SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.Heat shock protein 47 (HSP47), encoded by the  Heat shock protein 47 (HSP47) is essential for correct collagen folding. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta. The highly conserved p.R222 residue is located within the collagen interacting surface and HSP47-R222S shows a significantly reduced affinity for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen. In contrast to other types of OI, this overmodification is not caused by prolonged exposure of procollagen to modifying enzymes, since the intracellular folding rate of type I procollagen appears to be normal. We show significant upregulation of several molecular chaperones and collagen-modifying enzymes and increased binding of several of these molecules to unfolded type I collagen chains upon abnormal HSP47-R222S binding. This suggests a compensatory mechanism for aberrant HSP47-R222S binding, eventually leading to overmodification of type I procollagen chains, and underscores the importance of HSP47 for proper posttranslational modification. COL1A1 and pro-α2(I)-chains encoded by COL1A2). These precursor chains typically consist of a long helical domain, which contains multiple Gly-Xaa-Yaa repeats, flanked by globular carboxy- (C-) and amino- (N-) terminal domains. In the rough endoplasmic reticulum (rER) two pro-α1(I)-chains and one pro-α2(I)-chain associate at their C-propeptide and fold into an elongated triple helix, which propagates in a zipper-like fashion towards the N-terminus. Prior to this helix formation several posttranslational modifications occur, including prolyl 4-hydroxylation, prolyl 3-hydroxylation and lysyl hydroxylation. Galactose or glucosyl-galactose sugars are additionally attached to some of the hydroxyl-lysine residues. The enzymes responsible for these modifications can only act on non-triple helical pro-α-chains, and posttranslational modifications cease once triple helix formation is completed. 8]Escherichia coli. Differential Scanning Fluorimetry showed a similar melting point for both wild-type (54.50°C) and R222S (54.00°C) protein, confirming that the variant does not cause structural instability  for wild-type (0.409 ± 0.004 μM) and R222S (11.479 ± 0.154 μM) HSP47 revealed an approximately 25-fold lower affinity of HSP47-R222S for the GPR-foldon with BLI  to type I collagen compared to wild-type HSP47 (0.06 ± 0.006 μM) (off) was relatively similar between wild-type and R222S HSP47 in both BLI and SPR, the association rate constant (kon) was altered, suggesting that HSP47-R222S also shows a slower association with the GPR-foldon  using Biolayer Interferometry (BLI) and full length pepsinized type I collagen from mouse tail tendon using Surface Plasmon Resonance (SPR) analysis. Wild-type HSP47 was shown to interact in a concentration dependent manner with both immobilized GPR-foldon  and immowith BLI , while K.006 μM) . While tR-foldon  and typeR-foldon .D values of 139 nM and 56 nM, respectively. For HSP47-R222S a shift in the curves was seen, confirming that the binding of HSP47-R222S to both the GPR-foldon and to full length type I collagen was severely reduced compared to wild-type HSP47. This is reflected by KD values of approximately 1.5 μM (GPR-foldon) and 3.6 μM (type I collagen), which were roughly 25 times worse than for the wild-type HSP47 protein (These findings were subsequently confirmed using an enzyme-linked immunosorbent binding assay (ELISA-like binding assay). Analysis of wild-type HSP47 showed a slightly higher affinity for type I collagen compared to GPR-foldon, with K protein .Collectively, these results demonstrate that the affinity of HSP47-R222S for the collagen triple helix is severely reduced, though not completely abolished, in comparison with wild-type HSP47. As such, these findings confirm a significant role of the p.R222 residue for binding of HSP47 to type I collagen.Next, the consequences of the p.(R222S) substitution on HSP47 chaperone functionality and its effects on the biosynthesis and properties of type I (pro)collagen were evaluated. To this end, biochemical analysis of metabolically labeled (pro)collagen chains was performed. Steady-state analysis of pepsinized collagen separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the bands representing the type I collagen α-chains showed a decreased electrophoretic mobility in both medium and cell layers of patient fibroblasts compared to control fibroblasts . These fSince HSP47 is required for correct folding of type I procollagen , the effOverall, these findings show that the reduced binding of HSP47-R222S to type I procollagen chains is associated with type I (pro)collagen overmodification, which leads to a mildly increased thermal stability. Despite the observed overmodification, folding and secretion of type I collagen was normal.In order to quantitate and characterize the posttranslational modifications of type I collagen, amino acid analysis and mass spectrometry was performed to determine the modification on selected residues in the triple helical region.Overall, amino acid analysis revealed a significant increase in 3-hydroxylated proline and 4-hydroxylated proline levels, with an increase of 65% and 2.5%, respectively, in the patient sample compared to control . The relCRTAP and LEPRE1 mutations, where an increase in hydroxylysines is seen ranging between 31.3–35.1% and 32.4–35.3%, respectively. ;[p.?] variants. –17,21,25of HSP47 , underscHSP47 binding prevents local or reverse unfolding of the procollagen α-chains during triple helix formation, and prevents procollagen aggregate formation, which facilitates the transport of procollagen to cis-Golgi. ,10 SeverCOL1A1 and COL1A2 and bi-allellic pathogenic variants in CRTAP, LEPRE1 (P3H1) and PPIB delay procollagen folding, leading to prolonged exposure of the nascent α-chains to hydroxyl- and glycosyltransferases in the ER. methionine  and incubated with shaking at 37°C for 30 min. Cells were then spun out briefly and resuspended in chase medium (standard Dulbecco’s modified Eagle’s medium with cold methionine and ascorbate) and aliquoted for separate time points . Cells were incubated at 37°C with shaking, removed at each time point, and spun out at high speed. Medium was transferred to a labeled fresh tube, and cell pellet and medium were frozen. After thawing 1 ml of lysis buffer  was added to each cell pellet and mixed. Cell lysates and medium were precipitated with ammonium sulfate . Precipitates were centrifuged at high speed for 20–30 min at 4°C and the resultant pellet was resuspended in 0.5 M acetic acid with 0.4 mg/ml pepsin. Samples were digested overnight at 4°C with shaking. Pepsinized collagen extracts were precipitated with 0.7 M NaCl final concentration at 4°C and spun out at high speed for 30 min. Pellets were resuspended in 0.5 M acetic acid and SDS-PAGE sample buffer, run on 6–8% acrylamide gels, dried, and exposed to film for autoradiography. A similar protocol was performed in duplicate using 14C-proline and alternative time points .Patient and control fibroblasts were grown to confluency as described in the ‘Acid- and base-hydrolyzed collagen was analyzed as reported previously.  Acid hydTo digest type I collagen in trypsin, lyophilized samples were resuspended in equal volume as the pre-lyophilisate of 1M Tris-HCl pH 8.0 with 20 ng/μL trypsin and digested for 18 hours at 37°C. Identification of peptides produced by proteolytic digest was performed on a Q-TOF Micromass spectrometer  equipped with an electrospray ionization source. Data were collected with the MassLynx (version 4.1) data acquisition software (Waters) and processed using Mascot Distiller . High performance liquid chromatography was performed with nanoACQUITY (Waters) system using a 75 μm x 100 mm 3 μm Atlantis dC18 column as the analytical column and a 180 μm x 20 mm 5 μm Symmetry C18 column as the trapping column. Chromatographic mobile phases consisted of solvents A (0.1% formic acid and 99.9% water (v/v)) and B (0.1% formic acid and 99.9% acetonitrile (v/v)). Peptide samples were loaded onto the trapping column and equilibrated 4 min in 99% solvent A followed by a 180 min gradient to 60% solvent A, 40% solvent B at a constant flow rate of 0.8 μl/min. Analysis was performed in survey scan mode. Tryptic peptides were identified from MS/MS spectra by a Mascot search against the National Center for Biotechnology Information (NCBI) database .™ Protease Inhibitor Cocktail, EDTA-Free (Thermo Fisher Scientific) at 4°C. For detection of ATP6, the culture medium was replaced with and without 50 mM DTT after the cells grew to over 80% confluency. After incubation for 90 minutes, the cells were washed twice with PBS and lysates were extracted using RIPA buffer (Thermo Fisher Scientific) containing Halt™ Protease Inhibitor Cocktail, EDTA-Free (Thermo Fisher Scientific) at 4°C. After centrifugation, soluble proteins in the extract were mixed with SDS sample buffer with reducing agents. These protein solutions were separated on Bolt 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) then transferred to PVDF membranes and Western blots were performed using antibodies listed in Patient and control dermal fibroblasts were cultured in 60 mm dishes. Cell lysates were extracted using M-PER (Thermo Fisher Scientific) containing HaltPatient and control dermal fibroblasts were cultured in 150 mm dishes and cell lysates were prepared with M-PER as described above. Six ml of cell lysates in which the total protein concentration was normalized to 1.0 OD at 280 nm measured in a Cary 4 Series UV-Vis spectrophotometer  were incubated with 1.5 ml of gelatin Sepharose 4B  for 2 h at 4°C. After washing twice with 10 ml TBS buffer followed by a wash with 25 mM Tris buffer, pH 7.4, containing 1 M NaCl and additionally twice with TBS buffer, sepharose beads were eluted with 2X SDS-PAGE sample buffer containing β-mercaptoethanol by heating to 95°C for 5 min. The eluted proteins were separated by SDS-PAGE followed by transfer to PVDF membrane and Western blotting and analyses were performed as described earlier. Gels were also stained with SilverXpress Silver Staining Kit (Thermo Fisher Scientific).P value of less than 0.05 was considered statistically significant.For comparisons between patient and control, we performed unpaired t test to determine whether differences were significant using ORIGIN Pro ver. 9.1  or GraphPad Prism software . A S1 Fig(A) and SYPRO Orange binding (B) showed a similar melting point for both wild-type (WT) (59.35°C and 54.50°C) and R222S (57.48°C and 54.00°C) protein, indicating that the variant does not cause structural instability.Differential Scanning Fluorimetry based on intrinsic tryptophan fluorescence (TIFF)Click here for additional data file.S2 Fig(A) Quantification of the folding rate of type I collagen (n = 4) presented in (B) Calculation of the area under the curve (AUC). Each dot represents a separate experiment. Results are shown as mean ± SEM. ns: not significant by unpaired t test. Although the folding appears slightly faster in patient versus control fibroblast, no significant difference could be observed. C: control, P: patient.(TIFF)Click here for additional data file.S3 Fig(A) Quantification of the secretion rate of type I collagen presented in (B) Additional secretion rate assay of type I collagen performed using an alternative experimental setup. (C) Quantification of the secretion rate of type I collagen in the medium using the alternative experimental setup (n = 2). Data presented are means ± SEM. Both setups show a near-normal secretion rate for patient compared to control. C: control, P: patient.(TIFF)Click here for additional data file.S4 FigCircular dichroism measurements of type I collagen extracted from control (black) and patient (pink) fibroblasts performed at a heating rate of 1°C/min. C: control, P: patient.(TIFF)Click here for additional data file.S5 Fig(A) Amino acid analysis of type I collagen after acid hydrolysis shows apparently normal levels of prolyl 4-hydroxylation and slightly increased levels of prolyl 3-hydroxylation in patient samples (insert). 