Mammary tumors are common in cats, presenting an aggressive behavior with high tumor recurrence. Therefore, new and efficient therapeutic protocols are urgent. Monoclonal antibodies  are widely used in human breast cancer therapy, inhibiting the HER2 dimerization and leading to cell apoptosis. Furthermore, drug combinations, with tyrosine kinase inhibitors (TKi) are valuable in patients’ therapeutic protocols. In this study, two mAbs, and an ADC, as well as combined protocols between mAbs and mAbs plus lapatinib (TKi) were tested to address if the drugs could be used as new therapeutic options in feline mammary tumors. All the compounds and the combined treatments revealed valuable antiproliferative effects, and a conserved cell death mechanism, by apoptosis, in the feline cell lines, where the mutations found in the extracellular domain of the HER2 suggest no immunotherapy resistance.her2 ECD (subdomains II and IV) was analyzed in 40 clinical samples of FMC, in order to identify mutations, which can lead to antibody resistance or be used as prognostic biomarkers. Results obtained revealed a strong antiproliferative effect in all feline cell lines, and a synergistic response was observed when combined therapies were performed. Additionally, the mutations found were not described as inducing resistance to therapy in breast cancer patients. Altogether, our results suggested that anti-HER2 mAbs could become useful in the treatment of FMC, particularly, if combined with lapatinib, since drug-resistance seems to be rare.Feline mammary carcinoma (FMC) is a highly prevalent tumor, showing aggressive clinicopathological features, with HER2-positive being the most frequent subtype. While, in human breast cancer, the use of anti-HER2 monoclonal antibodies (mAbs) is common, acting by blocking the extracellular domain (ECD) of the HER2 protein and by inducing cell apoptosis, scarce information is available on use these immunoagents in FMC. Thus, the antiproliferative effects of two mAbs (trastuzumab and pertuzumab), of an antibody–drug conjugate compound (T-DM1) and of combined treatments with a tyrosine kinase inhibitor (lapatinib) were evaluated on three FMC cell lines . In parallel, the DNA sequence of the  Similarly to human breast cancer , the felher2 [The epidermal growth factor receptor 2 (HER2) is a common target in patients with HER2-overexpression tumors . HER2 isher2 ,13, the Nowadays, the use of mAbs are widely common in HER2-positive breast cancer patients, decreasing its downstream pathways activation, such as AKT and mTOR, responsible for the antiapoptotic mechanisms , cell cyIn human breast cancer, 50% of the HER2-positive patients show immunotherapy resistance , the comher2 ECD (subdomains II and IV); (3) describe the cell death mechanism induced by the mAbs and T-DM1, in the carcinoma cell lines; (4) evaluate the synergistic antiproliferative effects by the combination of mAbs (trastuzumab plus pertuzumab) and assess the increase in the cytotoxic response by the use of mAbs with the TKi (lapatinib); (5) identify genomic mutations in the feline her2 gene ECD (subdomains II and IV) in FMC clinical samples, in order to recognize possible therapy resistant animals, or prognostic factors.An early diagnosis and individual therapy is crucial for the improvement of survival time  and prevv/v) CO2 , in Dulbecco’s Modified Eagle Medium , for CAT-MT and SKBR-3, whereas FMCm and FMCp cell lines were maintained in Roswell Park Memorial Institute 1640 Medium , both supplemented with heat-inactivated 20% (v/v) fetal bovine serum . Periodically, all cell lines were inspected to control their morphology and proliferation rate, being tested for Mycoplasma .In this study, three feline cell lines  ,51 and a3 cells/well for CAT-MT and FMCp, 15 × 103 cells/well for FMCm and 10 × 103 cells/wells for SKBR-3), and then exposed to increasing concentrations of each antibody , using the Cell Proliferation Reagent WST-1  and following the manufacturer’s instructions. Briefly, cell lines were seeded in 96-well plates to obtain a confluency of 90%, after 24 h  and pertuzumab plus lapatinib, a similar methodology was used, testing concentrations that covers different cytotoxic responses .Expression analysis of HER2 was performed, as reported by us ,52, in tHER2 immunoreactivity was scored as recommended by the American Society of Clinical Oncology’s (ASCO) , and as 4 cells/well for CAT-MT and FMCp, 10 × 104 cells/well for FMCm, and 7 × 104 cells/well for SKBR-3 cell lines), and then were exposed to mAbs and to the ADC for 72 h, at a concentration close to the EC50 value. Control wells were left unexposed, with the PBS used as a vehicle, in three independent experiments. The percentage of apoptotic cells after drug exposure was calculated by using the APC Annexin V Apoptosis Detection Kit with Propidium Iodide  and following the manufacturer’s instructions. Briefly, supernatants were harvested, and the remaining attached cells were trypsinized  and added to the correspondent supernatants. Then, samples were centrifuged for 5 min at 500 g at RT, washed with PBS and resuspended in 500 µL of Annexin V Binding Buffer (BioLegend), with a maximum concentration of 1 × 107 cells/mL. Afterwards, 100 µL of each sample were passed to a new tube, and 5 µL of APC Annexin V (BioLegend) and 10 µL of PI (BioLegend) was added. Then, samples were vortexed and incubated for 15 min at RT, protected from the light. Finally, 400 µL of Annexin V Binding Buffer (BioLegend) were added to the samples, before acquisition in a BD LSR Fortessa X-20 , at Champalimaud Foundation, Lisbon, Portugal. Data were analyzed using FlowJo software , considering double negative staining cells, as living cells, annexin positive cells in early apoptosis and double positive cells (both for annexin and PI) in late apoptosis phase.The flow cytometry assay was performed as already reported by us . For thiher2 gene amplification was not observed [The 40 tumor tissue samples used in this study were collected from cats that underwent mastectomy, at the Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon, with all the procedures consented by the owners, showing no interference in the animals’ well-being. The clinical history of the cats was recorded , includiher2 ECD (NC_018736.3), previously identified by comparison with the genomic human her2 sequence (NC_000017.11), different primers were designed (Genomic DNA extraction was performed in 5 mg of 44 frozen tissue samples (4 breed control and 40 tumor tissues) and in the three feline cell lines , collected after grown till confluency in a T25 culture flask, as previously described ,55, usindesigned  in the Pher2 (NC_018736.3), using the ClustalW tool (BioEdit Alignment Editor software) [her2 sequence (NC_000017.11) and searched in National Cancer Institute, International Cancer Genome Consortium and Catalogue of Somatic Mutations in Cancer (COSMIC) databases for putatively induced resistance to mAbs, and the ADC tested in this study.The feline sequenced samples were aligned with the identified feline oftware) , while toftware) . Proteinp-values below 0.05 being considered statistically significant, and a 95% confidence interval . For the cytotoxicity assays, outliers with more than two standard deviations were removed from the analysis and the EC50 value for each drug, in all the cell lines was calculated using Log (agonist) vs. Response (Variable slope) function. In the drug conjugation assays, the two-way ANOVA test was performed. Regarding the mutations found in tissue samples, associations between groups were assessed using the nonparametric Fisher’s exact test.Statistical analysis was carried out using the GraphPad Prism software , with two-tailed 50 = 3047.89 µg/mL ± 1.43; 50 = 528.45 µg/mL ± 1.14) and FMCp  cell lines. In parallel, pertuzumab showed similar results on CAT-MT and FMCm cell lines . Regarding the ADC T-DM1, the highest antiproliferative effect, at the range of concentrations tested, was reported on CAT-MT and FMCp cells  = 52.84 µg/mL ± 1.50).Trastuzumab, pertuzumab and T-DM1 exhibited a dose-dependent antiproliferative effect on feline mammary carcinoma cell lines. Indeed, trastuzumab exerted a more potent antiproliferative effect on CAT-MT cell line  or ADC (T-DM1) allowed to confirm that the observed antiproliferative effects were related to apoptosis induction . Deepenip < 0.001; p < 0.001; p = 0.018; p < 0.001; The combination