Variants in MME are associated with autosomal-recessive distal hereditary motor neuropathy

© 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. Objective: To identify a new genetic cause in patients segregating distal hereditary motor neuropathy (dHMN) with an autosomal recessive pattern. Methods: Whole-exome sequencing was conducted in two siblings and was combined with segregation analysis. Additionally, 83 unrelated dHMN patients with unknown genetic cause were screened. RNA analysis was performed using blood lymphocytes and HEK293 cells transfected with mutant plasmids. Immunohistochemistry and Western blot analysis was applied to the nerve tissue. The enzymatic activities of mutant proteins were measured in the cultured cells to verify the pathogenicity of variants. Results: The clinical features of the patients showed late-onset phenotype of distal motor neuropathy without sensory involvement. We identified that compound heterozygous variants of c.1342C\u27T and c.2071_2072delGCinsTT in the membrane metalloendopeptidase (MME) gene co-segregated with the phenotype in a dHMN family. In an additional group of 83 patients with dHMN, compound heterozygous variants of c.1416+2T\u27C and c.2027C\u27T in MME were identified in one patient. The splice site variant c.1416+2T\u27C results in skipping of exon 13. The stop variant c.1342C\u27T induces mRNA degradation via nonsense-mediated mRNA decay. Transcript levels of MME in the lymphocytes showed no significant differences between the patients and controls. We also identified that MME variants were associated with mild decrease in protein expression in the sural nerve and significant impairments of enzymatic activity. Interpretation: Variants in the MME gene were associated with not only a Charcot-Marie-Tooth neuropathy phenotype but also with an autosomal-recessive dHMN phenotype. Loss of function may play a role in the pathogenesis of dHMN

Antimicrobial activity and molecular mechanism of the CRES protein.

Cystatin-related epididymal spermatogenic (CRES) protein, a member of the cystatin superfamily of cysteine protease inhibitors (also known as CST8), exhibits highly specific, age-dependent expression in mouse testis and epididymis. The CRES protein possesses four highly conserved cysteine residues which govern the overall conformation of the cystatins through the formation of two disulfide bonds. Previous studies have revealed that other cystatin family members, such as cystatin 3 and cystatin 11, show antibacterial activity in vitro. This prompted us to investigate the potential antimicrobial activity of the CRES protein. Colony forming assays and spectrophotometry were used to investigate the effects of recombinant CRES protein on Escherichia coli (E. coli) and Ureaplasma urealyticum (Uu), respectively, in vitro. After incubation of E. coli with CRES recombinant protein fused with glutathione-S-transferase (GST), a substantial decrease in colony forming units was observed, and the effect was dose and time dependent. Furthermore, it took longer for Uu to grow to plateau stage when incubated with GST-CRES recombinant protein compared with the control GST. The antibacterial and Anti-Uu activities were not impaired when the cysteine residues of CRES protein were mutated, indicating that the antimicrobial effect was not dependent on its disulfide bonds. Functional analysis of three CRES polypeptides showed that the N-terminal 30 residues (N30) had no antimicrobial activity while N60 showed similar activity as full-length CRES protein. These results suggest that the active center of CRES protein resides between amino acid residues 31 and 60 of its N-terminus. Mechanistically, E. coli membrane permeabilization was increased in a dose-dependent manner, and macromolecular synthesis was inhibited on treatment with GST-CRES. Together, our data on the antimicrobial activities of CRES protein suggest that it is a novel and innate antimicrobial protein which protecting the male reproductive tract against invading pathogens

Permeabilization of bacterial membranes mediated by CRES.

<p>* <i>p</i><0.05, ** <i>p</i><0.01.</p

Macromolecular synthesis of <i>E. coli</i> D5α affected by CRES.

<p>(A) DNA synthesis. (B) RNA synthesis. (C) Protein synthesis. * <i>p</i><0.05, ** <i>p</i><0.01.</p

Cloning and expression of CRES.

