The Transcriptional Corepressor NAB2 Inhibits NGF-induced Differentiation of PC12 Cells

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta 1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21WAF1, a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21WAF1 restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF

Differential regulation of NAB corepressor genes in Schwann cells

<p>Abstract</p> <p>Background</p> <p>Myelination of peripheral nerves by Schwann cells requires not only the Egr2/Krox-20 transactivator, but also the NGFI-A/Egr-binding (NAB) corepressors, which modulate activity of Egr2. Previous work has shown that axon-dependent expression of Egr2 is mediated by neuregulin stimulation, and NAB corepressors are co-regulated with Egr2 expression in peripheral nerve development. NAB corepressors have also been implicated in macrophage development, cardiac hypertrophy, prostate carcinogenesis, and feedback regulation involved in hindbrain development.</p> <p>Results</p> <p>To test the mechanism of NAB regulation in Schwann cells, transfection assays revealed that both <it>Nab1 </it>and <it>Nab2 </it>promoters are activated by Egr2 expression. Furthermore, direct binding of Egr2 at these promoters was demonstrated in vivo by chromatin immunoprecipitation analysis of myelinating sciatic nerve, and binding of Egr2 to the <it>Nab2 </it>promoter was stimulated by neuregulin in primary Schwann cells. Although Egr2 expression activates the <it>Nab2 </it>promoter more highly than <it>Nab1</it>, we surprisingly found that only <it>Nab1 </it>– but not <it>Nab2 </it>– expression levels were reduced in sciatic nerve from Egr2 null mice. Analysis of the <it>Nab2 </it>promoter showed that it is also activated by ETS proteins (Ets2 and Etv1/ER81) and is bound by Ets2 in vivo.</p> <p>Conclusion</p> <p>Overall, these results indicate that induction of <it>Nab2 </it>expression in Schwann cells involves not only Egr2, but also ETS proteins that are activated by neuregulin stimulation. Although <it>Nab1 </it>and <it>Nab2 </it>play partially redundant roles, regulation of <it>Nab2 </it>expression by ETS factors explains several observations regarding regulation of NAB genes. Finally, these data suggest that NAB proteins are not only feedback inhibitors of Egr2, but rather that co-induction of Egr2 and NAB genes is involved in forming an Egr2/NAB complex that is crucial for regulation of gene expression.</p

Regulation of the PMP22 gene through an intronic enhancer

Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as Peripheral Myelin Protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in 3 copies of PMP22, is the most common cause of the peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we find a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites for not only Egr2 but also Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression

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

Statistical-mechanical lattice models for protein-DNA binding in chromatin

Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibriums measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.Comment: 19 pages, 7 figures, accepted author manuscript, to appear in J. Phys.: Cond. Mat

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 wildtype 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

SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer

<p>Abstract</p> <p>Background</p> <p>The <it>ERBB3 </it>gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of <it>ERBB3 </it>or the factors that bind them.</p> <p>Results</p> <p>In this study we identified three transcriptional enhancers at the <it>ERBB3 </it>locus and evaluated their regulatory potential <it>in vitro </it>in NC-derived cell types and <it>in vivo </it>in transgenic zebrafish. One enhancer, termed <it>ERBB3</it>_MCS6, which lies within the first intron of <it>ERBB3</it>, directs the highest reporter expression <it>in vitro </it>and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within <it>ERBB3</it>_MCS6 and demonstrate, <it>in vitro</it>, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous <it>Erbb3 </it>locus is dependent on Sox10. Further we demonstrate <it>in vitro </it>that Sox10 physically interacts with that <it>ERBB3</it>_MCS6. Consistent with its <it>in vitro </it>activity, we also show that <it>ERBB3</it>_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages <it>in vivo </it>in zebrafish in a manner consistent with <it>erbb3b </it>expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for <it>ERBB3</it>_MCS6 expression <it>in vivo </it>in zebrafish.</p> <p>Conclusions</p> <p>Taken collectively, our data suggest that <it>ERBB3 </it>may be directly regulated by SOX10, and that this control may in part be facilitated by <it>ERBB3</it>_MCS6.</p

Transmembrane protease serine 5: a novel Schwann cell plasma marker for CMT1A

OBJECTIVE: Development of biomarkers for Charcot-Marie-Tooth (CMT) disease is critical for implementing effective clinical trials. The most common form of CMT, type 1A, is caused by a genomic duplication surrounding the PMP22 gene. A recent report (Neurology 2018;90:e518-3524) showed elevation of neurofilament light (NfL) in plasma of CMT1A disease patients, which correlated with disease severity. However, no plasma/serum biomarker has been identified that is specific to Schwann cells, the most directly affected cells in CMT1A. METHODS: We used the Olink immuno PCR platform to profile CMT1A patient (n = 47, 2 cohorts) and normal control plasma (n = 41, two cohorts) on five different Olink panels to screen 398 unique proteins. RESULTS: The TMPRSS5 protein (Transmembrane protease serine 5) was elevated 2.07-fold (P = <0.0001) in two independent cohorts of CMT1A samples relative to controls. TMPRSS5 is most highly expressed in Schwann cells of peripheral nerve. Consistent with early myelination deficits in CMT1A, TMPRSS5 was not significantly correlated with disease score (CMTES-R, CMTNS-R), nerve conduction velocities (Ulnar CMAP, Ulnar MNCV), or with age. TMPRSS5 was not significantly elevated in smaller sample sets from patients with CMT2A, CMT2E, CMT1B, or CMT1X. The Olink immuno PCR assays confirmed elevated levels of NfL (average 1.58-fold, P < 0.0001), which correlated with CMT1A patient disease score. INTERPRETATION: These data identify the first Schwann cell-specific protein that is elevated in plasma of CMT1A patients, and may provide a disease marker and a potentially treatment-responsive biomarker with good disease specificity for clinical trials

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