trastuzumab plus pertuzumab showed synergistic antiproliferative effects in all feline cell lines. In CAT-MT cells the exposure to trastuzumab at 125 µg/mL plus pertuzumab at 2000 µg/mL increased the cytotoxicity from 13.6% to 40.0%  combined with lapatinib showed an improvement of the antiproliferative effects induced by mAbs. Accordingly, the combination of trastuzumab plus lapatinib, in the CAT-MT cell line, revealed a valuable synergistic response, for all the concentrations tested, with the highest increase of 78.4% of cytotoxicity, by the combination of trastuzumab at 125 µg/mL plus lapatinib at 7.26 µg/mL  presented at least one mutation  of the mutations were located in exon 3 (11/40 tumor samples), no mutations were found in the exon 4. Additionally, exon 3 presented 11 synonymous and 10 missense mutations, the remaining four being silent mutations. Regarding the subdomain IV, which is the her2 region recognized by trastuzumab, few mutations were detected, namely one heterozygous missense mutation at exon 10 , in two tumor samples; five mutations in exon 11 , one being a heterozygous synonymous mutation and four missense mutations, occurring in three tumor samples; two mutations at exon 12 , occurring in two tumor samples, one of them synonymous; three mutations at exon 13 , in two tumor samples, one synonymous, one missense and one frameshift mutation (c.14406 Ins. C).Previous studies on somatic mutations in the mutation , with alher2 ECD sequence, and searching in the referred databases, the mutations found were never reported as inducing resistance to anti-HER2 immunotherapy in breast cancer patients.Additionally, any correlation was reported between animals’ clinicopathological features and the described mutations. Moreover, after comparing the feline mutations with the genomic human The FMC is a common disease, showing similar features with human breast cancer ,2. Prese50 = 8756 nM ± 83), and a maximum of 79.4% of cytotoxicity in the FMCp cell line, by exposure to neratinib [Currently, only one felinized mAb is known to be in use as a therapeutic option for cats ,60, witheratinib , no clineratinib . Thus, teratinib , inducineratinib , and theeratinib , a crucieratinib ,68, beineratinib  decreaseeratinib . This reeratinib . Furthereratinib ,72. Perteratinib , with a eratinib  and ERK eratinib , which ieratinib . Finallyeratinib , which ieratinib ,40,77, beratinib . In facteratinib , suggesteratinib ,78,80, oeratinib ,82. Unfoeratinib ,83.The tested mAbs and the ADC cell death mechanism occur by apoptosis, suggesting a Bcl-2 dependent mechanism , conservAs for human breast cancer, combined protocols reveal to be a valuable tool ,86,87 inher2 ECD could explain the resistance patterns obtained with the use of anti-HER2 molecules, although mutations in the her2 gene are described as more common in the TK domain [her2 ECD mutations occurred in exon 3 (69.4%), subdomain II, which encode for the region recognized by pertuzumab, suggesting that if resistance occurs in cat, it might be more common in therapeutic protocols using this mAb. Additionally, 38.9% (14/36 mutations) of the mutations were synonymous and 61.1% (22/36 mutations) corresponds to missense mutations, conditioning 59.1% (13/22 mutations) of them a change in the polarity of the codified codon, which could modify the 3D arrangement of the protein [her2 ECD is S310F/Y, related to a resistant pattern to trastuzumab [her2 ECD were described as inducing resistance to therapy, according to National Cancer Institute, International Cancer Genome Consortium and COSMIC databases, and none of them were related to the cat’s clinicopathological features.Beyond the acquired therapy resistance, somatic mutations in the K domain , and in K domain . In the  protein . In breastuzumab  and pertstuzumab ,102 therThis study tested two mAbs (trastuzumab and pertuzumab) and an ADC compound (T-DM1) revealing valuable in vitro antiproliferative effects in feline primary (CAT-MT and FCMp) and metastatic (FMCm) cell lines, as well as a conserved cell death mechanism, by apoptosis, comparing to the human breast cancer cell line, SKBR-3. Furthermore, promising synergistic antiproliferative effects were obtained by combining both mAbs (trastuzumab plus pertuzumab), and mAbs with the TKi, lapatinib, already referred to as a valuable therapeutic protocol for humans. Additionally, mutations found in the clinical samples were never reported as inducing resistance to therapy, in breast cancer patients. Concluding, the use of anti-HER2 mAbs and combined protocols are proposed as a new targeted therapy for cats with different mammary carcinoma subtypes, suggesting that mAbs-resistant FMCs are rare. Still, more studies are needed to produce felinized anti-HER2 monoclonal antibodies, preventing cat immune reactions. Moreover, the similarities between the FMC and human breast cancer were demonstrated, reinforcing the utility of the cat as a breast cancer model.


GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient’s cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient’s motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient’s motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation. Mutations in the ganglioside-induced differentiation associated protein 1 ( PMP22 gene, responsible of the so-called CMT1A, remains the main genetic cause of this pathology.Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of sensory-motor disorders belonging to the larger class of genetic neuropathies. With an estimated prevalence of 1:2500, it is considered as the most frequent inherited pathology of the peripheral nervous system. It indifferently affects both sexes, of any geographical origin and age, and it is clinically defined by muscular weakness and atrophy, foot deformities such as pes cavus, and sometimes sensory loss and balance issues [GDAP1 (ganglioside-induced differentiation protein 1) gene have been reported for the first time in 2001 [GDAP1, located on chromosome 8 of the human genome, encodes a 358 aa protein, expressed on the outer mitochondrial membrane of neurons and, at lower levels, of myelinating Schwann cells [GDAP1 CMT-inducing mutations have been largely described in their clinical aspects and associated phenotypes, fewer studies have deeply investigated the molecular mechanisms altered in motor neurons and responsible for the neural degeneration.Mutations in  in 2001 ,4, and a in 2001 ,6. GDAP1nn cells ,8,9. Eve2+ homeostasis, as shown in SH-SY5Y and transfected HeLa cells [Three main model organisms have been developed to elucidate GDAP1 role in cellular physiology, through its up- or down- regulation, in Drosophila melanogaster ,11, its La cells ,24. MoreLa cells ,26. HoweNELF, MFN2 [HSPB1 [PMP22 duplication [GDAP1 are reported in three studies [Human induced-pluripotent stem cells (hiPSCs), created for the first time in 2006 , have beLF, MFN2 ,30, HSPB2 [HSPB1 , but alslication . HiPSCs  studies ,29,33, a studies .GDAP1, underlining the role of mitochondria and oxidative stress in this human GDAP1-defective CMT disease.Here, we report the first functional study on human hiPSCs-derived motor neurons from a CMT2H patient, carrying the homozygous c.581C>G (p.Ser194*) mutation in GDAP1 gene genetic analyses detected the c.581C>G (p.Ser194*) mutation, homozygous in the patient and his affected brother, and heterozygous in the other family members. No other mutation in CMT- and peripheral neuropathies-associated genes was detected by targeted Next Generation Sequencing (NGS) , as well as the informed consent of all participants. The study was conducted in accordance with the Declaration of Helsinki. The family of the CMT-patient presented two cases . The proGS) , with 10% Fetal Bovine Serum (FBS) . After two weeks, once fibroblasts (FBs) have migrated from the skin fragment and grew in the culture dish, they were isolated using trypsin. In the first three days, fibroblasts were cultivated combining the Chang Medium® D (25%) with the RPMI 1640 medium (75%) , supplemented with 10% FBS. Then, Chang medium D was completely replaced by RPMI medium and FBS.Skin biopsies were obtained from patient, Ctrl-1, and Ctrl-2, and incubated in CHANG Medium2 density. The second day, 600,000 fibroblasts, from patient and controls, were reprogramed with three plasmids , through the Nucleofector II device . Reprogramed cells were cultured in DMEM+GlutaMAX medium , supplemented with 10% FBS, 