3-Hyp: 3-hydroxyproline, 4-Hyp: 4-hydroxyproline, Pro: unmodified proline. (B) Amino acid analysis of type I collagen after acid hydrolysis shows increased levels (30%) of lysyl hydroxylation in patient samples. Hyl: hydroxylysine, Lys: unmodified lysine. (C) Amino acid analysis of type I collagen after base hydrolysis shows significantly increased glycosylation in patient samples. Glucosyl-galactosyl hydroxylysine content is increased by approximately 50% in patient samples, while no change in galactosyl hydroxylysine is observed. GGH: glucosyl-galactosyl hydroxylysine standard in blue, GH: galactosyl hydroxylysine standard in green. (D) Quantification of proline and lysine modifications after acid hydrolysis show significantly increased 3-Hyp, 4-Hyp and Hyl levels in patient versus control samples. Data presented are means ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001, unpaired t test. (E) Calculation of the percentage and increase in modification. The percentage was calculated as the number of hydroxylated lysine/proline residues divided by the total number of lysine/proline residues. Mean is represented with SEM between brackets. C: control, P: patient.(TIFF)Click here for additional data file.S6 Fig 3-Hydroxylation of proline residues is normal at the A1 site (P986) (A) and increased at the A3 site (P707) (B) of the type I collagen α1-chain. (C-D) Additional sugar attachment is observed at lysyl hydroxylation sites at residues K174 (C) and K531 (D) of the type I collagen α1-chain from patient cells. (E) Increased lysyl hydroxylation at residue K756 of the type I collagen α1-chain from patient cells. The peptide sequence is shown above the mass spectra and the examined residue is depicted in red. C: control, P: patient.Type I collagen was extracted from conditioned culture media of control and patient dermal fibroblasts. (TIFF)Click here for additional data file.S7 FigThe cell lysate extracted from patient and control dermal fibroblast cultures were electrophoresed on a Bolt 4–12% Bis-Tris Plus Gel followed by transfer to PVDF and Western blotting. Antibodies used to the blotting are listed in (TIFF)Click here for additional data file.S8 FigRT-qPCR was performed to assess expression levels. Protein names are indicated and the corresponding HUGO-approved gene name is included between brackets. Data presented are means ± SEM and individual data points represent independently prepared total RNA. *: p<0.05, **: p<0.01, ***: p<0.001 and ns: not significant by unpaired t test. C: control, P: patient.(TIFF)Click here for additional data file.S9 Fig(A) bind to gelatin and (B) do not bind gelatin. The cell lysate extracted from control and patient dermal fibroblasts and the eluted fractions from gelatin sepharose are depicted. The red color on the gel bands indicates that a membrane was overexposed and the signals were saturated. The asterisk (*) in the LH3 blot highlights the predicted migration of LH3. The additional, non-specific bands in the input lysate of the LH3 blot, compared to Western blots for the gelatin sepharose elution fraction of proteins that (TIFF)Click here for additional data file.S10 Fig(A) Cells were treated with tunicamycin (+Tu), thapsigargin (+Th) or left untreated (-) as indicated and evaluated for the presence of BiP, XBP1 spliced and CHOP. (B) Cells were either treated with DTT (+) or left untreated (-) as indicated. ATF6 (N) and ATF6 (C) indicate the predicted size of native full length and cleaved form by ER stress, respectively. ATF6 (C) is overlapping with a non-specific protein. The black line spacing denotes irrelevant lanes that were eliminated from the image. (C) Endogenous levels of eIF2α and phospho-eIF2α were evaluated. (D) Endogenous levels of the autophagy marker LC3 were evaluated. LC3-I and LC3-II represent the cytosolic form and the autophagosome-bound form of LC3, respectively. β-tubulin was used as loading control. C: control, P: patient.(TIFF)Click here for additional data file.S11 FigCrystal structure of the HSP47-collagen interaction with the collagen trimer depicted centrally, surrounded by two HSP47 molecules depicted in light and dark grey. Missense variants identified in humans and dog (with asterisk) are indicated in different colors on the HSP47 crystal structure. While residues L50, L78, L326  and R405 are located further away from the collagen interaction surface, R222 (present study) and M237 are located close to the interaction surface.(TIFF)Click here for additional data file.S1 Table1 personal communication with the authors.NA: not assessed, (XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


The risk loci for juvenile idiopathic arthritis (JIA) consist of extended haplotypes that include functional elements in addition to canonical coding genes. As with most autoimmune diseases, the risk haplotypes for JIA are highly enriched for H3K4me1/H3K27ac histone marks, epigenetic signatures that typically identify poised or active enhancers. In this study, we test the hypothesis that genetic risk for JIA is exerted through altered enhancer-mediated gene regulation.in vitro reporter assays to establish as proof-of-concept the idea that genetic variants in linkage disequilibrium with GWAS-identified tag SNPs alter enhancer function.We mined publically available HiC and other chromatin conformation data to determine whether H3K27ac-marked regions in 25 JIA risk loci showed physical evidence of contact with gene promoters. We also used in vitro assays, we found that a 657 bp, H3K4me1/H3K27-marked region within the first intron of IL2RA shows enhancer activity in reporter assays, and this activity is attenuated by SNPs on the IL2RA haplotype that we identified using whole genome sequencing of children with JIA. Similarly, we identified a 1,669 bp sequence in an intergenic region of the IL6R locus where SNPs identified in children with JIA increase enhancer function in reporter assays.All 25 loci examined showed multiple contact sites in the 4 different cell lines that we queried. These regions were characterized by HiC-defined loop structures that included 237 immune-related genes. Using These studies provide evidence that altered enhancer function contributes to genetic risk in JIA. Further studies to identify the specific target genes of genetically altered enhancers are warranted. After an overnight incubation at 16°C, the ligation mixtures were transformed into competent E. coli JM109 cells (Promega). Plasmid DNA was isolated by Miniprep kit (Qiagen) according to the manufacturer’s instructions. All recombinant plasmid sequences were confirmed by Sanger sequencing.The PCR amplified fragments were digested using XhoI and BglII  and ligated into a pGL4.23[http://www.genomics.agilent.com). The wild-type (wt) construct pGL4.23-IL2RA enhancer (chr10:6092661–6093317) was used as template to generate 4 enhancer genetic variants. The primer oligonucleotide sequences were: rs117119468, chr10:6092989, C>T, FP: 5'-AATAAAAGAGCCATCTCTCCAGACATCTGTCTGACG-3’, RP: 5'-CGTCAGACAGATGTCTGGAGAGATGGCTCTTTTATT-3’; rs12722502, chr10: 6093139, C>T, FP: 5'-GTGGTCTCTGCTTCCAGTCCTTGTGGTAGCA-3’, RP: 5'-TGCTACCACAAGGACTGGAAGCAGAGACCAC-3’; rs370928127,chr10: 6092686, A>G, FP: 5'-TGCGTGCTCTGCTTCTGCGATCTTACAGATTCCTC-3’, RP: 5'-GAGGAATCTGTAAGATCGCAGAAGCAGAGCACGCA-3’; rs552847047, chr10: 6093178, G>T, FP: 5'-GAAACCAGGCAGAGACTGCAACCCAGCTG-3’ and RP: 5'-CAGCTGGGTTGCAGTCTCTGCCTGGTTTC-3’.Mutations were introduced with the QuikChange II Site-Directed Mutagenesis Kit . The primers were designed according to the manufacturer’s protocol, utilizing the QuikChange Primer Design Tool  construct pGL4.23-IL6R enhancers (chr1:154357931–154358524 and chr1:154358954–154359600) were used as template to generate 2 enhancer genetic variants, respectively. The primer oligonucleotide sequences were: rs540546266, chr1:154358009, C>T, FP: 6) were tranfected with 1ug of the pGL4.23-constructs plus 1 ug of the control Renilla plasmid DNA  to normalize for transfection efficiency. Twenty-four hours after transfection, the cells were harvested, lysed and analyzed for luciferase activity. Luminescence was measured with the Dual-Luciferase® Reporter Assay System (Promega) in a microplate luminometer . Results from each construct and from the empty vector were normalized by dividing the luciferase reporter activity by the renilla reporter activity. Relative luciferase activities were calculated by dividing each normalized construct by the normalized empty vector. Relative luciferase activities are representative of at least three independent transfection experiments. Student’s t test was used to determine statistical significance.Cells were transiently transfected using electroporation following the protocol of the manufacturer . Cells  across the region of interest and tested each construct for luciferase production. Both constructs with brisk enhancer activity that was observable in Jurkat cells as well as myeloid HL60 cells. These results are summarized in IL6R gene (on JIA risk haplotype) that exhibited H3K4me1 / H3K27ac peaks and abundant TF binding (ENCODE and Roadmap Epigenomics data). We tested two constructs  in Jurkat cells. Each construct showed brisk enhancer activity, as shown in In order to corroborate the analysis derived from HiC data, we used a reporter assay to assess enhancer function within a 657 bp region within the first intron of the IL2RA intronic enhancer, rs117119468 (C->T), and rs12722502 (C->T)  , that we2 (C->T) . The conIL6R intergenic enhancer. These SNPs were either identified as having high likelihood of having regulatory effects using the SNiPA software program [We next tested the effects of 4 variants within the  program   not on the JIA haplotype . IL2RA was identified as a JIA risk locus through a candidate gene approach [IL2RA expression and adaptive immune function. IL6R encodes the alpha subunit of the IL6 receptor. IL6 is a known mediator of the pathogenic processes associated with a broad range of autoimmune and inflammatory diseases, and IL6R is the target of the therapeutic agent, tocilizumab, which has been shown to be efficacious in treating multiple autoimmune diseases, including JIA [It is important to note that enhancer function can’t be determined on the basis of chromatin structure/features alone. Thus, to corroborate our analyses of HiC data, we experimentally tested enhancer function within the JIA-associated approach  and valiceptor CD. IL2RA wapproach , it has ding JIA .IL2RA, and, furthermore, demonstrated that genetic variants that we had previously identified on WGS of patients with polyarticular JIA [IL6R gene. This is the first instance, as far as we are aware, of the identification of specific, non-coding functions that are altered by JIA-associated genetic variants. We note, as well, that although many intronic enhancers regulate the transcriptional efficiency of the genes in which they are located [IL2RA locus Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Correction to: J Biomed Sci 27, 64 (2020)https://doi.org/10.1186/s12929-020-00653-yDictyostelium presents an excellent system to dissect the roles of SLC25A1 and SLC25A13 in NCL biologys.’ on Page 8 is incorrect. The correct sentence should be ‘Therefore, Dictyostelium presents an excellent system to dissect the roles of SLC25A1 and SLC25A13 in NCL pathology.’In the original publication of this article , the wor


Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5′ end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in  RNA editing is a post-transcriptional modification of pre-mRNAs that can introduce codon changes in mature mRNAs. The A (adenosine)-to-I (inosine) deamination in pre-mRNAs is the most abundant RNA editing in mammals . BecauseGria2 regulates alternative splicing of exon 14 (flop) and exon 15 (flip)  , 23, 24.5 (flip) , 26.Tmem63b mRNA from mouse brain in the current study. We identified four isoforms of Tmem63b resulting from an A-to-I RNA editing that changes glutamine to arginine at exon 20 and an alternative splicing of exon 4. The editing was almost exclusively detected in the spliced isoform lacking exon 4, suggesting a linkage between the two post-transcriptional events. Using Adar2 cKO mice and cultured cerebellar granule neurons (CGNs), we found that the isoform containing exon 4 suppressed the Q/R editing efficiency in a cis manner. Functional analysis demonstrated that the splicing and the editing coordinately regulated hypoosmolarity-induced Ca2+ influx. Together, these results reveal a long-distance coupling between alternative RNA splicing and RNA editing in Tmem63b, in which the splicing plays a dominant role. These post-transcriptional modifications may enable the osmosensitive Tmem63b channel to play diversified roles in the brain.We have recently found that Tmem63b serves as an osmosensor in the inner ear and is required for survival of outer hair cells and hearing . To expaTmem63b cDNAs from mouse brain tissues, we obtained one cDNA sequence that was different from the documented mRNA sequence NM_198167 in the NCBI database (A). The nucleotide c.1856 in this cDNA was guanosine, instead of the published adenine at the same site in exon 20. This finding suggested an A-to-I RNA editing that results in the substitution of glutamine codon CAG to arginine codon CGG at position 619 of the protein sequence (B). In addition, exon 4 was spliced out in the cDNA (C). Thus, the Tmem63b transcript we obtained was a new isoform with two post-transcriptional modifications, i.e. A-to-I editing at c.1856 in exon 20 and alternative splicing of exon 4.Recently, the osmosensitive cation channels in TMEM63 family have attracted research interests , 28, 29.database A. The nusequence B. In addthe cDNA C. Thus, A). Specific primer pairs were designed to amplify sequences containing the editing site from mouse genomic DNAs (311 bp) and brain cDNAs (327 bp) (A). The majority of PCR products amplified from brain cDNAs of adult mice were digested by BsaWI and showed two additional bands (208 bp and 119 bp), whereas the PCR products amplified from mouse genomic DNAs were not recognized by BsaWI (B). BsaWI failed to digest Tmem63b cDNA fragments from tissues other than brain, including lung, kidney, heart, liver, and spleen (E), suggesting that this editing is brain-specific. In addition, the editing efficiency varied in brain sub-regions, from ∼40% in the hypothalamus to ∼60% in other brain regions in adult mice  A. The maby BsaWI B. BsaWI d spleen E, suggesTmem63b, we transfected mouse Adar1 or Adar2 cDNA together with a Tmem63b minigene containing the genomic sequence from exon 19 to exon 21 (the editing segment) in HEK293 cells and evaluated the editing efficiency (A). To avoid putative contamination from endogenous Tmem63b in HEK cells, the exogenous RNAs were reverse-transcribed into cDNAs by a primer RTIRES  paired to vector sequence (A). Adar2 but not Adar1 transfection resulted in partial digestion of PCR products (403 bp) into two bands (284 bp and 119 bp) by BsaWI (B). To verify that the transfected Adar1 was functional, we coexpressed Adar1 with an Htr2c minigene containing the “A” editing site at exon 5 that is preferentially edited by Adar1  and bred them with Nestin-cre line  mice (C). The Adar2 cKO mice were postnatally lethal, likely because of inability to edit the Gria2 Q/R site (Tmem63b in P0 mouse brains. The Q/R editing of Tmem63b was completely eliminated in Adar2 cKO brains (D and E).In mammals, Adar1 (Adar) and Adar2 (Adarb1) catalyze A-to-I editing. To determine whether Adar1 or Adar2 catalyzed the Q/R editing in ficiency A. To avosequence A. Adar2 by BsaWI B. To verby Adar1  and founby Adar1 . To examcre line  to obtaire) mice C. The AdQ/R site . We therO brains , D and ETmem63b, we analyzed the secondary structure of RNA sequence surrounding editing site using the bioinformatics software RNAstructure . We then introduced a series of mutations on the predicted ECS and analyzed their effects on Q/R editing in Adar2 coexpressed HEK 293 cells. The editing efficiency in WT sequence was 62%, whereas deletion of 14 consecutive nucleotides from the center of ECS abolished Q/R editing . In addition, mutations on single or multiple nucleotides that destabilized the dsRNA duplex also decreased the editing efficiency , whereas introduction of the compensatory mutations around the editing site restored the editing efficiency , indicating the importance of the hairpin structure formed by the intronic ECS and the exonic editing region. Taken together, the intronic sequence at the proximal 5′ end of intron 20 was demonstrated to be the ECS for Tmem63B Q/R editing.To dissect the critical ECS for Q/R editing of tructure . A dsRNAA). Tmem63b cDNAs with (long-form) or without (short-form) exon 4 were amplified into PCR products of different lengths, 223 bp and 184 bp, respectively. The efficiency of the splicing was calculated as the percentage of short form to total Tmem63b. The short form was detected in brain of adult mouse but not in lung, kidney, liver, and spleen (B). Notably, a weak splicing occurred in about 2% of total heart Tmem63b mRNAs. In the whole brains, ∼80% of the Tmem63b mRNAs were the short form. The splicing efficiency in different brain regions mildly varied from 70 to 80% . The results showed that the long form (exon 3-4-5) and the short form (exon 3-5) were 18.7% and 77.5%, respectively, of the total Tmem63b mRNAs (exon 5) (E). This experiment further verified that around 80% of mRNAs from adult mouse brain were short isoforms. Taken together, the alternative splicing of Tmem63b exon 4 is brain-specific. The splicing efficiency is constant among different brain regions and relatively stable at different development stages.Above data suggested that up to ∼80% of time PCR E. The re(exon 5) E. This eTmem63b pre-mRNA are also coupled, 40 Tmem63b clones originated from mouse brain were subjected to Sanger sequencing with full length, of which 36 were short cDNA clones lacking exon 4 and the majority of them (33/36) contained the edited R at the Q/R site. In contrast, all four long cDNA clones were not edited at the Q/R site (A). These data suggested that Q/R editing efficiency in long and short isoforms was biased. We further analyzed this phenotype using BsaWI digestion. A pair of primers, with the forward primer base-paired to exon 4 (LFw) and the reverse one downstream of the Q/R site (CRe), was used to amplify the long isoform (2255 bp). The forward primer, with 4 bp of the 3′ end base-paired to exon 5 and the rest of sequence base-paired to exon 3 (SFw), was designed to amplify the short isoform (2246 bp) (B). We found that the fragments amplified from short isoform were largely digested by BsaWI (1593 bp and 653 bp) whereas the digestion bands were not seen in long-form Tmem63b fragments (C and D). The Q/R editing was also limited to the short-form mRNAs of P7 and E18.5 mouse brains (E). The editing efficiency in short-form Tmem63b was further analyzed in different brain regions of P7 and adult mice. In adult, the editing was ∼60% in hypothalamus and ∼80% in other brain regions, whereas in P7, the editing in short-form mRNAs was generally lower in all brain regions as compared with those in adult  of the clones amplified from the short isoform were edited to R at the Q/R site . Taken together, these results indicated that the alternative splicing of exon 4 and Q/R editing at exon 20 in Tmem63b pre-mRNA were coupled.Previous studies demonstrated that A-to-I editing in exons and splicing of nearby downstream introns could affect each other , 25, 26.Q/R site A. These 2246 bp) B. We fouragments , C and De brains , indicate brains E. The edAdar2 cKO mice. However, we found that the efficiency of alternative splicing was not changed (A and B), suggesting that the editing did not affect the splicing.We then studied which of the post-transcriptional modifications, the editing or the splicing, was dominant. We reasoned that if the editing determined the splicing efficiency as reported previously , 23, 24, changed , A and BTmem63b in these neurons at DIV5, a time point at which we examined the transfected constructs (C). The splicing efficiency . The editing efficiency of endogenous Tmem63b in cultured CGNs was 28.7 ± 2.1% . Importantly, the editing was only detected in short isoforms using BsaWI digestion, with editing efficiency of 37% (Tmem63b minigenes transfected in cultured CGNs. The editing segment of Tmem63b (A) was efficiently edited  when transfected into CGNs . The alternative splicing might modulate the editing through a trans mechanism, where the introns spliced out from long- or short-form Tmem63b regulate the editing, or a cis mechanism, where the mRNA sequence around the splicing region affects the editing. For the long-form Tmem63b, the introns spliced out are intron 3 (I3) and intron 4 (I4), whereas in short form, the intronic segment spliced out is I3E4I4 (D). We found that all these intronic segments had no effects on the editing efficiency (E and F), arguing against a trans mechanism. Then we constructed minigenes by fusing the editing segment with DNA segments containing exon 3∼5 varied by alternative splicing of exon 4 . The LiQi (genomic sequence from exon 3 to exon 5 linked with the editing segment) was largely (∼65%) spliced to short form . The editing efficiency in LiQi  was close to SQi  but higher than LQi , indicating that the splicing regulates the editing in a cis manner. We then wondered whether the editing efficiency can be modified by changing splicing. We first made splicing site mutant (SpM), 5′ splice site (GU) at intron 4 and 3′ splice site (AG) at intron 3 mutated to complimentary nucleotides, which disrupts the inclusion of exon 4 . As expected, only the short-form transcript was observed. The editing in SpM  was the same as SQi (H and I). In neurons, multiple splicing factors such as Nova  which could be involved in Nova-mediated exon skipping . As expected, LiQi harboring these mutations significantly reduced the short isoform (∼40% in NvM versus ∼65% in WT LiQi). The editing efficiency was lower in NvM  as compared with WT LiQi (H and I). Taken together, these data indicate that the splicing of exon 4 regulated Q/R editing through a cis mechanism.To test the possibility that the splicing regulates the editing, we used primary culture of CGNs. We first examined the coupling of splicing and editing of endogenous nstructs C. The sp% n = 3, , close t% n = 3, D. Importy of 37% . We then Tmem63b A was effGNs CTL, , D–F. Ths I3E4I4 D. We fouficiency , E and F as Nova , Rbfox  (A and B). A charged arginine residue at this position might interfere with the conductance of polyvalent cations, such as Ca2+. To test this possibility, we examined the Ca2+ conductance relative to Na+ for these four isoforms of Tmem63b in Neuro2a (N2a) cells (D and E). We recorded the currents using Na-gluconate in both extracellular and pipette solutions; gluconate was used to avoid the contamination from the endogenous Cl− currents (+ solution and reached the amplitudes of ∼600 pA (measured at −70 mV), the extracellular solution was switched to 170 mOsm/liter Ca2+ solution. Because most reacting cells burst with continuous exposure to hypotonic solution, we did not try to record the currents at plateau in this experiment , −2.8 ± 2.8 mV for QS , −2.7 ± 2.2 mV for RL , and −2.9 ± 1.7 mV for RS . The reversal potential in Ca2+ solution was −10.4 ± 4.9 mV for QL (n = 13), −10.1 ± 7.6 mV for QS (n = 9), −23.4 ± 12.6 mV for RL (n = 7), and −20.3 ± 12.4 mV for RS (n = 8) (F and G). The calculated Ca2+ permeability relative to Na+ (Ca/PNaP) was QL (0.64) ∼ QS (0.72) > RL (0.38) ∼ RS (0.45) (H). These data thus demonstrated that Q/R editing at the intracellular mouth of Tmem63b channel reduces Ca2+ permeability.Tmem63 family are osmosensitive (or mechanosensitive) cation channels , 39. TheA–C) . The TmeA–C) , A and Ba) cells . Tmem63ba) cells , D and Ecurrents . The osmperiment . The rev (n = 8) , F and GS (0.45) H. These Tmem63b encoded amino acids in the first intracellular loop 1 between M0 and M1 (A and C). In AtOSCA1.2, the intracellular segment intracellular loop 2 