SWI/SNF chromatin remodeling complex, as critical for the integration of axonal caliber recognition with the transcriptional program of myelination. Nuclear levels of ACTL6A in SC are increased by contact with large caliber axons or nanofibers, and result in the eviction of repressive histone marks to facilitate myelination. Without Actl6a the SC are unable to coordinate caliber recognition and myelin production. Peripheral nerves in knockout mice display defective radial sorting, hypo-myelination of large caliber axons, and redundant myelin around small caliber axons, resulting in a clinical motor phenotype. Overall, this suggests that ACTL6A is a key component of the machinery integrating external signals for proper myelination of the peripheral nerve.

Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for PMP22 Gene-Dosage Disorder Drug Discovery.

Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.

Adeno-associated virus gene therapy to the rescue for Charcot-Marie-Tooth disease type 4J.

The genetic peripheral neuropathy known as Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive mutations in the FIG4 gene. The transformational success of adeno-associated virus (AAV) gene therapy for spinal muscular atrophy has generated substantial interest in using this approach to create similar treatments for CMT. In this issue of the JCI, Presa et al. provide a preclinical demonstration of efficacy using AAV-directed gene therapy for CMT4J. The study showed a dramatic improvement in both survival and neuropathy symptoms in a severe mouse model of CMT4J after administration of AAV gene therapy at several time points. The authors' approach advances the technique for delivering treatments to individuals with CMT, for which FDA-approved therapies have not yet come to the clinic.

Revisiting the pathogenic mechanism of the GJB1 5' UTR c.-103C > T mutation causing CMTX1.

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5' untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5' UTR was compared to the GJB1 P2 5' UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5' UTR IRES activity were observed between the negative control, the wild-type P2 5' UTR, the c.-103C > T 5' UTR or the c.-102G > A 5' UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5' UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5' UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.

H3K27 demethylases are dispensable for activation of Polycomb-regulated injury response genes in peripheral nerve.

The induction of nerve injury response genes in Schwann cells depends on both transcriptional and epigenomic reprogramming. The nerve injury response program is regulated by the repressive histone mark H3K27 trimethylation (H3K27me3), deposited by Polycomb repressive complex 2 (PRC2). Loss of PRC2 function leads to early and augmented induction of the injury response gene network in peripheral nerves, suggesting H3K27 demethylases are required for derepression of Polycomb-regulated nerve injury genes. To determine the function of H3K27 demethylases in nerve injury, we generated Schwann cell-specific knockouts of H3K27 demethylase Kdm6b and double knockouts of Kdm6b/Kdm6a (encoding JMJD3 and UTX). We found that H3K27 demethylases are largely dispensable for Schwann cell development and myelination. In testing the function of H3K27 demethylases after injury, we found early induction of some nerve injury genes was diminished compared with control, but most injury genes were largely unaffected at 1 and 7 days post injury. Although it was proposed that H3K27 demethylases are required to activate expression of the cyclin-dependent kinase inhibitor Cdkn2a in response to injury, Schwann cell-specific deletion of H3K27 demethylases affected neither the expression of this gene nor Schwann cell proliferation after nerve injury. To further characterize the regulation of nerve injury response genes, we found that injury genes are associated with repressive histone H2AK119 ubiquitination catalyzed by PRC1, which declines after injury. Overall, our results indicate H3K27 demethylation is not required for induction of injury response genes and that other mechanisms likely are involved in activating Polycomb-repressed injury genes in peripheral nerve.

MicroRNAs as Biomarkers of Charcot-Marie-Tooth Disease Type 1A.