1% MEM non-essential amino acids (Thermo Fisher SCIENTIFIC), 1% sodium pyruvate (Thermo Fisher SCIENTIFIC), and, at day 1, with 0.1% gentamycin (Thermo Fisher SCIENTIFIC). Culture medium was replaced every day. After 14–21 days, colonies were selected, using a needle, and transferred in new gelatin/MEF coated dishes. HiPSCs colonies grew in a KO-DMEM medium , with 20% KnockOut Serum Replacement , 1% MEM non-essential amino acids, 1% Glutamine , 0.1% β-mercaptoethanol , and 0.1% gentamycin. It was replaced every day, extemporaneously supplemented with 20 ng/mL Fibroblast Growth Factor (FGF2) . After hiPSCs amplification, all quality controls were performed  protocol. First day, CF-1 Mouse Embryonic Fibroblasts (MEF), Mitomycin-C treated , were seeded on gelatin coating , at 25,000 cells/cmerformed .2, using the same coating and the same culture medium.Differentiation protocol was applied as previously described by Faye et al. . After n®, Venlo, The Netherlands). After verifying RNA integrity with the Bioanalyzer 2100 system (Agilent Technologies), it was converted in cDNA with the QuantiTect® Reverse Transcription kit (QIAGEN®). For the quantitative PCR , primers were designed between the fifth and the sixth exon of GDAP1, and between the fifth and the sixth exon of TBP (TATA-Box Binding Protein), chosen as reference gene. Reactions were prepared with the Rotor-Gene SYBR-Green PCR Kit (400) (©QIAGEN) and performed on the Corbett Rotor-Gene 6000 Machine (© QIAGEN). All qPCR reactions were performed four times.Total RNA was extracted from fibroblasts, hiPSCs, NPs and MNs of Ctrl-1 and patient, using the miRNeasy Mini kit   for 10 min and permeabilized with 0.1% Triton X-100  for one hour. They were incubated overnight at 4 °C with the primary antibody, prepared in 3% BSA, and then the next day with the secondary antibody for one hour at room temperature. PFA, Triton X-100 and BSA were diluted in Dulbecco’s phosphate-buffered saline 1X (DPBS) . Nuclei were stained with 2 µg/mL 4′,6′-diamidino-2-phénylindole dihydrochloride (DAPI) . Images captures were performed using a fluorescence microscope  or a confocal microscope , while their processing and analysis were made with NIS Element BR software, Zen Black and Zen Bleu software, and Image J software. All antibodies’ dilutions and references are reported in ® Elite ABC kit  was used for the avidin-biotin/peroxidase detection. The DAB+ chromogen, i.e., the peroxidase substrate solution, was added to induce the formation of the brown precipitate, visualized with a light microscope.The chromogenic 3,3′-Diaminobenzidine (DAB) staining was used as complementary ICC method to immunofluorescence, by reason of its high sensitivity. For the DAB staining, MNs were fixed, permeabilized, and incubated with the GDAP1 primary antibody overnight. Next day, the VECTASTAINAll manipulations for the electron microscopy were performed in Neurology and Anatomic Pathology departments at University Hospital of Limoges. Cells were fixed in 2.5% glutaraldehyde, then incubated 30 min, at RT, in 2% OsO4 . After washing them with distilled water, they were dehydrated 10 min in a series of ethanol dilutions  and three times in 100% ethanol. At the end, they were embedded overnight in Epon 812. Thin blocks were selected and stained with uranyl acetate and lead citrate and examined using a Jeol 1011 electron microscope.® Luminescent Cell Viability Assay kit , and the luminescent signal was recorded with the Fluoroskan Ascent®FL  following manufacturer instructions. DAPI staining was used to normalize luminescence’s values to the number of cells. Reactions were performed in triplicate, and experiments were repeated three times.ATP was dosed using CellTiter-GloSuccinate dehydrogenase activity was measured using the Cell Proliferation Kit I , following manufacturer conditions. Absorbance of formazan crystals, at 595 nm, was recorded with the Multiskan™ FC Microplate Photometer (Thermo Fisher SCIENTIFIC), and normalized to the number of cells, measured with the DAPI staining. Reactions were performed in triplicate, and experiments were repeated three times.2O2 solution, prepared in culture medium. After the treatment, 5 µM MitoSOX™ Red mitochondrial superoxide marker  was added to the whole of the plate and incubated for 10 min at 37 °C. Fluorescent signal was detected using the Leica DM IRB microscope and normalized to the number of cells, measured with the DAPI staining. Reactions were performed in triplicate, and experiments were repeated three times.Fibroblasts and MNs were analyzed in basal conditions as well as in stressed conditions. Stressed wells were treated two hours with 1 mM Hp < 0.05 was considered significant.All statistical analyses were performed using the GraphPad Prism 5 software . Data were expressed as mean ± SEM (Standard Error of the Mean). They were compared using the nonparametric Mann–Whitney U test; Fibroblasts of Ctrl-1, Ctrl-2, and the CMT2H patient were reprogramed in hiPSCs. After validating hiPSCs of the three subjects for all quality controls , our difGDAP1 mRNA, from fibroblasts to hiPSCs, neural progenitors, and motor neurons, of patient and Ctrl-1 subject. In Ctrl-1, we showed that GDAP1 is differently expressed in the four cell types , and slightly more expressed in hiPSCs . In contrast, GDAP1 mRNA levels significantly increased in neural cell types, associated with the progression of neural differentiation. In particular, GDAP1 expression was around 33-fold higher in NPs , and 56-fold higher in MNs , compared to fibroblasts’ levels. In patient’s cells, the same expression trend was observed in the four cell types, thus GDAP1 mRNA was nearly absent in patient’s fibroblasts (0.01 ± 0.003), and significantly higher in hiPSCs , NPs , and MNs  .In order to investigate GDAP1 functions and GDAP1-associated mechanisms, we evaluated the expression of To complete the expression study, we evaluated GDAP1 protein expression on MNs, the cellular type known to express GDAP1, in comparison with fibroblasts. The experiment was performed for the two control subjects and the CMT patient .For the immunofluorescence analysis, MNs were stained with GDAP1 antibody (red), and Tuj-1 antibody (green), specific of neural cells. As shown in In fibroblasts, GDAP1 staining (red) was associated to Prolyl 4-hydroxylase subunit-β antibody (P4HB) (green). As expected, according to the RNA measurement, a weak GDAP1 signal was observed, both in controls’ and patient’s fibroblasts B.These results were supported by the DAB staining showing the higher expression of GDAP1 protein in MNs of Ctrl-1 compared to patient .p < 0.01). At d4, proliferating cells were slightly lower than 60% in Ctr-1 (57.7 ± 1.37%) and Ctrl-2 (59.38 ± 3.69%), and considerably reduced to 40% in patient’s cells   B.p < 0.001) or Ctrl-2   .HiPSCs-derived motor neurons of controls and patient were analyzed by electron microscopy. Surprisingly, in the cytoplasm of multiple patient’s MNs, we observed the emergence of several round structures, of various sizes, suspected to be lipid droplets (LDs) F. These Given the mitochondrial localization of GDAP1 protein, we investigated mitochondrial morphology and structure. Looking at MNs’ ultrastructure, any difference in mitochondrial size and shape was remarked between controls and patient. Moreover, MNs of both subjects presented elongated and fragmented mitochondria. However, focusing on mitochondrial cristae, we observed that their organization was altered in mitochondria of patient’s MNs. In particular, cristae’s regular distribution and thickness were perturbed, preventing to discriminate their structure in the internal mitochondrial compartment. Swollen cristae were also observed F. This dp < 0.05)  complexes and the ATP production. Given the limited availability of hiPSCs-derived MNs, we performed the MTT test to evaluate the activity of the succinate dehydrogenase (complex II). In both fibroblasts and motor neurons, succinate dehydrogenase activity seemed to be slightly increased in patient’s cells, compared to Ctrl-1 and Ctrl-2, and it reached significant difference in fibroblasts  . In cont < 0.05) .p < 0.05). This significant difference was even observed in fibroblasts . The same trend emerged in the comparison with Ctrl-2 cells, but significant difference was not reached.Mitochondria are also the main producers of reactive oxygen species (ROS), such as superoxide anion, inducing oxidative stress. A