paralleling plasma membrane is predicted to be involved in mechanosensation (2+ influx (2+]i elevation was detected in cells expressing Tmem63b (I), but the ratios of responsive cells varied among four isoforms (J). Control cells that do not express Tmem63b failed to respond to hypotonic stimuli . Hypoosmolarity-induced Ca2+ influx occurred more frequently in cells expressing the short form of Tmem63b (41% of QS and 34% of RS transfected cells) than in cells expressing the long forms (29% of QL and 17% of RL), indicating that exclusion of exon 4 enhanced the osmosensitivity of Tmem63b channel (J). These data also showed that Q-form Tmem63bs appeared to be more sensitive to osmolarity change than R-forms. Thus, the alternative splicing of exon 4 and the Q/R editing in Tmem63b coordinately regulate Ca2+ influx induced by hypoosmolarity.The exon 4 of 0 and M1 , A and Censation , 43. We + influx . We have+ influx . Interes+ influx , indicat+ influx . GCaMP6f Tmem63b I, but thisoforms J. Contro stimuli I; 1/199. channel J. These Tmem63b pre-mRNAs in the mouse brain. Our study reveals that it displays the following properties. First, the occurrence sites for above post-transcriptional modifications are remote. Second, the alternative RNA splicing plays a dominant role.In this study, we have identified a coupling between the A-to-I editing at the Q/R site of exon 20 and the alternative splicing of exon 4 in Tmem63b mRNAs are edited at the Q/R site in the adult mouse brain, consistent with a previous study . A positively charged residue at this site reduces the permeability of divalent cation Ca2+ (H). A similar situation happens in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 and kainate receptors GluK1 and GluK2, where the Q/R editing occurs at the center of the channel pore and dictates the Ca2+ permeability (A and C). The structure studies on OSCA channels suggest that the α-helices in intracellular loop 2 paralleling membrane may involve mechanodetection and channel gating . The founder mice were backcrossed with C57BL/6J mice for over five generations followed by breeding with Nestin-Cre mice . Genotypes were determined by PCR with the following primers: 5′-AGTCATTCCTCCTAGCCTTTT-3′ (forward) and 5′-TTATCACCTTGGCATCTTTG-3′ (reverse) to confirm the presence of the loxP site, 5′-ATTTGCCTGCATTACCGGTC-3′ (forward) and 5′-ATCAACGTTTTCTTTTCGG-3′ (reverse) for the presence of Cre.All animal studies were approved by the Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University and performed in accordance with guidelines for humane treatment of animals. All animals were housed in 12 h of light and 12 h of dark cycles at 25 ± 1 °C, with water and food obtained Cre mice  to obtaiTmem63b (NM_198167.3), Adar1 , and Adar2  were amplified from mouse brain cDNAs, using specific primers  for 2 days. At DIV 2, primary cultured neurons were transfected with Tmem63b minigenes using lipofectamine 2000, and the medium was replaced with MEM-based medium containing 5 mg/ml d-glucose (Sigma-Aldrich), 1% ITS (insulin-transferrin-sodium selenite) (Sigma-Aldrich), 2 mm l-glutamine, and 1% penicillin-streptomycin. CGNs were collected for analysis 48–72 h after transfection.HEK 293 cells were cultured in DMEM with 10% FBS (Gibco). The minigenes were transfected into cells using lipofectamine 2000 according to the manufacturer's instruction. The cells were collected for analysis 24–48 h after transfection. CGNs were acutely dissected and cultured following the protocols reported in previous studies . In brieTmem63b in tissues, total RNA samples from mouse tissues were extracted by TRIzol (Invitrogen) and reverse-transcribed into cDNAs using HiScript® 1st Strand cDNA Synthesis Kit  and oligo(dT)18 primer. For cultured cells transfected with Tmem63b or Htr2c minigenes, the extracted total RNAs were digested by RNase-free DNaseI  at 37 °C for 1 h to remove the endogenous genomic and transfected DNAs. Then the RNAs were reverse-transcribed into cDNAs by specific reverse transcription primer, 5′-GAATGCTCGTCAAGAAGAC-3′ (RTIRES), using HiScript® 1st Strand cDNA Synthesis Kit . Only the RNAs from exogenous constructs could be reverse-transcribed. The RT (minus) controls of each experiment were carried out to assess the amount of DNA contaminations in RNA preparations.For cloning and quantification of mRNA isoforms of Tmem63b isoforms were quantified by Applied Biosystems StepOnePlus Real-Time PCR system (Life Technologies) using AceQ® qPCR SYBR® Green Master (High ROX Premixed)  reagents with specific primer pairs: exon 3-4-5, 5′-ACAGATGCAGACAGGCTTC-3′ and 5′-TTGGTCAAAGTCGACAGAGC-3′; exon 3-5, 5′-TGACAGATGCAGACAGTGTG-3′ and 5′-TTGGTCAAAGTCGACAGAGC-3′; exon 5, 5′-ATCAGCTATGCATGGGGAC-3′ and 5′-CCAGGAACAGAAGCCATTG-3′; and Gapdh, 5′-TGAACGGGAAGCTCACTGG-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′.For quantitative real-time PCR, the RNA samples from mouse brains were reverse-transcribed into cDNAs using HiScript Q RT SuperMix for qPCR (+gDNA wiper) Kit . cDNAs of different Tmem63b minigene (403 bp) were amplified by primers pCAGFw (forward) and EDIRe2 (reverse). The cDNA sequence of transfected Htr2c minigene (340 bp) was amplified by primers pCAGFw (forward) and pCAGRe (reverse). In the experiment to study the coupling between alternative splicing of exon 4 and Q/R editing at exon 20 in mouse brain cDNAs, the primers LFw (forward) and CRe (reverse) were designed to amplify the long-form (2255 bp) Tmem63b, whereas the short-form (2246 bp) Tmem63b fragments were amplified by SFw (forward) and CRe (reverse). In the experiment to explore the regulation of splicing to editing in CGNs, the cDNA sequences of transfected Tmem63b minigene were amplified by following primers: pCAGFw and EDIRe2 for the study of trans regulation, resulting in the PCR a product of 403 bp; and E5Fw (forward) and EDIRe2 (reverse) for the study of cis regulation, resulting in the PCR a product of 334 bp. The primers used in above amplification were shown in For quantification of Q/R editing in mouse tissues, the genomic sequences spanning the editing site (311 bp) were amplified by primers EDIFw1 (forward) and EDIRe1 (reverse). The cDNA sequences spanning the Q/R site (327 bp) were amplified by primers EDIFw2 (forward) and EDIRe2 (reverse). In the study of A-to-I editing enzyme and ECS in HEK293 cells, the cDNA sequences of transfected Tmem63b, the amplified fragments were incubated with BsaWI endonuclease at 60 °C for 1 h and resolved by agarose gel electrophoresis. For Htr2c, the amplified fragments were incubated with BtsI endonuclease at 55 °C for 1 h and resolved by agarose gel electrophoresis. The gels were scanned by Gel Imaging System (Tanon). The abundance of each band was analyzed by ImageJ software . The efficiency of editing was displayed by the ratio of integrated densities of BsaWI enzymed bands to control bands. In analysis of ECS, because the BsaWI recognition site is disrupted when the mutations are nearby Q/R site, the editing efficiencies of these mutants were quantified by measuring the ratio of peak heights obtained from sequencing chromatograms.For Tmem63b cDNAs with (long-form) or without (short-from) exon 4 were amplified into different lengths (223 bp and 184 bp), followed by agarose gel electrophoresis. The long (309 bp) and short (270 bp) isoforms from LiQi minigenes were amplified by pCAGFw (forward) and E5Re (reverse), followed by agarose gel electrophoresis (A pair of primers with forward (SPFw) and reverse (SPRe) were designed to amplify the cDNA sequence near the splicing region . Tmem63bphoresis . The gelm): 65 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES . The isotonic solution was exchanged to 170 mOsm/liter hypotonic solution without changing the ionic concentrations by a peristaltic pump  at a constant speed. The osmolarity was measured by a vapor pressure osmometer (Wescor Vapro Model 5600). The cytoplasmic calcium fluorescence was recorded at 1 Hz for 10 min by laser scanning confocal microscope  at room temperature (24 ± 2 °C) using 488-nm illumination. The change of fluorescence was normalized by the ratio of real-time intensity (Ft) relative to the initial value (F0). The cells with Ft/F0 > 1.5 were considered as positive cells responding to osmolarity changes.The cytoplasmic calcium concentration was monitored by free calcium indicator GCaMP6f . Tmem63bm) 80 Na-gluconate, 130 mannitol, and 10 HEPES . The cells were perfused with isotonic extracellular solution containing (in mm) 80 Na-gluconate, 1 Ca-gluconate, 10 HEPES, and 130 mannitol . The hypotonic extracellular solution without changing the ionic concentration contains (in mm) 80 Na-gluconate, 1 Ca-gluconate, and 10 HEPES . For measuring the Ca2+ permeability, the Na+-rich hypotonic extracellular solution was changed to Ca2+-rich solution containing (in mm) 40 Ca-gluconate, 40 mannitol, and 10 HEPES (pH 7.4 with Ca(OH)2, 170 mOsm/liter). The currents were collected using an Axopatch 200B amplifier and Digidata 1550 digitizer (Molecular Devices) at the sampling rate of 10 kHz and were low-pass filtered at 1 kHz. The current data were analyzed using pClamp 10 software. The cells were holding at 0 mV before application of 100-ms ramp from −80 mV to 80 mV every 1 s. The cells with a membrane resistance below 800 MΩ or series resistance above 10 MΩ were discarded. The Ca2+ permeability relative to Na+ (PCa/PNa) was calculated as PCa/PNa = [Na+]iexp(ΔVrevF/RT) (1+exp(ΔVrevF/RT)) / 4[Ca2+]o, where the ΔVrev is measured the shift in reversal potential, [Na+]i is the intracellular Na+ concentration, and [Ca2+]o is the concentration of the extracellular substituting Ca2+. The reversal potential was measured in 7–13 cells for each isoform.Whole-cell path clamp recordings were performed on transfected N2a cells under ramp protocol. The recording electrodes had the resistance of 8∼10 MΩ when filled with the pipette solution composed of (in mThe secondary structures of RNA around the editing site and the ECS were predicated by RNAstructure software (Version 6.0.1). The sequence 200 bases downstream and upstream of the editing site was imported for analysis. The lowest free energy structure and a set of low free energy structures were predicted according to following restrictions on parameters: Maximum % Energy Difference, Maximum Number of Structures, and Window Size. The parameters were set as previously reported . The MaxAtOSCA1.2, the plant homolog of Tmem63b with Cryo-EM structure solved (AtOSCA1.2 (PDB ID: 6mjv). The model was depicted by PyMol program.Protein sequence of mouse Tmem63b was aligned to e solved , 43. Thee solved  with thet test if not otherwise stated. p values less than 0.05 were considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. p ≥ 0.05 was denoted as “ns.”All data are presented as mean ± standard deviation (S.D.) in at least three independent experiments. Statistical analyses were performed using GraphPad Prism version 6.0c software and analyzed using one-way analysis of variance (ANOVA) or unpaired All data supporting our conclusions are contained within this article and in the


After this article  was publThe authors apologize and note that these duplications arose due to errors in figure preparation. The results in A corrected The underlying data for other results reported in this article are provided in S1 FileWestern blots of AccD5 and Kas A. Detection was performed using anti-AccD5 or anti-KasA antibodies elicited in rabbit. A) Raw data of AccD5 Western Blot. Lane 1, WT control, lanes 2 is empty and lanes 3 to 8 corresponding to T1 to T3 samples, with and without ATc shown in . C Raw d(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(JNB)Click here for additional data file.S4 File(JNB)Click here for additional data file.S5 File(JNB)Click here for additional data file.S6 File(JNB)Click here for additional data file.S7 File(JNB)Click here for additional data file.S8 File(JNB)Click here for additional data file.S9 File(JNB)Click here for additional data file.S10 File(TIF)Click here for additional data file.S11 File(XLS)Click here for additional data file.


