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

The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-β (TGF- β) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-β responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-β signaling is unknown. Here we show that TGF-β caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-β-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-β responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-β signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma

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

Coordinated Sumoylation and Ubiquitination Modulate EGF Induced EGR1 Expression and Stability

Abstract: Background: Human early growth response-1 (EGR1) is a member of the zing-finger family of transcription factors induced by a range of molecular and environmental stimuli including epidermal growth factor (EGF). In a recently published paper we demonstrated that integrin/EGFR cross-talk was required for Egr1 expression through activation of the Erk1/2 and PI3K/Akt/Forkhead pathways. EGR1 activity and stability can be influenced by many different post-translational modifications such as acetylation, phosphorylation, ubiquitination and the recently discovered sumoylation. The aim of this work was to assess the influence of sumoylation on EGF induced Egr1 expression and/or stability. Methods: We modulated the expression of proteins involved in the sumoylation process in ECV304 cells by transient transfection and evaluated Egr1 expression in response to EGF treatment at mRNA and protein levels. Results: We demonstrated that in ECV304 cells Egr1 was transiently induced upon EGF treatment and a fraction of the endogenous protein was sumoylated. Moreover, SUMO-1/Ubc9 over-expression stabilized EGF induced ERK1/2 phosphorylation and increased Egr1 gene transcription. Conversely, in SUMO-1/Ubc9 transfected cells, EGR1 protein levels were strongly reduced. Data obtained from protein expression and ubiquitination analysis, in the presence of the proteasome inhibitor MG132, suggested that upon EGF stimuli EGR1 sumoylation enhanced its turnover, increasing ubiquitination and proteasome mediated degradation. Conclusions: Here we demonstrate that SUMO-1 modification improving EGR1 ubiquitination is involved in the modulation of its stability upon EGF mediated induction

A polar barrier to transcription can be circumvented by remodeler-induced nucleosome translocation

Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, we have shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3–H4 dimer. Here, we show that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3–H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. However, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. We suggest that interactions between PBSs and chromatin remodelers might have significant regulatory potential

CXCR4/CXCL12 expression and signalling in kidney cancer

CXCL12 (SDF-1), a CXC-chemokine, and its specific receptor, CXCR4, have recently been shown to be involved in tumourgenesis, proliferation and angiogenesis. Therefore, we analysed CXCL12α/CXCR4 expression and function in four human kidney cancer cell lines (A-498, CAKI-1, CAKI-2, HA-7), 10 freshly harvested human tumour samples and corresponding normal kidney tissue. While none of the analysed tumour cell lines expressed CXCL12α, A-498 cells were found to express CXCR4. More importantly, real-time RT–PCR analysis of 10 tumour samples and respective adjacent normal kidney tissue disclosed a distinct and divergent downregulation of CXCL12α and upregulation of CXCR4 in primary tumour tissue. To prove that the CXCR4 protein is functionally active, rhCXCL12α was investigated for its ability to induce changes of intracellular calcium levels in A-498 cells. Moreover, we used cDNA expression arrays to evaluate the biological influence of CXCL12α. Comparing gene expression profiles in rhCXCL12α stimulated vs unstimulated A-498 kidney cancer cells revealed specific regulation of 31 out of 1176 genes tested on a selected human cancer array, with a prominent stimulation of genes involved in cell-cycle regulation and apoptosis. The genetic changes reported here should provide new insights into the developmental paths leading to tumour progression and may also aid the design of new approaches to therapeutic intervention