perturbation of mitochondrial cristae could promote redox imbalance. As expected, and shown in GDAP1 gene have already been reported to be responsible for demyelinating, axonal, and intermediate forms of Charcot-Marie-Tooth disease, and associated to heterogeneous phenotypic manifestations [GDAP1 role in cellular functions and processes has not been clearly elucidated, although several relevant studies have been performed [GDAP1 mutations surely represent the most accessible and easier tool to mimic GDAP1-induced pathophysiology. Murine and human GDAP1 proteins share 94% of amino acid homology supporting the relevance of this rodent model in expression and localization studies [GDAP1-ortholog gene (CG4623) [GDAP1 [GDAP1  [GDAP1, notably neurons and Schwann cells. These cell types cannot be obtained from humans, and used in vitro as cellular models. Given the inability to culture human neural cells, the only model available in these conditions was represented by human fibroblasts [GDAP1 [GDAP1, to investigate GDAP1 functions and GDAP1-associated mechanisms in CMT disease.More than 80 mutations in stations . Howevererformed ,16,22,38 studies ,9, struc studies , functio studies ,13. Furt(CG4623) ,11, or y) [GDAP1 ,40. The ) [GDAP1 ,9,15, or5, HT22) ,8,22,24,5, HT22) ,21. Inderoblasts ,18,22, wGDAP1 expression. In animal models, such as mice and rats, GDAP1 has been shown to be largely expressed in neurons. In particular, the higher expression was detected in cerebellum, cerebral cortex, hippocampus, olfactory bulb, spinal nerve, but also in sciatic nerve, and motor and sensory neurons [GDAP1 expression in Schwann cells was controversial, whereas, in non-neural tissues, it was poorly explored [GDAP1 mRNA expression in four human cell types of the same control subject (Ctrl-1): fibroblasts, hiPSCs, NPs, and MNs. Our study revealed, on Ctrl-1 cells, that GDAP1 is weakly expressed in fibroblasts and hiPSCs, while its expression was significantly higher in NPs, and, above all, in MNs. It is interesting to note that GDAP1 mRNA in fibroblasts represented only 3% of NPs-GDAP1 mRNA, and 1.8% of MNs-GDAP1 mRNA. This is in agreement with Noack et al. work, who showed that control human fibroblasts expressed only 2.6% of GDAP1 mRNA compared to embryonic stem cells-derived motor neurons [GDAP1 mRNA was only 10–20% of mRNA estimated in each cell type of Ctrl-1, up to 6- and 8-fold smaller than those assessed in Ctrl-1 NPs and Ctrl-1 MNs. Our data seem to suggest that GDAP1 mRNA is degraded in patient’s cells. As mutated GDAP1 mRNA contains a premature termination codon (PTC), the nonsense-mediated mRNA decay (NMD) system could be activated and induce its degradation, preventing the synthesis of a truncated, and maybe non-functional, protein [GDAP1 neural expression, we chose MNs and NPs, as cellular models to investigate its functions, and evaluate its role in Charcot-Marie-Tooth disease development.We first questioned about  neurons ,8,9. GDAexplored ,8,9. Her neurons . In cont protein . In any GDAP1 expression detected in fibroblasts does not exclude a GDAP1 role in this cell type, and the possibility of conducting functional studies on them [However, the weak  on them ,17,18. IGDAP1-associated pathophysiology. First, we investigated mitochondrial morphology. Electron microscopy revealed, only in patient’s MNs, a general disorganization of mitochondrial cristae, which could affect the inner mitochondrial space and subsequent metabolism. The disruption of cristae structure has already been associated to other pathological conditions induced by mutations or lack of proteins involved in mitochondrial dynamics, such as optic atrophy 1 (OPA1) protein [GDAP1 [GDAP1 mutations [On the other hand, morphological and oxidative stress analyses, allowed highlighting, exclusively in MNs, two main mechanisms which could play a key role in CMT disease progression: the deregulation of mitochondria morphology and dynamics, and the redox imbalance. These aspects support the relevance of our MNs cellular model in the study of  protein , or mito protein . Cristaen [GDAP1 , but alsutations .GDAP1 seems to be expressed, even if at a low level. Moreover, we have demonstrated that patient’s neural cells present a lower proliferation rate, compared to controls. Both these aspects could potentially be related to GDAP1 involvement in mitochondrial dynamics. Indeed, it is now well known that the proper preservation of mitochondrial dynamics is a fundamental condition for cell cycle progression, and fragmentation of mitochondrial network is required during the mitosis phase [GDAP1 knockout, altering the fission machinery, impairs OSKM (Oct4/Klf4/Sox2/cMyc) reprogramming, and cell cycle progression, in murine iPSCs [Interestingly, some additional findings of our cell culture need to be pointed out. We observed in patient’s hiPSCs carrying the p.Ser194* mutation a higher spontaneous differentiation and a reduced maintenance of stemness compared to controls’ hiPSCs, where is phase . Thus, tne iPSCs . Based oGDAP1 knockout mice [GDAP1 p.Ser194* mutation. In fact, GDAP1 has been suggested to have an antioxidant role in cellular homeostasis [GDAP1 is weakly expressed and LDs lacking. The overproduction of superoxide anion, in GDAP1-mutated fibroblasts, has also been reported in a recent work [GDAP1-mutated fibroblasts presented also a reduced expression of Sirtuin 1 (SIRT1) enzyme, which activates the PPARgamma coactivator-1alpha (PGC1 α) [In the cytoplasm of patient’s MNs, round electron-dense structures were observed, suspected to be lipid droplets. LDs could be considered as an accumulation of energetic substrate, such as triglycerides, linked to a defect of mitochondrial beta oxidation, or a hallmark of cellular stress, previously observed in nutrient imbalance, inflammation and oxidative stress . Moreoveout mice , in accoout mice . This pheostasis ,22. Consent work . Moreove(PGC1 α) . PGC1 α (PGC1 α) ,52,53,54GDAP1 impairment in Charcot-Marie-Tooth pathophysiology through mitochondrial dysfunction and oxidative stress development was underlined in an original human model of motor neuron from patient’s fibroblasts, carrying the homozygous codon-stop c.581C>G (p.Ser194*) mutation. The results underlined that GDAP1 is mostly expressed in neural cell types such as MNs and PNs, but also, at lower levels, in fibroblasts and hiPS cells. In patient’s cells, 80–90% of GDAP1 mRNA would be degraded by the NMD system, leading to the considerable reduction of GDAP1 protein. Taken together, these results demonstrated that hiPS cells can be a powerful tool to recreate any suitable cellular model from patients carrying mutations and are essential for understanding the pathophysiological role of the altered protein, but also necessary to develop new therapeutic strategies.In conclusion, the role of


The inositol 1,4,5- triphosphate receptor (IP3R) and ryanodine receptor (RyR) are large homotetrameric channels associated with the ER and serve as two major actors in ER-derived Ca2+ supply. Most of the knowledge on these receptors derives from mammalian systems that possess three genes for each receptor. These studies have inspired work on synonymous receptors in insects, which encode a single IP3R and RyR. In the current review, we focus on a fundamental, common question: “why do insect cells possess two Ca2+ channel receptors in the ER?”