Vitamin D: Overview of Renal and Extra-renal metabolism and regulation.Vitamin D: Overview of molecular mechanism and multifaceted functions beyond skeletal homeostasis.Anti-viral infection.Anti-malaria and anti-systemic lupus erythematosus (SLE).Vitamin D: Overview of local immunomodulation in human infectious diseases.Vitamin D might aid in preventing SARS-CoV-2 infection:Vitamin D: Suppression of key pro-inflammatory pathways including nuclear factor kappa B (NF-kB), interleukin-6 (IL-6), and tumor necrosis factor (TNF).Vitamin D might act as a strong immunosuppressant inhibiting cytokine release syndrome in COVID-19:Vitamin D: Induction of key neurotrophic factors.Vitamin D might prevent loss of neural sensation in COVID-19 by stimulating expression of neurotrophins like Nerve Growth Factor (NGF):.The coronavirus disease 2019 (COVID-19) pandemic has led to a declaration of a Public Health Emergency of International Concern by the World Health Organization. As of May 18, 2020, there have been more than 4.7 million cases and over 316,000 deaths worldwide. COVID-19 is caused by a highly infectious novel coronavirus known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leading to an acute infectious disease with mild-to-severe clinical symptoms such as flu-like symptoms, fever, headache, dry cough, muscle pain, loss of smell and taste, increased shortness of breath, bilateral viral pneumonia, conjunctivitis, acute respiratory distress syndromes, respiratory failure, cytokine release syndrome (CRS), sepsis, etc. While physicians and scientists have yet to discover a treatment, it is imperative that we urgently address 2 questions: how to prevent infection in immunologically naive individuals and how to treat severe symptoms such as CRS, acute respiratory failure, and the loss of somatosensation. Previous studies from the 1918 influenza pandemic have suggested vitamin D’s non-classical role in reducing lethal pneumonia and case fatality rates. Recent clinical trials also reported that vitamin D supplementation can reduce incidence of acute respiratory infection and the severity of respiratory tract diseases in adults and children. According to our literature search, there are no similar findings of clinical trials that have been published as of July 1st, 2020, in relation to the supplementation of vitamin D in the potential prevention and treatment for COVID-19. In this review, we summarize the potential role of vitamin D extra-renal metabolism in the prevention and treatment of the SARS-CoV-2 infection, helping to bring us slightly closer to fulfilling that goal. We will focus on 3 major topics here:  COVID-19 is an infectious disease caused by SARS-CoV-2, a newly discovered coronavirus that primarily spreads between people during close contact and through respiratory droplets when infected individuals cough, sneeze, or talk . FurtherThe last century’s influenza pandemic (1918–1919) claimed between 40 and 50 million lives . A subst3) and ergocalciferol (Vitamin D2), precursors of 1,25(OH)2D3 (the active form of vitamin D) [Vitamin D is a group of fat-soluble steroids, and the most common forms of vitamin D supplementation are cholecalciferol (Vitamin Dtamin D) . New devtamin D) . Vitamintamin D) , 19, 20.tamin D) , 22. Vittamin D) , 24. Vittamin D) , 27. Bestamin D) . Furthertamin D) , 30. The2D3), the active form of vitamin D. Systemically, vitamin D plays a critical role in calcium homeostasis and bone metabolism by binding the vitamin D receptor (VDR) to form vitamin D response element (VDRE) to regulate expression of the down-stream genes of vitamin D [In humans, the main source of vitamin D is derived from dermal synthesis with a small proportion coming from food sources . To becoitamin D . Parathy2D3 is critical for the immunomodulatory function of vitamin D in local tissues [2D3 and expression of CYP27B1 are not fully understood because of the lack of tools to acutely measure 1,25(OH)2D3 levels in their microenvironment under physiological or pathogenic conditions and different regulatory mechanisms of extra-renal CYP27B1 between tissues [Accumulating evidence demonstrates that extra-renal synthesis of 1,25(OH) tissues . However tissues .Many cellular and molecular targets of vitamin D have been identified in cancer studies during the past decades . MechaniThe immune system is divided into two branches: innate and adaptive immunity. The innate response is the first line of defense relying largely on mucosal barriers, monocytes, neutrophils, macrophages, and dendritic cells, which also function as antigen presenting cells that activate the B and T lymphocytes of the adaptive immune response. The expression of CYP27B1 and VDR were found in most immune cells including macrophages, dendritic cells, and activated B and T lymphocytes . EmerginVitamin D is known to exert direct antibacterial and antiviral actions through cathelicidin (LL37), an antimicrobial peptide that promotes the induction of reactive oxygen species and the inhibition of phospholipids’ synthesis , 53. VitLow levels of vitamin D have been reported to be involved in the pathogenesis of many infectious diseases including bacterial or viral infections of the respiratory tract such as tuberculosis and influenza, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Hepatitis C Virus (HCV), parasitic infections of the gastrointestinal tract, and systemic fungal infection .Fig. 3Me2D3 then leads to downstream changes in gene expression, ultimately reducing inflammation while maintaining antiviral function [Vitamin D has been found to be a component of innate responses needed to maintain respiratory tract health. Mechanistically, acute viral respiratory tract infections have been found to upregulate CYP27B1 in respiratory epithelial cells, converting local stores of vitamin D into 1,25-dihydroxyvitamin D leading to the induction of cathelicidin . This infunction . Supplemfunction . Vitaminfunction .TLRs play a key role in the mediation of early pathogen recognition during early stages of viral infections. TLR1/2 heterodimers can activate human macrophages and induce expression of CYP27B1 and VDR, leading to the induction of antimicrobial peptides . A recenThe induction of autophagy and apoptosis are new mechanisms by which vitamin D may exert its potential effects against viral infections including HIV-1, rotavirus (Rota), and kaposi sarcoma associated herpesvirus . There aPrevious in vitro studies have shown that vitamin D has antiplasmodial activity through VDR dependent and independent pathways . The onsLow serum levels of 25(OH)D have been associated with TLR-driven amplification of autoimmunity and disease severity in SLE . Hydroxy2D3 rather than systemic production is responsible for many of the immune effects of vitamin D in respiratory diseases [2D3 locally to act in an autocrine or paracrine fashion to modulate cell proliferation, cell differentiation and inflammation [It is increasingly recognized that localized synthesis of 1,25(OH)diseases . Extra-rammation , 73. At ammation . During ammation . Downregammation . Additioammation . IL-6 waammation . Anti-ILSome COVID-19 patients have neurological symptoms—loss of taste and smell, headaches, etc. How SARS-CoV-2 affects the nervous system has not been well studied because few autopsies of these patients have been done due to the highly contagious virus. Previous evidence from studies of SARS-CoV have demonstrated the presence of SARS-CoV in neurons of the brain from SARS patients , and sugNTR in neurons, glial cells, and schwann cells. Neurotrophin induction underlies the neuroprotective effect of vitamin D in brain ischemia and neurodegenerative disorders. For example, vitamin D can decrease the progression of Alzheimer’s disease through the induction of NGF [Previous studies have revealed that vitamin D has a potent neuroprotective effect through several independent mechanisms . The neun of NGF .The immunomodulating properties of vitamin D represent another mechanism of its activity as an immunosuppressant to protect neurons . Vitamin2D3 with increased age is a major factor of weakened immune response with age (immunosenescence). In a previous study in Norway patients, 1,25(OH)2D3 was found to decrease from 140 pmol/l for those aged 20–39 years to 98 pmol/l for those > 80 years, despite an increase in serum 25(OH)D from 24 ng/ml (59.9 nmol/L) for those 20–39 years to 27 ng/ml (67.4 nmol/L) for those > 80 years [2D3 as well. The concentration of 1,25(OH)2D3 should be considered on several levels: 1) 25(OH)D substrate level; 2) renal expression of 1α-hydroxylase or CYP27B1; 3) extra-renal expression of CYP27B1 and 4) local expression of 25-hydroxyvitamin D-24α-hydroxylase [2D3 in renal or extra-renal tissues. In our recent study of acute myeloid leukemia (AML) cells, we observed significantly lower levels of CYP27B1 proteins within bone marrow (BM) cells when compared to non-AML controls [Low levels of 25(OH)D are common and have been associated with a variety of disease states, including susceptibility to respiratory infections . Clinica80 years . Thus, vroxylase . Vitamincontrols . In simicontrols .2D3 were significantly lower in non-survivors compared to survivors among sepsis patients, and that complex mechanisms underlie low 1,25(OH)2D3 in critically ill patients [Additionally, we recently demonstrated that serum levels of 1,25(OH)patients .Our hypothesis is that vitamin D supplementation can reduce the risk of COVID-19 incidence, symptoms, and severity. It should be investigated in a well-designed clinical trial as vitamin D is in general safe and there is a large body of data supporting the use of higher doses of Vitamin D3 in the setting of vitamin D deficiency.2D3) monitored and replaced to meet the baseline requirement.We propose that COVID-19 patients have vitamin D levels (25(OH)D and 1,25(OH)3) and ergocalciferol (Vitamin D2), precursors of 1,25(OH)2D3, although administration of calcitriol is limited because of the potential hypercalcemia-related complications.Guidelines recommended a target level of 30–40 ng/mL (75–100 nmol/L) for patients with risk of fractures, falls, autoimmune conditions, and cancers . The End2D3, high doses of vitamin D supplementation may still cause systemic hypercalcemia, another major obstacle of vitamin D therapy. The concentrations of 1,25(OH)2D3 required to induce differentiation in vitro are typically in the range of 10–100 nM (nM); however, a serum level of such a concentration would result in hypercalcemia in humans as the typical concentration of 1,25(OH)2D3 is around 0.1 nM. One potential strategy is to develop a tissue-specific approach to deliver therapeutic levels of 1,25(OH)2D3 locally in the absence of hypercalcemia [2D3 can be achieved by focused stimulation of locally regulated CYP27B1 in inflammatory cells to exert autocrine/paracrine actions [In a previous study of vitamin D supplementation in those with SLE, 50,000 IU of vitamin D weekly for 128 weeks were prescribed for those patients with levels less than 40 ng/ml (100 nmol/L) and the results suggest that high doses of vitamin D can reduce disease activity . It is icalcemia . There icalcemia . A similcalcemia . Our dat actions .2D3 and pathogenesis of a wide variety of infectious and autoimmune diseases is warranted for further investigation of vitamin D supplementation (readily available and affordable) as a potential agent to prevent viral infection and improve the survival outcome. Hence, we hypothesize that vitamin D supplementation will help COVID-19 patients maintain sufficient serum levels of vitamin D as guideline recommended [2D3 to inhibit CRS and disease progression. Serum calcium level should be monitored and if significantly elevated, it can be mitigated by hydration or a dose adjustment of the vitamin D supplement. There is no current Food and Drug Administration (FDA)-approved vitamin D treatment for diseases. However, section 101.72 of FDA  mentioned that adequate calcium and vitamin D may reduce the risk of osteoporosis. In the era of overwhelming COVID-19 disease burden, the risk and benefit of such trials (Additional file There is a plethora of evidence that vitamin D is required for normal immune function for fighting pathogens and preventing autoimmune diseases , 97–101 ommended , 103, alAdditional file 1: Table S1. Ongoing Clinical Trials as of July 2nd, 2020, of vitamin D in the Prevention and Treatment of COVID-19.


TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown.Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated.Histopathological, swimming behavior, and muscle endurance were monitored in TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, l-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by l-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by l-carnitine treatment.While TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.These results demonstrate that  TPM3) cause CM with nemaline myopathy, cap myopathy, and CFTD [TPM3-related CM were reported to exhibit lowered Ca2+-sensitivity and damaged acto-myosin cross-bridge cycling in slow fibers [Congenital myopathy (CM) is a group of genetic muscle disorders characterized clinically by generalized hypotonia and muscle weakness present at birth and pathologically by the presence of specific morphological features on muscle biopsy, with estimated prevalence about 1:25,000 [w fibers  and distw fibers . Althougw fibers . Currentw fibers .l-Carnitine is an amino acid that is widely present in human tissues and can be produced from essential amino acids lysine and methionine. It is responsible for sending fatty acids into the mitochondria to burn smoothly and produce energy. l-Carnitine significantly reduced statin-induced myopathy [l-Carnitine improved exercise performance in human patients with mitochondrial myopathy [l-Carnitine may prevent age-associated muscle protein degradation and regulate mitochondrial homeostasis [l-tyrosine supplementation was reported to improve bulbar function, activity levels, and exercise tolerance in some nemaline myopathy patients [l-tyrosine supplementation was not able to attenuate the skeletal muscle dysfunction in zebrafish and the dominant skeletal muscle α-actin nemaline myopathy in mouse models [myopathy  and skelmyopathy . l-Carnimyopathy . l-Carnieostasis . Althougpatients . Howevere models .TPM3 at nucleotide 452 from A to G, resulting in amino acid 151 changing from glutamic acid (E) to glycine (G). Previously, a TPM3 mutation changing nucleotide 452 from A to C resulting in amino acid 151 changing from glutamic acid (E) to alanine (A) was reported in a CM patient with cap myopathy [TPM3(E151G) and CM, we generated transgenic zebrafish overexpressing the human wild type (WT) and two mutant TPM3 genes in the muscle of zebrafish for functional analysis and potential drug screening. Here, we show that TPM3(E151G) transgenic zebrafish display much severe CM phenotypes than those from TPM3(E151A) and TPM3(WT) transgenic zebrafish. Remarkably, l-carnitine supplementation ameliorates the swimming speed and muscle endurance as well as rescues the downregulation genes involved in sodium, potassium, and calcium channels in TPM3(E151G) transgenic zebrafish by RNAseq. This study provides insights in the pathogenicity of the novel TPM3(E151G) mutation and a drug screening platform to uncover potential therapies for this rare disease.A seven-year-old boy patient who had been suffering from generalized hypotonia, muscle weakness with myopathic face, high-arched palate, funnel chest, hyporeflexia, poor feeding, and motor delay since early infancy. He presented severe motor delay having head control at 8 months, was able to sit alone at 12 months, walk alone at 20 months, and still cannot run until seven years of age. As his parents refused the invasive clinical examination such as electromyogram or muscle biopsy for him, thus Dr. Yuh-Jyh Jong diagnosed with his serum DNA by using target gene capture/deep sequencing approach . It reveAll zebrafish were maintained at the Zebrafish Core Facility in National Health Research Institute . Adult fish were maintained under an automated 14:10 h light/dark cycle and a constant temperature of 28 °C. All experiments involving zebrafish were approved by the Institution Animal Care and Use Committee (IACUC) of the NHRI (protocol No. NHRI-IACUC-103122-AE). The Taiwan Zebrafish Core Facility at NHRI or TZeNH is a government-funded core facility, and since 2015, it has been accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) .TPM3(WT);myl7:EGFP), Tg(MLC2:TPM3(E151A);myl7:EGFP), and Tg(MLC2:TPM3(E151G);myl7:EGFP) were generated in this study as described previously using Tol2 Gateway cloning Toolkit [AB wild-type was used in this study. Other transgenic fish lines, including Tg and pME-TPM3(E151G) were created by QuickChange II site-directed mutagenesis  from pME-TPM3(WT). All primers for mutagenesis are listed in Additional file TPM3(E151A):pA/CG2 was generated using Gateway® LR reaction  using p5E-MLC2, pME-TPM3(E151A), p3E-polyA, and pDestTol2CG2 vectors. The final expression construct pTol2-MLC2:TPM3(E151G):pA/CG2 was generated by Gateway® LR reaction  using p5E-MLC2, pME-TPM3(E151G), p3E-polyA, and pDestTol2CG2 vectors.pME-TPM3E51A and pTPM3 transgenic zebrafish were examined by qRT-PCR, which confirmed all three TPM3 transgenic zebrafish expressed human TPM3 gene.Three constructs were created by Tol2-Gateway cloning, and confirmed by real-time reverse transcription-PCR (qRT-PCR) and sequencing, and microinjected into AB wildtype zebrafish embryos. Expressions of transgenes were using by heart specific promoter myl7-driven green florescence protein (GFP) expression in 48 hours-post-fertilization (hpf) embryos and sequencing confirmed with genomic DNA extracted from adult fin. The mRNA level of 2, and 0.33 mM MgSO4). We maintained the embryos in an incubator at 28 °C.Embryos were collected once per week with the best results produced with a regular schedule. The night before embryo collection, adult male and female zebrafish were placed into a mating tank with a clapboard after the last feeding. Next morning, the clapboard was removed to start mating. Embryos were collected after 1 hour, and we removed the dead and unfertilized embryos. We transferred healthy embryos to a clean petri dish containing E3 medium . For DNA and RNA co-injection, we injected 2.3 nL containing 0.05% phenol red into an embryo at the one-cell stage. We ensured the embryos did not develop past the two-cell stage. After injection, embryos were placed in E3 medium and incubated at 28 °C.Two days after microinjection, we used a fluorescent microscope to screen the hearts of zebrafish embryo for green fluorescence. When the transgenic zebrafish were 3 months old, DNA was extracted by cutting the fins. After amplification of the target fragment by PCR, we entrusted the DNA Sequencing Core Lab at the National Health Research Institutes to sequence and confirm the target sequence.Before examination, the fish was anesthesia using 0.0125% buffered MS-222 solution . Total length and standard length were measured using Vernier calipers with 0.1 mm accuracy. Fish body weight was determined to an accuracy of 0.01 g. For morphological analysis, a digital CMOS X-ray detector , set to 45 kV and 120 mA, with 2.5 seconds of exposure, was used to view vertebrae of the fish .The zebrafish larvae swimming behavior data were tracked and acquired by using DanioVision equipment . Before the indicated times of development, zebrafish larvae were placed individually into a 48-well plate with 1200 µL E3 medium overnight. According to DanioVision manual, we used light-induced visual motor response analysis. The swimming behavior test for zebrafish larvae involved the following conditions: the first 30 min was required for their adaptation, then we used a light switch (20 min: 10 min in dark and 10 min in light) for 2 cycles . The watA three armed T-maze was used for our experiment. The stem of the maze (length 36 cm × width 11 cm × height 20 cm) included the start box (11 × 11 × 20 cm) and each arm of the maze (25 × 11 × 20 cm). Blue or red cellophane was attached to the end of the arm. The T-maze was filled with system water and the temperature was maintained at 28 ± 1 °C.For the zebrafish adult behavior test, we used T-maze for memory testing and velocity testing. The T-maze behavior test is divided into three phases: pre-training, training, and testing. In the pre-training phase, adult zebrafish were placed in a 3 L fish tank when they were 3 months old with a white sponge filled with food to induce the fish to find the white sponge twice each day (10:00 and 15:00) for one week. In the training phase, adult zebrafish were moved to the behavioral room and placed into an individual tank. All fish were acclimatized for 1 h before placement into the T-maze. During each trial, a zebrafish was placed in the start box for 1 min with the door closed. Then, the door was raised and closed after the fish had left. A stopwatch was used to measure the time required for the fish to find the white sponge filled with food. If the zebrafish did not leave the start box or find the target within 3 min, the experiment was terminated. In training phase, each fish was trained once each day for one week. In the testing phase, adult zebrafish were moved to the behavioral room and placed into an individual tank. All fishes acclimatized for 1 h before placement into the T-maze. During each trial, a zebrafish was placed in the start box for 1 min with the door closed. Then, the door was raised and closed after the fish had left. A stopwatch was used to measure the time required for the fish to find the white sponge. If the zebrafish did not leave the start box or find the target within 3 min, the experiment was terminated. In testing phase, each fish was tested once per day for one week.The zebrafish adult behavior test and larvae swimming behavior test were analyzed using EthoVision XT 13 . We evaluated the velocity, latency, and distance movement of individual zebrafish.(Ucrit) is defined as the maximum speed an adult fish can sustain over a period of time [Ucrit was calculated using [Critical swimming speed  of time . Criticaed using .