<p>(A) Design of the oligonucleotide primers used to construct the cysteine mutants of CRES. (B) Schematic diagram of the recombinant plasmid PGEX-4T-1/CRES. (C) RT-PCR products of the CRES fragments on 1.5% agarose gel. Lane M, marker; lane N, negative control (PCR without cDNA); lane 1, CRES-N30 fragment; lane 2, CRES-N60 fragment; lane 3, CRES full length fragment. (D) RT-PCR products of the mutant CRES gene on 1.5% agarose gel. Lane M, marker; lane N, negative control; lane 1, CRES C105 mutant; lane 2, CRES C139 mutant; lane 3, CRES C105 & C139 mutant. (E) Prokaryotic expression and purification of CRES and GST recombinant protein. Lane M, marker; Lane 1, cells carrying the GST vector without the insert after Isopropylthio-β-D1-galctopyranoside (IPTG) induction; Lane 2–4, cells carrying the GST vector with the insert (CRES-N30,CRES-N60, full length CRES) after IPTG induction. Lane 5, purified GST recombinant protein. Lane 6–7, purified GST-CRES recombinant protein (N30, N60, N90). (F) Prokaryotic expression and purification of cysteine mutants of CRES. Lane M, marker; Lane 1, cells carrying the GST vector before IPTG induction; Lane 2–5, cells carrying the GST vector with the insert (wild type, C105 mutant, C139 mutant, C105 & C139 mutant) after IPTG induction. Lane 6–9, purified GST-CRES recombinant protein (wild type, C105 mutant, C139 mutant, C105 & C139 mutant). (G) The purified CRES recombinant proteins were analyzed by western blot using anti-GST monoclonal antibody.</p

Tocilizumab promotes repair of spinal cord injury by facilitating the restoration of tight junctions between vascular endothelial cells

Abstract Background Our previous study demonstrated that M1 macrophages could impair tight junctions (TJs) between vascular endothelial cells by secreting interleukin-6 (IL-6) after spinal cord injury (SCI). Tocilizumab, as a humanized IL-6 receptor (IL-6R) monoclonal antibody approved for the clinic, has been applied in the treatment of neurological diseases in recent years, but the treatment effect of Tocilizumab on the TJs restoration of the blood-spinal cord barrier (BSCB) after SCI remains unclear. This study aimed to explore the effect of Tocilizumab on the restoration of TJs between vascular endothelial cells and axon regeneration after SCI. Methods In this study, the mouse complete spinal cord crush injury model was used, and Tocilizumab was continuously injected intrathecally until the day of sample collection. A PBS injection in the same location was included as a control. At 14 days postinjury (dpi) and 28 dpi, spinal cord tissue sections were examined via tissue immunofluorescence. The Basso Mouse Scale (BMS) scores and footprint analysis were used to verify the effect of Tocilizumab on the recovery of motor function in mice after SCI. Results We demonstrated that depletion of macrophages has no effect on axon regeneration and motor functional recovery after SCI, but mice subjected to Tocilizumab showed a significant increase in axon regeneration and a better recovery in motor function during the chronic phase after SCI. Moreover, our study demonstrated that at 14 and 28 dpi, the expression of claudin-5 (CLDN5) and zonula occludens-1 (ZO-1) between vascular endothelial cells was significantly increased and the leakage of BSCB was significantly reduced in the injured core after daily intrathecal injection of Tocilizumab. Notably, the infiltration of CD68+ macrophages/microglia and the formation of fibrotic scar were decreased in the injured core after Tocilizumab treatment. Tocilizumab treatment could effectively reduce the IL-6 expression in macrophages in the injured core. Conclusion The application of Tocilizumab to antagonize IL-6R can effectively reduce the expression of IL-6 in macrophages and facilitate TJs restoration of the BSCB, which is beneficial for axon regeneration and motor functional recovery after SCI. Hence, Tocilizumab treatment is a potential therapeutic strategy for SCI

Determination of the epididymal epithelial cells viability treated with CRES using MTT assay.

<p>** <i>p</i><0.01.</p

Antibacterial activities of CRES.

<p>(A) <i>E. coli</i> D5α were incubated with 1 ng/mL (purple), 10 ng/mL (blue), 100 ng/mL (orange) GST-CRES and 100 ng/mL GST (green) for 0.5 h, 1 h, 2 h, and 4 h. (B) <i>E. coli</i> D5α incubated with 100 ng/mL GST (purple), C105 mutant (green), C139 mutant (blue), C105 & C139 mutant (orange), GST-CRES (brown) for 0.5 h, 1 h, 2 h, and 4 h. (C) <i>E. coli</i> D5α incubated with 100 ng/mL GST (purple), GST-N30 (green), GST-N60 (blue), GST-CRES (orange) for 0.5 h, 1 h, 2 h, and 4 h. ** <i>p</i><0.01.</p

The primer sequences used to amplify the CRES protein fragments and to construct the cysteine mutants of CRES.

<p>The base in red is the mutational site. <u>CTCGAG:</u> XhoI site, <u>GAATTC:</u> EcoRI site.</p

Transmission electron micrographs of <i>E. coli</i> D5α.

<p>(A–C) <i>E. coli</i> D5α incubated with buffer. (D–I) <i>E. coli</i> D5α incubated with GST-CRES.</p