. Through a comparative approach, this review covers the discovery of RyRs and IP3Rs, examines their structures/functions, the pathways that they interact with, and their potential as target sites in pest control. Although insects RyRs and IP3Rs share structural similarities, they are phylogenetically distinct, have their own structural organization, regulatory mechanisms, and expression patterns, which explains their functional distinction. Nevertheless, both have great potential as target sites in pest control, with RyRs currently being targeted by commercial insecticide, the diamides.Calcium (Ca There is evidence indicating the N-terminal cytoplasmic domain modulates the gating of the channel pore located in the C-terminus similar to that in mammalian RyRs . Ne. Ne3Rs a species ,124. Thi3Rs , which pumps Ca2+ from the cytosol into the ER lumen. There are other players involved in SOCE, for example, the stromal interaction molecule (STIM)-Orai1 complex. STIM is normally located in the ER transmembrane and senses luminal Ca2+ depletion, which leads to its translocation to junctions between the ER and plasma membrane where it couples with the plasma membrane Ca2+ channel protein Orai1 [2+ release-activated Ca2+ (CRAC) channels in the plasma membrane, allowing Ca2+ influx from the extracellular pools to the cytosol and then from the cytosol to the ER through SERCA [3R upon depletion of ER Ca2+ resulting in enhanced SOCE activity [2+ to certain levels inactivates CRAC channels thereby terminating Ca2+ influx into the cell, a process known as Ca2+-dependent inactivation (CDI) [2+-binding protein, calmodulin, is involved in CDI by binding to STIM, leading to disruption of the STIM-Orai1 complex [3R is initiated by various external  or internal  signals that are adjusted based on the biology of insects and associated physiological processes. Activation of the channels might be specific to an organ or cell requiring either the RyR or the IP3R.Although RyRs and IPifferent . Regardlchondria ,137,138,chondria ,140,141,chondria . The othin Orai1 . This cogh SERCA . Notablyactivity ,147,148;on (CDI) . It is n complex . The act3Rs are expressed in most cells, in particular in the ER of neurons [3R signaling pathway is integrated with several other signaling pathways, such as the insulin/target of rapamycin (TOR) pathway [2+ activate IP3R, while high concentrations (above 300 nM) inhibit channel activity [2), into secondary messengers diacylglycerol (DAG) and IP3. IP3 binds to IP3-binding sites in the N-terminus of the tetrameric IP3R to initiate conformational changes that are transmitted down to the transmembrane region leading to opening of the Ca2+-permeable pore ~7 nm away from the IBC to release the Ca2+ from the ER [3 binding are conserved in IP3Rs, but not in RyRs [3Rs revealed that IP3 binding alone is not sufficient to activate IP3Rs [3 binding primes IP3Rs to bind Ca2+ and Ca2+ binding triggers channel opening [3Rs might also require binding of both IP3 and Ca2+ to open the channel; however, this has not been demonstrated. It is also noteworthy that IP3 must bind to each of the four subunits of IP3R; the 4- and 5-phosphates of IP3 moiety are essential for binding, while the 1-phosphate enhances affinity [3R propagates regenerative Ca2+ signals by Ca2+-induced Ca2+ release (CICR) leading to generation of cell-wide Ca2+ spikes, oscillations or localized Ca2+ “puffs” arising from simultaneous opening of a small cluster of IP3Rs [3R are the main event leading to differential gene expression [3Rs is also regulated through post-translational modifications, primarily by phosphorylation and dephosphorylation via protein kinases and phosphatases, respectively [3R resulting in an increase in Ca2+ release in mammals [D. melanogaster IP3R lacks PKA sites indicating that it is not regulated by PKA [2+/calmodulin-dependent protein kinase II (CaMKII), might be involved in the phosphorylation of insect IP3Rs similar to that in mammalians [3 is deactivated by phosphorylation to IP4 or dephosphorylation to IP2 thereby terminating the IP3R signaling pathway [2+-binding motifs, such as calmodulin, can also regulate the activity of the IP3Rs. Calmodulin has been shown to inhibit the binding of IP3 to IP3Rs in mammals in a dose-dependent manner [3R [3Rs; however, in D. melanogaster, IP3R and calmodulin compete for binding to transient receptor potential (TRP) proteins, which are known to form plasma membrane channels [IP neurons , fat bod neurons  ; submicromolar levels of Ca2+ act on the RyR channel by increasing open channel probability [2+ in the cytosol near the receptor causes it to release even more Ca2+; however, as the concentration of intracellular Ca2+ rises to millimolar concentrations, RyR channel activation becomes inhibited, preventing the total depletion of SR Ca2+ [2+, adenine nucleotides also have a biphasic effect on (3H)ryanodine binding [P. xylostella RyR, PKA phosphorylation sites have been detected in the Repeat34 domain, which might regulate the interaction with the neighboring SPRY3 domain [2+ binding protein, calmodulin has different effects depending on the Ca2+ levels and the type of the RyR in mammalians. Calmodulin activates (at low Ca2+ levels) or inhibits (at high Ca2+ levels) the RyR1 and RyR3 channels, while only inhibitory effects were reported for RyR2 [Drosophila melanogaster calmodulin mutants with a single amino acid change (V91G) were found to possess abnormal Ca2+ release in response to depolarization of muscles, which was linked to failed regulation of the RyR [2+ release from internal stores in photoreceptor neurons, indicating calmodulin is involved in the termination of the light response [D. melanogaster visual cascade also includes the cation influx channels transient receptor potential (TRP) protein, which also requires IP3R signaling [RyRs have a more restricted distribution compared to IP neurons . RyR actng sites ,175. RyRbability ,177,178. SR Ca2+ ,180,181. binding ; however binding ,183. Thi binding . In P. x3 domain . The pho3 domain . Notablyfor RyR2 ,185,186.for RyR2 ,51, the  the RyR . Inhibitresponse ,189,190.response ,189. As ignaling , the intD. melanogaster. These studies revealed RyR is expressed in muscles of the body wall, visceral muscles around the gut, central nervous systems, and optic lobe and retina in the embryonic, larval, and adult stages [D. melanogaster adults, RyR mRNA was detected in tubular muscles and at a lower level in neuronal tissues [D. melanogaster. For example, the highest expression level of RyR was detected in the thorax compared with the head and abdomen in adult B. dorsalis [P. rapae [2+ levels for locomotory activities. Similarly, RyR expression was higher in the adult thorax compared to the abdomen; however, the highest expression was detected in the head in D. citri [H. armigera larvae [P. rapae adults [L. decemlineata larvae [S. furcifera nymphs [T. citricida adults [RyR expression levels between the head, thorax, and abdomen were detected in the fourth instar larva of P. xylostella [L. decemlineata larvae indicated that RyR expression level was highest in foregut, at moderate levels in the hindgut and epidermis, and to a lower extent in the fat body, midgut, ventral ganglia, and Malpighian tubules [P. rapae, RyR was primarily expressed in the epidermis, at moderate levels in nerve cords, hemocytes, the midgut, and least in the fat body and Malpighian tubules [C. suppressalis, RyR was primarily expressed in the head (including brain and muscle), at moderate levels in the integument and the haemolymph, and least in the fat body, Malpighian tubules, the midgut, and the silk gland [RyR mRNAs is not unexpected considering that more muscles are distributed around the foregut, the hindgut, and attached to the epidermis [RyR expression was highest in larval or adult stages and lowest in eggs in O. furnacalis [B. dorsalis [H. armigera [L. decemlineata [T. castaneum [RyR expression was lowest in eggs; however, it was higher in nymphs than adults in D. citri [S. furcifera, RyR expression in the fifth instar nymph was significantly higher than in the eggs or female adults; however, no significant difference was detected between the eggs and female adults [C. suppressalis with the highest expression in the third instar larvae, but with similar expression in eggs, pupae, and adults [N. lugens, RyR transcript levels in female adults were significantly higher than in first to fifth instar nymphs; however, the lowest expression was still in eggs [RyR in T. citricida adults were also found to be significantly higher than those in nymphs [RyR was found between nymphs and adults [RyR expression levels in eggs, larvae, and adults were all found to be similar in the lepidopteran P. xylostella [RyR is higher in adult or juvenile stages (larva or nymph) than in eggs, suggesting involvement of RyRs in locomotory activities. Notably, the immobile pupal stages can also have high expression of RyR [RyR expression might fluctuate during pupal transition and be elevated depending on the timing of sampling [RyR expression in pupae might be related to factors other than muscle formation. Notably, RyR expression patterns might also be different between sexes. For example, RyR expression was found to be significantly higher in males in S. furcifera [N. lugens [G. molesta [RyR genes is not currently known. Nevertheless, the higher RyR expression in the thorax compared to the abdomen is in accordance with the primary function of RyRs in the mediation of excitation-contraction coupling in muscles, which is primarily located in the thorax for mobility [RyR in the head is in accordance with the involvement of this body part in nerve conduction, hormone secretion and sensory activities, processes that are regulated by RyR activity. It is noteworthy that expression levels of different RyR mRNA splicing variants vary between different developmental stages and tissues [M. persicae RyR mRNA lacks an alternative splicing event, which might be related to its asexual reproduction phase [RyR mRNAs is common in mammalians with more than 12 distinct splice variants identified to date, leading to important differences in their channel functioning [2+ release, while some contribute to distinct Ca2+ release patterns [T. citricida RyR mRNA splicing occur by intron retention [T. citricida RyR mRNA, encoding a truncated protein [RyR gene.RyRs mediate many cellular and physiological activities, such as muscle contraction, neurotransmitter release, and hormone secretion  Table 2Table 2. t stages ,33,195.  