\documenUi is the highest velocity the fish maintained for the whole 5 min (cm/s), Uii is the velocity increase, Ti is the time elapsed at the tired velocity, and Tii is the time between velocity changes (5 min).where Principal component analysis (PCA) was performed using XLSTAT 2014.1  to identify the main cause of induced responses and the relationship between these parameters. A biplot was graphed of both the measured parameters and observations.l-tyrosine, Taurine, l-carnitine, creatine, Terazosin and ATP, were used to test their effects. Four chemicals: l-tyrosine , Taurine , l-carnitine  and creatine  were dissolved in water at a concentration of 10 µM, 1 mM, 10 µM and 100 µM respectively. The concentration were determined according to a previous report on dosage analysis [TPM3 transgenic zebrafish model, TPM3 transgenic adult F2 zebrafish were in-crossed to produce the F3 embryos. Following a previous method [Six chemicals, including analysis . Terazosanalysis . ATP  were used to measure critical swimming speed for the restoration effect of swimming behavior of TPM3 transgenic zebrafish after l-carnitine treatment.The larva treated TPM3 transgenic zebrafish were immersed in 10 µM l-carnitine from 3 months of age from 20:00 to 08:00 for one month. The body weight and body length were measured after treatment. Swimming tunnel  were used to measure the critical swimming speed for the rescue effect of l-carnitine treatment.TPM3(WT) and TPM3 mutant embryos. Final concentrations of drugs were as follows: TPM3(WT) oligomycin 50 μM, TPM3 mutant oligomycin 25 μM, TPM3(WT) FCCP 20 μM, and TPM3 mutant FCCP 3 μM. Antimycin and rotenone were added to a final concentration of 2 μM for both.Zebrafish embryos develop until 48 hpf. Oxygen consumption rate (OCR) was measured from embryos using a V17 Islet capture plate in the Seahorse XFe24 Analyzer . E3 medium was used in wells, and two dechorionated embryos were placed in each well. All measurement cycles for the run were set to 3 min mix, 2 min wait, and 3 min measure. Oligomycin was then added followed by 3 measurement cycles. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was then added followed by another 3 measurement cycles. Finally, a mixture of antimycin and rotenone was added followed by 10 measurement cycles. Optimal doses of drugs were optimized separately for Before examination, the fish was anesthetized by 0.025% buffered MS-222 solution  following a previously reported method [The muscle tissue was grounded using Microtube Pellet Pestle Rods . TRI Reagent®  was mixed with sample and homogenized using a homogenizer. 1/5 of chloroform was added, mixed, and later the supernatant containing RNA was transferred to a new Eppendorf. After extracting total RNA from muscle tissue, the total RNA was applied in NucleoSpin® RNA kit  for the cleanup. RNA was isolated using a NucleoSpin® RNA column  per manufacturer‘s protocol. Finally, for all the RNA samples, we measured the concentration using a NanoDrop ND-1000 , which were then stored in a − 80 °C freezer. Complementary DNA (cDNA) was synthesized using iScript™ cDNA synthesis kit . cDNA was diluted 100-fold for qRT-PCR analysis using SYBR Green . Primers for qRT-PCR are listed in Additional file Next generation sequencing experiment was performed by NHRI Genomics Core Facility. Sequencing libraries 1 µg total RNA were generated using RNA HyperPrep kit with RiboErase , and sequenced on NovaSeq 6000 instrument . All RNA sample preparation procedures were carried out according to the Illumina's official protocol. Agilent's SureSelect Strand-Specific RNA Library Preparation Kit was used for library construction followed by AMPure XP beads  size selection. Illumina's bcl2fastq program v2.20 used for basecalling. De-multiplexed sequencing data was processed with Trimmomatic (version 0.36) to remove any adapter and poor-quality sequences with a sliding-window approach. Resulting read pairs were aligned with a HISAT2 aligner against zebrafish GRCz11 reference genomes (Ensembl version 99) to obtain reads count and fragments per kilobase per million (FPKM) data of genes and transcripts.p-value < 0.05, ANOVA method: Ebayes. The gene ontology analysis was performed using gene ontology analysis via WEB-based GEne SeT AnaLysis Toolkit , pathway analyses were performed by NetworkAnalyst , and activated pathways were selected and matched according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.The expression analysis settings were as follows: Gene-level fold change <  − 2 or > 2, gene-level bGestalt , http://t-test. All data are expressed as mean ± standard error of the mean (SEM). A P-value less than 0.05 was considered statistically significant. For all figures: NS, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Data were collected and analyzed using Excel  and GraphPad Prism 9 software . Individual tests were described in the main text. All the statistical analyses were conducted using two-tailed Student’s TPM3(WT), TPM3(E151A), and TPM3(E151G) under the promoter of myosin light chain 2 (MLC2) were generated as described previously [TPM3 transgenic zebrafish were examined by qRT-PCR and confirmed all three TPM3 transgenic zebrafish expressed human TPM3 gene  or TPM3(E151A) mutant, those fish displayed normal appearance, however, overexpressed E151G mutant TPM3, the fish showed abnormal appearance with no tail as early as F0  and TPM3(E151A) transgenic fish also exhibited a higher proportion of abnormalities , TPM3E51A, and TPM3(E151G) fish, 9.8% of TG1, 5.9% of TG2, and 86.7% of TG3 displayed a crooked body appearance. Even for the TPM3(E151A) fish, 4.2% of TG1 and 68% of TG2 had crooked body appearance , and TPM3(E151G) had a much lower survival rate than TPM3(E151A)  fish were all normal  F1 adult fish all had normal aligned vertebrae  F1 fish, we observed four out of seven fish (57.1%) and two out of seven fish (28.5%) respectively with malformations in the vertebrae  F1 adult fish exhibited skeleton abnormalities, with malformations in two out of eight (25%), four out of seven (57.1%), two out of four (50%), and two out of eight fish (25%) for TG1 to TG4, respectively  was more severe than in TPM3(E151A).As the mutant detector . The nonmal Fig. f. Four iTPM3 transgenic zebrafish in F1 adults, the non-transgenic AB(WT) control has compared to TPM3 transgenic fish for the muscle pathology. Both AB(WT) and TPM3(WT) fish all had normal muscle cell from all three staining. By HE stain, we observed that the muscle fibers of TPM3(WT) were arranged neatly and consistent, TPM3(E151A) fish shows smaller-sized fibers and TPM3(E151G) shows variation in fiber size  fish had rod-like structures in the muscle fibers resembled nemaline myopathy on Gomori trichrome stain  fish have both type 1 (darker) and type 2  fibers similar to non-transgenic control AB(WT)  transgenic fish also showed both types. On the other hand, TPM3(E151G) has increased smaller-sized and darkly stained fibers  displayed small and dark-stained type 1 muscle fibers and disproportionate arrangement, similar to CFTD , wildtype and mutant TPM3 transgenic fish. Both TPM3(WT) and TPM3(E151A) expressed higher level of type 1, and lesser type 2 myosin heavy chain compared to AB(WT), so there is fiber type conversion. On the other hand, TPM3(E151G) expressed lower levels of both type 1 and type 2 myosin heavy chain , but generally losing both muscle type.To further elucidate whether expression of WT or mutant ebrafish , we measTPM3 mutants may lead to swimming disability, the swimming velocity and latency of TPM3 transgenic fish were measured by using DanioVision for larva and T-maze for adult zebrafish. The AB(WT) fish control were compared to the wildtype or mutant TPM3 transgenic fish for the swimming velocity, and there is no difference between AB(WT) and wildtype TPM3 transgenic fish larva  at 7 dpf all exhibited significant lower velocity compared to AB(WT) fish  TG2 swimming velocity was also significantly lower compared with the TPM3(WT) fish at 7 dpf  larvae exhibited lower velocity compared to AB(WT)  adult fish after day 3 to day 5 exhibited significantly lower swimming velocity compared to TPM3(WT), we found no difference between TPM3(WT) and TPM3(E151A)  and TPM3(E151A) required significantly longer time to find the target compared with TPM3(WT) after day 3 to day 5  and TPM3(E151G) F1 adults still exhibited significantly lower swim velocity compared with TPM3(WT) F1 adult  TG1 line, with or without tails , all swam slower than TPM3(WT). In latency, all F1 adult fish of the TPM3(E151G) and TPM3(E151A) required significantly longer time to find the target compared with TPM3(WT) F1 adult  to measure muscle endurance in a swimming tunnel for a total of 13 groups of F1 adult zebrafish including AB(WT) zebrafish, four independent lines for TPM3(WT), TPM3(E151A), and TPM3(E151G) was determined. A non-transgenic AB(WT) fish control has compared to the wildtype TPM3 transgenic fish for the critical swimming speed, no differences in muscle endurance between AB(WT) and TPM3(WT) adult fish were detected  exhibited significantly debilitated muscle endurance compared with AB(WT)  showed significantly weaker muscle endurance compared with AB(WT)  TG3, and TPM3(E151G) TG1 and TG2 had significantly lower body weight compared to AB(WT)  TG3 and TG4, TPM3(E151A) TG1, and TPM3(E151G) TG1 and TG2, were significantly shorter, whereas TPM3(E151G) TG4 was significantly longer compared with AB(WT) , sex, body weight, and standard length, were analyzed by using principal component analysis. When two principle factors were selected, the total explanatory power was 80%. The first principal component (PC) and the second PC accounted for 52.3% and 27.7% of the variance, respectively. When the two PC factors represented all the factors, only 20% of the variation was ignored. We observed that AB(WT) and TPM3(WT) almost overlap, which we interpreted as the same group. TPM3(E151G) is separated from AB(WT) and TPM3(WT), which is obviously different from the AB(WT) and TPM3(WT) group  mutant zebrafish exhibited weaker muscle endurance and slower swimming behavior, resembling human patients. As no treatment exists for CM, we used the TPM3(E151G) zebrafish to screen for potential therapeutic drugs. TPM3 transgenic fish (F3) were treated with five chemicals  from 28 h-post-fertilization (hpf) to 7 day-post-fertilization (dpf), and we measured the swimming velocity by using DanioVision. Amazingly, we found that only l-carnitine significantly improved the swimming velocity of TPM3(E151G) larvae, but the other four chemicals did not  to improve the swimming velocity, since the swimming speeds of TPM3(E151A)  larvae, but not the swimming velocity of TPM3(E151A) and AB(WT) larvae.Based on the results above, we found that not Fig. a. In add1A) Fig. b and AB(1A) Fig. c fish wel-Carnitine can store and regulate the supply of ATP to enhance muscle endurance [TPM3(E151G) F3 larvae but had no beneficial effect to TPM3(WT) F3 larvae  were treated with l-carnitine from 28 hpf for 30 days and measured for the muscle endurance using a swimming tunnel at 30 dpf. As expected, the muscle endurance of TPM3(E151G) fish was restored by l-carnitine treatment  transgenic fish  and TPM3(E151G) + l-carnitine overlapped, indicating l-carnitine effectively restored the TPM3(E151G) muscle endurance to resemble the muscle endurance of TPM3(WT)  transgenic zebrafish starting from the age of three months failed to restore the muscle endurance  for all the fish in this study, and found there is no difference between them  Fig. i. Howevehem Fig. j. This dl-carnitine and ATP treatments both restored the muscle endurance of TPM3(E151G) transgenic fish, we wondered whether mitochondria respiration and ATP production were impaired in the TPM3(E151G) transgenic fish. The TPM3(WT), TPM3(E151A), TPM3(E151G), and