OBJECTIVE: To determine whether microRNAs (miRs) are elevated in the plasma of individuals with the inherited peripheral neuropathy Charcot-Marie-Tooth disease type 1A (CMT1A), miR profiling was employed to compare control and CMT1A plasma. METHODS: We performed a screen of CMT1A and control plasma samples to identify miRs that are elevated in CMT1A using next-generation sequencing, followed by validation of selected miRs by quantitative PCR, and correlation with protein biomarkers and clinical data: Rasch-modified CMT Examination and Neuropathy Scores, ulnar compound muscle action potentials, and motor nerve conduction velocities. RESULTS: After an initial pilot screen, a broader screen confirmed elevated levels of several muscle-associated miRNAs (miR1, -133a, -133b, and -206, known as myomiRs) along with a set of miRs that are highly expressed in Schwann cells of peripheral nerve. Comparison to other candidate biomarkers for CMT1A (e.g., neurofilament light) measured on the same sample set shows a comparable elevation of several miRs (e.g., miR133a, -206, -223) and ability to discriminate cases from controls. Neurofilament light levels were most highly correlated with miR133a. In addition, the putative Schwann cell miRs (e.g., miR223, -199a, -328, -409, -431) correlate with the recently described transmembrane protease serine 5 (TMPRSS5) protein biomarker that is most highly expressed in Schwann cells and also elevated in CMT1A plasma. CONCLUSIONS: These studies identify a set of miRs that are candidate biomarkers for clinical trials in CMT1A. Some of the miRs may reflect Schwann cell processes that underlie the pathogenesis of the disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a set of plasma miRs are elevated in patients with CMT1A.

Failures of nerve regeneration caused by aging or chronic denervation are rescued by restoring Schwann cell c-Jun.

After nerve injury, myelin and Remak Schwann cells reprogram to repair cells specialized for regeneration. Normally providing strong regenerative support, these cells fail in aging animals, and during chronic denervation that results from slow axon growth. This impairs axonal regeneration and causes significant clinical problems. In mice, we find that repair cells express reduced c-Jun protein as regenerative support provided by these cells declines during aging and chronic denervation. In both cases, genetically restoring Schwann cell c-Jun levels restores regeneration to control levels. We identify potential gene candidates mediating this effect and implicate Shh in the control of Schwann cell c-Jun levels. This establishes that a common mechanism, reduced c-Jun in Schwann cells, regulates success and failure of nerve repair both during aging and chronic denervation. This provides a molecular framework for addressing important clinical problems, suggesting molecular pathways that can be targeted to promote repair in the PNS.

Neurofilament light plasma concentration positively associates with age and negatively associates with weight and height in the dog.

Plasma neurofilament light chain (pNfL) concentration is a biomarker for neuroaxonal injury and degeneration and can be used to monitor response to treatment. Spontaneous canine neurodegenerative diseases are a valuable comparative resource for understanding similar human conditions and as large animal treatment models. The features of pNfL concentration in healthy dogs is not well established. We present data reporting basic pNfL concentration trends in the Labrador Retriever breed. Fifty-five Labrador Retrievers were enrolled. pNfL concentration was measured and correlated to age, sex, neuter status, height, weight, body mass index, and coat color. We found increased pNfL with age (P < 0.0001), shorter stature (P = 0.009) and decreased body weight (P < 0.001). These are similar to findings reported in humans. pNfL concentration did not correlate with sex, BMI or coat color. This data further supports findings that pNfL increase with age in a canine population but highlights a need to consider weight and height when determining normal pNfL concentration in canine populations.

CTCF loss mediates unique DNA hypermethylation landscapes in human cancers.

BACKGROUND: The chromatin insulator CCCTC-binding factor (CTCF) displays tissue-specific DNA binding sites that regulate transcription and chromatin organization. Despite evidence linking CTCF to the protection of epigenetic states through barrier insulation, the impact of CTCF loss on genome-wide DNA methylation sites in human cancer remains undefined. RESULTS: Here, we demonstrate that prostate and breast cancers within The Cancer Genome Atlas (TCGA) exhibit frequent copy number loss of CTCF and that this loss is associated with increased DNA methylation events that occur preferentially at CTCF binding sites. CTCF sites differ among tumor types and result in tissue-specific methylation patterns with little overlap between breast and prostate cancers. DNA methylation and transcriptome profiling in vitro establish that forced downregulation of CTCF leads to spatially distinct DNA hypermethylation surrounding CTCF binding sites, loss of CTCF binding, and decreased gene expression that is also seen in human tumors. DNA methylation inhibition reverses loss of expression at these CTCF-regulated genes. CONCLUSION: These findings establish CTCF loss as a major mediator in directing localized DNA hypermethylation events in a tissue-specific fashion and further support its role as a driver of the cancer phenotype.

Pmp22 super-enhancer deletion causes tomacula formation and conduction block in peripheral nerves.

Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene's proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.