tissues ,188 but  tissues ,197. Amo tissues . Subsequdorsalis  and P. rP. rapae , suggestD. citri . Similara larvae , P. rapae adults , L. decea larvae , S. furca nymphs  and T. ca adults  with higlostella . A more  tubules . In the  tubules . In the lk gland . Such dipidermis . Neverthrnacalis , B. dorsdorsalis , H. armiarmigera , L. decemlineata , and T. astaneum . SimilarD. citri . In anote adults . This trd adults . In N. l in eggs . The expn nymphs , while nd adults . In contlostella . In brielostella , indicatn of RyR ,41,46. An of RyR , suggestsampling . It is nurcifera , N. luge. lugens , and G. mobility . On the  tissues ,52,55,65on phase . Alternactioning ,199,200.patterns ,127,128.3Rs are involved in the key events related to the gene expression, development, learning, memory, neuronal signaling, and sensory transduction [3R are expressed in many cell types, but primarily associated with the ER of neurons. IP3R1 is the predominant neuronal isoform and present in endothelial cells, while IP3R2 is the predominant isoform in contractile myocardial cells and the sinoatrial node and IP3R3 in the intestinal crypt, ovary cells, villus epithelial cells, and the microvillous cells in the olfactory system [3R gene. The first D. melanogaster IP3R gene was reported by Yoshikawa et al. [3R is present in all tissues of adult D. melanogaster and at more homogeneous in levels than RyR [3RIP mRNA was also abundant in the thorax. Among the head, eyes, antennae and legs, the highest expression was detected at antennae [3RIP revealed that the gene is expressed at the highest levels in adults, at moderate levels in eggs, followed by early and mid stage pupa, and least in larvae [3Rs are restricted to only a few insects. Liu et al. [3RIP were detected in 1-day-old larvae and 3-day-old eggs, respectively, in T. castaneum. In B. tabaci, 3RIP was primarily expressed in larvae, unlike D. melanogaster, while expression was moderate in pseudopupa and female adults, and least in eggs [RyR genes and is in accordance with the possible involvement of IP3R in locomotor activities [3R genes was reported from a single insect species. The trend was in favor of females, contrasting to those reported for RyR genes [RyR mRNA, alternative splicing of 3RIP mRNA is also common in mammalians [D. melanogaster [IPsduction ,136 , which is involved in hyperphagia, is elevated [3R-mediated Ca2+ signals modulate neural circuits for feeding [3Rs in neurons [D. melanogaster as manipulation of cytosolic Ca2+ levels in the larval fat body does not have a significant effect on larval fat stores [L. decemlineata, accumulate greater amounts of lipid at the larval stage, which show impaired lipid metabolism upon silencing Ca2+-signaling genes [2+ might be different depending on the species.Most of the data on the involvement of insect ER Car adults . In linelt flies . The liplt flies . The cAMlt flies . In para+ levels . Therefoipocytes . While tot known ,220,225.ot known  reportederphagia . IP3R-meelevated ,220,223  feeding ,226,227  neurons . In briet stores ,229. In ng genes ,216. TheD. melanogaster adults, fat body-specific knockdown of RyR leads to an increase in lipid droplet size and triglyceride levels, suggesting a lipolytic role for RyRs [seipin gene in D. melanogaster adults leads to reduction in triglyceride storage and lipid droplet size, which is linked to impaired SERCA activity, suggesting seipin and SERCA function together to promote fat storage in adipose tissue [RyR partially restores fat storage in seipin mutants, while 3RIP silencing did not rescue this phenotype [2+ homeostasis in fat body adipocytes. It is noteworthy that opposite effects were reported on the levels and cellular sites of Ca2+ on fat storage in hepatocytes compared to adipocytes in mammals. Increased cytosolic and reduced ER calcium levels induce triglyceride accumulation leading to lipogenesis, whereas reduced cytosolic and increased ER calcium levels reduce triglyceride accumulation leading to lipolysis in hepatocytes and their orthologous cells in the insect fat body, oenocytes [3R acts as an obesity gene in hepatocytes or oenocytes [D. melanogaster and, therefore, this topic requires further investigation in other insect species.Knowledge on the involvement of RyRs in insect lipid metabolism is restricted to a single study. In for RyRs . On the e tissue ,230. Inthenotype . These fenocytes ,222,231.2+ is a major source for the stimulation of muscle cells in invertebrates from nematodes towards insects [2+ that binds to troponin, a regulatory protein on the thin filament. Troponin activate another regulatory protein, tropomyosin, which causes muscle contraction [2+ pump on the SR membrane transports Ca2+ ions back into the SR lumen. This raises the question as to whether RyR or IP3R or both are involved in Ca2+-related muscle excitation and contraction in insects.Calcium is an essential element in the excitation and contraction of muscles ,233. ER- insects ,236,237.traction ,239. In D. melanogaster indicated that RyR is mainly expressed in the muscles of the body wall, visceral muscles around the alimentary canal, as well as the central nervous system [RyR expression in muscles have been also reported from non-Drosophila insects, such as H. virescens [L. decemlineata [RyR led to impairment of hypodermal, visceral, and circulatory muscles, indicating RyR is essential for proper muscle function and EC coupling in larval body wall muscles [Drosophila melanogaster RyR mutants also have a severe defect in the ingestion and passage of food into the gut, confirming that the head and visceral muscles are impaired [2+ flux, which was found to be related to failed regulation of RyR [Gryllus bimaculatus (Orthoptera: Gryllidae) [2+ release from the SR, via RyR, plays a major role in hyperneural muscle contractions in Periplaneta americana (Blattodea: Blattidae), while IP3R-induced Ca2+ release has little impact [RyRs play a central role in the excitation/contraction (EC) coupling of cardiac and skeletal muscles in mammals ,240,241.s system ,65,242. irescens  and L. dmlineata . Partial muscles ,242. Dron of RyR . RyR actyllidae) . Similare impact .3Rs also play a role in the EC and regulation of skeletal, cardiac, and smooth muscle cell functions in mammals [3R in insect muscle activity has not been studied in detail. 