l-carnitine-treated TPM3(E151G) transgenic fish were subjected to Seahorse XF Extracellular Flux Analyzer , and the mitochondria respiration data revealed TPM3(E151G) mutation caused significant decreases in basal respiration and ATP production  transgenic fish, and they were similar to TPM3(WT)  and type II (myhz2) myosin fiber from TPM3 transgenic fish (F3) treated with l-carnitine from 28 hpf to 7 dpf, and found that the expression level of myh7 and myhz2 in TPM3(E151A) and TPM3(E151G) was significantly reduced. After l-carnitine treatment, the expression of myh7 and myhz2 is significantly recovered to the same as TPM3 (WT)  increased significantly indicated the fish became healthier  analysis. Downregulation of genes in two TPM3 mutant fish compared to TPM3(WT) were identified as involved in anatomy structure development, cell differentiation, and cell morphogenesis from the expression profiling of the muscle specimens  were found to be much more lower than TPM3(E151A) in adults . Comparing the differentially expressed genes (DEGs) from embryos and adults and verified with qRT-PCR, we found genes involved in anatomy structure development were both downregulated in embryos and adults  exhibits actin binding activity and myosin heavy chain binding activity.To understand the molecular mechanism of TPM3(E151G) mutant and TPM3(E151G) with l-carnitine treatment, and found 488 overlapped genes which were downregulated more than two-fold in TPM3(E151G) and upregulated in TPM3(E151G) with l-carnitine treatment, but only 9 overlapped genes whose expressions were upregulated in TPM3(E151G) and downregulated in TPM3(E151G) with l-carnitine treatment  and rescued by l-carnitine treatment involved in ion transmembrane transporter, especially potassium, calcium and sodium ion channels were enriched  and TPM3(E151G) show quite different phenotypes, from gene expression analysis, TPM3(E151A) and TPM3(E151G) also display differential gene expression, we have carry out Ingenuity Pathway Analysis (IPA) to find out the enriched pathways, diseases and functions as well as upstream regulators. In the embryos, TPM3(E151A) and TPM3(E151G) share few pathways, for example they both downregulated the synaptogenesis signaling pathways, GNRH signaling and insulin secretion signaling  dysregulated many different canonical pathways, upregulated Tol-like receptor signaling pathways and T cell exhaustions signaling pathways are only observed in TPM3(E151A)  and TPM3(E151G) embryos share many similar upstream regulators, for example they both downregulated the IFNA2, IRF3, PI3K, Creb, STAT1, CEBPB, and l-carnitine treatment can reverse those changes  and TPM3(E151G) are also different  but not TPM3(E151A) transgenic fish. However, release of Ca2 + , transport of molecule and hydrolysis of phospholipid are only downregulated in TPM3(E151A). Therefore, the TPM3(E151A) might need the Ca2 + transporter activator to rescue its muscle strength.From pathological findings, muscle functional analyses and pharmacological responses, ing Fig. . However1A) Fig. a. In theges Fig. b. The bient Fig. c. E151G ent Fig. d, but itTPM3(E151A) and TPM3(E151G) adult share some pathways, but, TPM3(E151G) exhibited more severe downregulated canonical pathways than in TPM3(E151A)  and TPM3(E151G) adult share some biological functions, such as transport of molecule, transport of cation, transport of ion. However, the majority biological functions are differentially regulated in TPM3(E151G) and TPM3(E151A)  and TPM3(E151G)-adult involved in movement disorders  Fig. a. The TP1A) Fig. b. Both ters Fig. c, d.Fig.TPM3 in muscles to study the pathogenicity of TPM3(E151G)-related CM. TPM3(E151G) fish displayed lower survival rate, severe skeletal abnormalities, and presented fiber type disproportion and exhibited weaker muscle endurance. Furthermore, we identified l-carnitine specifically improved the swimming velocity of TPM3(E151G) larvae and increased basal respiration and ATP production in mitochondria. The RNA-seq analysis from the muscles of TPM3(E151G) larvae revealed the downregulated genes involved in pathways of sodium, potassium and calcium ion channels which their downregulation can be reverted by l-carnitine treatment. TPM3(E151G) is a new novel mutation that has not been reported, and has much severe phenotypes than TPM3(E151A) mutation previously reported. Our study provides the evidence and prove that human TPM3(E151G) mutation was pathogenicity in TPM3-related CM by using zebrafish.In this study, we have generated transgenic zebrafish expressing wild type, E151A and E151G TPM3 transgenic zebrafish. In ethology, the T-maze is a simple maze used for animal cognitive experiments. The T-maze is widely used to measure the spatial memory and learning tasks in laboratory animals, such as rat or zebrafish. Zebrafish has quickly become an important model for studying vertebrate neural development [TPM3(WT) zebrafish can be trained, indicating that TPM3(WT) zebrafish has demonstrated good learning ability and memory in the T-maze test. However, TPM3 mutant zebrafish differed considerably from TPM3(WT), implying TPM3 mutant zebrafish might experience memory deficits and are unable to learn. To eliminate the influence of learning ability, we used a swimming tunnel to test exact muscle endurance without learning ability. The swimming tunnel uses different water flowing velocities to keep the fish in a state of swimming to measure the muscle endurance. We proved TPM3 mutation fish did reduce muscle endurance.For the behavioral test, we started with the T-maze to measure the swimming velocity and memory test related to learning in our elopment . Due to elopment . As in oTPM3 mutant transgenic fish as a drug screening platform, we found l-carnitine specifically can restore the muscle endurance and swimming speed. Most of l-carnitine is intracellular and is stored in liver, skeletal muscle, heart, and kidney [l-Carnitine promotes long chain fatty acyl CoAs into the mitochondria for β-oxidation and nonoxidative glucose disposal [l-carnitine uptake inside cells. Carnitine acyltransferases are a family of enzymes that catalyze the reversible transfer of acyl groups between coenzyme A and l-carnitine and the conversion of acyl-CoA esters to acylcarnitine esters [l-carnitine at super physiological concentrations. l-Carnitine was used to reduce lactate levels and elevate ATP levels upon administration [ACTA-1 gene mutation and carnitine deficiency was the first described with evidence of a disorder of mitochondrial fatty acid oxidation [Using the d kidney . l-Carnidisposal , 33. Orge esters . Neuroprstration . A nemalxidation .l-carnitine can enhance long chain fatty acyl-CoA into the mitochondria for β-oxidation to generate ATP to rescue muscle weakness.The pathogenesis of CM is diverse. Based on recent emerging evidence, however, five key pathophysiological areas have been proposed, including defects in sarcolemmal and intracellular membrane remodeling and excitation–contraction coupling; mitochondrial distribution and function; myofibrillar force generation; atrophy; and autophagy . We hypoTPM3 transgenic zebrafish platform, we used larvae and adult fish for drug screening; some results from the larvae and adult fish platform seem unconcordant. We found that l-carnitine improve the swimming velocity of larval TPM3(E151G) zebrafish treated from 28 hpf for seven days  zebrafish was rescued to near normal levels in adult treated l-carnitine from 28 hpf for one month  zebrafish if treated at 3 months age  was unable to be rescued by l-carnitine if treated at 3 months of age. The myogenic regulatory factors, such as Myod, Myf5, and Myogenin, are expressed during the segmentation period [l-carnitine enhances long chain fatty acyl-CoAs into the mitochondria for β-oxidation to generate energy for skeletal muscle satellite (stem) cells to regenerate. The TPM3(E151G) larva zebrafish was rescued to near normal levels if treated l-carnitine from 28 hpf for seven days or one month. However, the stem cells are fewer with lower activity in adult of 3 month of age. Therefore, the muscle endurance cannot be improved in adult TPM3(E151G) zebrafish even if provided enough energy.In our ays Fig. a. The munth Fig. f, but l-n period , 38 at 1n period , and decscn1a, scn2, scn4ba and scn8ab were upregulated by l-carnitine treatment  mutation and also can be reverted by l-carnitine treatment  larvae exhibits dysfunction of ion channels, l-carnitine treatment could reverse the gene expression of mainly sodium, potassium, and calcium channels, indicating that those ion channel genes may play key roles in pathophysiology of TPM3-related CM.The sarcolemmal ion channels are crucial for muscle contraction involving excitation–contraction coupling machinery, sodium channelopathies are critical for myopathy, epilepsy disorders and complex encephalopathies . Interesent Fig. d. Calciuent Fig. d. Mutatient Fig. d. The mul-carnitine can improve the swimming velocity of larvae and the muscle endurance of TPM3(E151G) zebrafish if treated in the early stage for one month, treatment was not effective if started at three months of age. Clinically, human patients have often completed muscle development at the time of diagnosis, which is similar to adult fish. As with all CM, no FDA-approved treatment exists to date. Early implementation of NGS to enhance diagnostic capabilities of clinical suspected CM, especially overcome historic limitations of histopathologic and clinical overlap, must be developed so potential treatment can be administered as early as possible.Although we found TPM3 de novo mutation for a clinically diagnosed boy who suffers from CM. It is the first study using transgenic zebrafish to prove the pathogenicity of a TPM3(E151G) novel mutation of CM. Moreover, we demonstrate that l-carnitine ameliorates the muscle endurance and pathophysiology of CM with TPM3(E151G) de novo mutation. This study highlights a role of potassium and calcium ion channels in TPM3-related CM and provides a potential mechanism of action of TPM3 pathophysiology.Our transgenic fish serve as translational model of Additional file 1: Table S1. Primer information.Additional file 2: Figure S1. The swimming velocity of larvae F1 TPM3 transgenic zebrafish at 3–6 dpf.Additional file 3: Movie S1. Swimming behavior of TPM3(E151A) and TPM3(E151G) compared with TPM3(WT) transgenic fish.Additional file 4: Figure S2. L-carnitine treatment of F3 adult TPM3 transgenic zebrafish starting at 3 months of age.Additional file 5: Figure S3. L-carnitine treatment increased the body weight and length of TPM3(E151G) larva.Additional file 6: Figure S4. A heatmap showing the deep sequencing data for TPM3 transgenic zebrafish.Additional file 7: Figure S5. The qRT-PCR validation of genes involved in anatomy structure development.