3RIP is expressed in D. melanogaster adult muscles, particularly in legs which contain tubular muscles, but to a lesser extent in the thorax, which contains the fibrillary muscles [3R has a possible role in tubular or fibrillar muscle function regulation in D. melanogaster. In G. bimaculatus, IP3R regulates the amplitude of rhythmic contractions of lateral oviduct muscles; however, the effect was considered minimal [2+ signaling modulation [3R inhibitors, is required. The involvement of Ca2+ in EC of lateral oviduct muscles via the action of several neurohormones was also reported in other studies. For example, octopamine, via the intracellular messenger cAMP, inhibits contraction of the oviducts, while proctolin, via the PLC/IP3R, stimulates contraction [Schistocerca gregaria (Orthoptera: Acrididae), ryanodine had no effect on proctolin-stimulated foregut muscle contraction, instead, gut muscle contraction was dependent on proctolin receptor-specific activation of the PLC signaling cascade leading to generation of IP3 [3-induced Ca2+ release from the SR, in contrast to the model of proctolin action on tonic muscle contractions of P. americana [2+ levels leading to muscle action, in particular related to locomotor activities such as flight, walking or climbing. For example, the mutations in 3RIP resulted in strong flight deficits in D. melanogaster [3RIP leads to significant defects in wing posture in Drosophila, indicating IP3R in neurons is necessary during pupal development for flight [2+ signals in cultured pupal neurons in D. melanogaster IP3R mutants also revealed high spontaneous Ca2+ influx and reduced SOCE, which might lead to loss of flight [3R signaling induced by neurohormones, primarily the amine-type, and their G-protein coupled receptors in the neurons  [3R in neurons can also be induced by other signaling molecules, such as neurotransmitters [3R-dependent Ca2+ release is essential for neuronal activity. Thus, expression of 3RIP in aminergic neurons during pupal development was found to rescue the adult flight deficit in D. 3Rmelanogaster IP mutants, suggesting the involvement of IP3R in flight is related to its role in development [2+ influx, are also necessary for normal flight activity [S. gregaria, the Ca2+ signal in such neurons is dependent on IP3R and PLC activation, but not on RyR [2+ stores in neurons are required for insect rhythmic motor functions which leads to muscle activity and IP3R signaling plays a central role in this supply. IP mammals ,244. Inv muscles ,61. Howe minimal . Notably minimal , or othedulation . Furthertraction ,250,251.n of IP3 . The autmericana . These fmericana . There anogaster ,254. Furr flight ,255. Exaf flight . These dneurons) ,258,259.smitters ,259, nevelopment ,254,256.activity . Insect activity ,262,263.t on RyR . In brie3R- [2+ levels in muscle cells is a requirement for muscle EC; this probably requires RyR and IP3R acting on neuronal pathways.The contradictory results on RyR-induced muscle EC ,243,265 3R- ,252 stilVisualization and olfactory responses play a crucial role in insect survival as they are involved in accessing food sources, protecting insects from threats, and finding mates to reproduce . This oc2 to a proton, and the secondary messengers hyrophilic IP3 and hyrophobic DAG [2 hydrolysis results in opening of light-sensitive, relatively Ca2+-selective, “transient receptor potential” (TRP) channels and TRP-like (TRPL) channels which mediate an ionic current responsible for generation of a quantum bump, known as the bump current [3R and RyR are limited. High expression of 3RIP in retina of adult D. melanogaster suggested a potential role for IP3R in visual transduction [D. 3Rmelanogaster IP mutants revealed that Ca2+ release via IP3R does not contribute to phototransduction [2+ channels in photoreceptors [D. melanogaster proposed that Ca2+ release via IP3R might have a critical role in light excitation. Silencing of 3RIP specifically in adult photoreceptor cells significantly reduced light-response amplitude in adult photoreceptor cells [3RIP silencing leads to a reduction in PLC catalytic activity, while elevation of intracellular Ca2+ rescued the suppressed light responsiveness phenotype. These findings suggest that Ca2+ release from internal stores is necessary to increase PLC activity required for bump current, and that functional cooperation between IP3R and PLC is necessary for light responsiveness [IP3R and phototransduction in previous studies [2+ from patchclamp recording electrodes, effectively replacing the Ca2+ that would have been released from IP3-sensitive stores. However, a more recent study using RNAi or 3RIP-null mutants [3R does not have a role in phototransduction. Bollepalli et al. [D. melanogaster is compromised by the Gal4 transcription factor used to regulate dsRNA in these experiments, which is not the case for the 3RIP knockdown or mutation in the study by Kohn et al. [3R in phototransduction. The role of RyR in Ca2+ regulation photoreceptor via RyR is equally ambiguous [D. melanogaster suggested a possible role for RyR in Ca2+ dependent-phototransduction [RyR expression was selectively eliminated in the adult eye demonstrated that this channel does not play a role in phototransduction [Phototransduction starts in ommatidia, units of the insect compound eye that contain sensory neurons known as retinal  cells. The rhabdomere is the central photoreceptive region in each retinal cell and contains photopigment molecules, called rhodopsins ,270. Absobic DAG . The rel current ,274,275.sduction ,61. Howesduction ,277, inseceptors . A subseor cells . Kohn etor cells  also repsiveness . This st studies ,277 was  mutants  challeng mutants  supportsi et al.  argues tn et al. . These cmbiguous ,189. Locsduction . Howeversduction . 2+. Other types of receptors are located in different types of sensilla  [3-signaling pathways [2+ stores were found to contribute to the ORN responses [3R and/or RyR are involved in odor transduction pathways. High expression of 3RIP in antennae in adult D. melanogaster suggests a potential role for IP3R in olfactory transduction [3R is present in the olfactory sensory neurons of a variety of species [D. 3Rmelanogaster IP mutant alleles [3RIP alleles, including a null allele, were found to exhibit a faster recovery after a strong odor pulse, suggesting that IP3R might be required for maintenance of olfactory adaptation in antennae [2+ response in ORNs was decreased upon targeting 3RIP and RyR by RNAi, as well as by specific blockers, such as thapsigargin or ryanodine [3RIP or RyR dsRNA were defective in odor-adaptation [2+-response was also found to be decreased in cAMP-defective flies based on silencing of the adenyl cyclase gene “rutabaga” and the phosphodiesterase gene “dunce” [RyR or 3RIP in combination with knock-down of rutabaga and/or dunce generated even stronger effects with smaller amplitudes and a shorter duration of Ca2+ response to various odors [3RIP or RyR expression is perturbed, perception of odorants (odor-acuity) is not affected, but adaptation to odorants is defective [3-signaling severely affects both the first and the second phase and they are unable to detect or adapt to odorants [2+-signaling pathway is important for odor-adaptation. Due to the limited number of studies, the mechanisms of insect odor transduction are still controversial [3) may be odor-specific [Calcium is also involved in olfactory sensory transduction ,284,285.ceptors) ,293,294.ceptors) . The trapathways ,303,304.esponses , raisingsduction ,61. Addi species ,307,308. alleles ,309. On aptation . The mag “dunce” ,311,312.oversial ,304,313.specific . 3R and RyR in phototransduction or olfactory responses is limited, and further research is required.In conclusion, evidence as to the role of IP3R have essential roles in development. This is in accordance with the fact that expression of either RyR [3RIP [D. melanogaster indicated that both genes are also necessary for embryonal development, in particular for development of nervous system and muscles [Both RyR and IPther RyR ,51,53,56yR [IP3R ,62 is up muscles ,189,206.3RIP in D. melanogaster leads to lethality in the second instar larvae accompanied by delays in molting from the first to the second instar and lower 20-hydroxyecdysone (20E) levels [PKA mutants [3R or cAMP pathway also delays second to third larval instar, third larval instar to pupal, and pupal to adult transitions [PKA and 3RIP mutant alleles have a synergistic negative effect on larval molting, suggesting IP3R signaling acts in parallel with the cAMP pathway to regulate molting [2+ release, leading to activation of PKC and CaMKII that are both involved in 20E-directed gene expression [D. melanogaster, silencing of 3RIP led to failures in molting and larval-pupal and pupal-adult metamorphosis in the beetle T. castaneum [D. melanogaster, which revealed that the larval-pupal transition requires IP3R/Ca2+ signaling in glutamatergic interneurons of the mid-ventral ganglion [3R signaling to allow pupariation on a protein-deficient diet [D. melanogaster larvae indicated that IP3R is essential in particular in aminergic cells for development and survival, and IP3R-mediated Ca2+ release is required to facilitate release of amine type hormones from aminergic cells or serotonergic and dopaminergic neurons [3RIP in aminergic neurons during pupal development rescues the onset adult flight deficit in 3RIP- D. melanogaster mutants [3RIP is also expressed in ovaries in contrast to RyR [2+ oscillations in ovaries [3R-mediated Ca2+ oscillations also occur in wing imaginal discs that give rise to wings in adults, conferring another role of IP3R signaling in development [Loss of ) levels ,276,315. mutants ,316. Disnsitions . Further molting . Exogeno molting ,315. Indpression ,319,320.astaneum . A relatganglion . The nutent diet . The aut neurons ,322,323. mutants ,254. As t to RyR ,197 and  ovaries , it may elopment . D. melanogaster RyR leads to formation of normal embryos that give rise to larvae with growth defects that die four–seven days during their first instar [RyR mutant allele rescue the calmodulin-lethal phenotypes, further indicating the vital role of RyR [T. castaneum, silencing of RyR does not cause any failure in molting or larval-pupal and pupal-adult metamorphosis, in contrast to 3RIP silencing in the same beetle; however, abnormalities in the folding of the hind wings and crawling behavior in adults occur, which might be related to impairment of muscle EC-coupling [Several studies have examined the role of RyR in insect development. Mutation of t instar . Heteroze of RyR . In T. ccoupling . 2+ levels are critical in this respect as lower Ca2+ concentrations induce autophagy, while higher Ca2+ concentrations switch autophagy to apoptosis [3R in these processes is a topic for future investigation.Developmental physiology also includes topics such as autophagy and the autophagic programmed cell death that play key roles in development, morphogenesis, and regeneration ,326. Intpoptosis . The rol2+ channels have great potential as target sites for the development of insecticides [3Rs, RyRs might be considered safer targets for insecticidal molecules [3Rs have been developed to date. The idea of targeting RyRs goes back to the discovery of the plant alkaloid ryanodine from the tropical American shrub, Ryania speciosa (Flacourtiaceae), which has high affinity to RyR and interferes with Ca2+ signaling in muscles; there receptors are aptly named RyR [2+ depletion. The insecticidal activity of R. speciosa extracts were first described by Rogers and co-workers in 1946 on a range of lepidopteran and hemipteran pests [Due to their essential roles, insect Cacticides ,330,331.olecules . While tamed RyR . Ryanodian pests ,335. Higan pests . Based o2+ channels to remain partially open leading to an excessive and uncontrollable release of stored Ca2+ ions from the ER into the cytosol of muscle cells [Liriomyza spp.), isopterans, such as sugar cane termites , and hemipterans, such as whiteflies (Bemisia spp.) [https://www.environmentalscience.bayer.us/turf-and-ornamentals-management/golf-course-management/portfolios-and-solutions/new-bayer-insecticide) (accessed on 4 April 2021).Diamides are derivatives of benzenedicarboxamide or phthalic acid  and anthranilic acid  ,344,346.ia spp.) ,349,350.ia spp.) , while tDiamide insecticides have low mammalian toxicity and are considered safe for beneficial insects and mites, which make them environmentally friendly ,344. The2+ release from intracellular Ca2+ stores in insect muscle cells [2+ release in isolated insect neurons [RyR in S. furcifera [L. decemlineata [2+ levels [B. mori RyR revealed that the binding region of flubendiamide is located in the transmembrane domain of the RyR comprising amino acid residues 4111–5084, while the region in the N-terminal cytoplasmic domain correspond to residues at 183–290 [2+ mobilization in response to flubendiamide, but not to caffeine [4610-A4655) in D. melanogaster RyR (GenBank accession number: D17389) C-terminal domain with that of a nematode RyR led to insensitivity to diamides [4659-A4703 in PxRyR, which is within the larger region examined by Kato et al. [D. melanogaster RyR (aa 4973–4982), where ryanodine binds, or the TM10, which plays a crucial role in human RyR channel activation and gating [4790 and G4946 (in P. xylostella RyR) are likely to be involved in forming the diamide binding site [H. virescens and P. americana muscles indicate that flubendiamide and chlorantraniliprole interact with a binding site that is distinct from the ryanodine binding site [4697, K4700, Y4701, L4704, I4790, Y4918, S4919, Y4922, D4942, V4943, G4946, and F4947 based on P. xylostella RyR) comprise the putative chlorantraniliprole binding pocket [Diamides act on RyR and induce Cale cells ,42,338,  neurons ,340,354.urcifera  or L. demlineata  greatly + levels ,355. Eff+ levels . Deletio 183–290 . HEK celcaffeine . A simildiamides . Notablyo et al. . However RyR aa 43–4982, wd gating ,356,357.ing site . On the ing site ,338,359.ing site . Recentlg pocket . Furtherg pocket . In brieg pocket ,360,362.Adoxophyes honmai (Lepidoptera: Tortricidae) against flubendiamide in Japan [Choristoneura rosaceana (Lepidoptera: Tortricidae) against chlorantraniliprole in the U.S.A. [Aphis gossypii (Hemiptera: Aphididae) against cyantraniliprole in Italy [P. xylostella [S. litura [S. exigua against clorantraniliprole in China [B. tabaci against both clorantraniliprole and cyantraniliprole in the U.S.A. [P. xylostella against flubendiamide and/or clorantraniliprole in Thailand [A. honmai in Japan [C. suppressalis in China [P. xylostella, C. suppressalis, T. absoluta, A. honmai, S. exigua, and S. frugiperda) and two hemipterans (A. gossypii and B. tabaci) from 11 countries including Brazil, China, Greece, Italy, Japan, Korea, Mexico, Phillippines, Puerto Rico, Spain, and Thailand have developed moderate to significant levels of resistance (relative ratio ≥10) to diamides  G4946E/V located at the interface between transmemrane domain 4 (TM4) and the TM4-TM5 linker (numbering is based on PxRyR), 2) I4790M/T within the upper TM2 or TM3, 3) E1338D at the N-terminus, and 4) Q4594L in a flexible loop located in DR1 before the pseudo voltage-sensor domain [P. xylostella RyR with the G4946E mutation, while affinity to other ligands, such as caffeine or ryanodine, did not change [S. exigua larvae with the G4946E mutation exhibited 223-, 336-, and >1000-fold increase in resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively [D. melanogaster flies with the G4946V mutation were also found to exhibit high levels of resistance against flubendiamide (RR: 91.3) and chlorantraniliprole (RR:195), but less so against cyantraniliprole (RR:5.4) [D. melanogaster RyR with G4946E mutation expressed in Sf9 cells revealed that this mutation confers a high degree of resistance also against pyrrole-2-carboxamides [Belgica antarctica, the mite Tetranychus urticae and the hemipteran mealybug Ferrisia virgata [D. melanogaster flies naturally wild-type for the I4790M mutation exhibit low to moderate resistance to diamides, while the M4790I mutation leads to higher levels of susceptibility to flubendiamide (RR: −15.3 fold), but less to chlorantraniliprole (RR: −7.5) and cyantraniliprole (RR: −2.3) [4946 in insects) as is a methionine in D. melanogaster and all other insects and arachnids, suggesting I4790 might be responsible for the differential sensitivities of the P. xylostella, T. absoluta, and possibly beetles and other insects to diamides [4594 is conserved amongst lepidopterans, while I4790 is lysine in D. melanogaster and coleopterans, hymentopterans and some other Dipterans; however, its involvement in diamide binding is not currently known, other than it being mutated in resistant populations [1338, which is located in the insect divergent region 2 (IDR2) between SPRY2 and SPRY3 domains and appears not to be conserved in insects [C. suppressalis resistant to chlorantraniliprole revealed a new mutation Y4667D/C (corresponding to Y4701 in PxRyR), which might confer to high levels of resistance [Detailed examination of RyRs from field-collected or lab-selected resistant strains revealed mutations that affected residues located in the C-terminal transmembrane spanning domains ,373,376,r domain ,393,405.r domain ,362. In t change . In a reectively . Similaroxamides . It is n virgata . The rep virgata ,331. On diamides ,373,405.diamides ,362. Thediamides  further diamides . Regardiulations ,373. The insects ,107. A rsistance . HoweverRyR. Metabolic resistance against inseciticides develops through elevated levels of detoxification enzymes, such as cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST) and esterases. The synergistics, piperonyl butoxide (PBO) an inhibitor of P450, diethyl maleate (DEM) a depletor of glutathione, S,S,S-tributylphosphorothioate (DEF) an esterase inhibitor, and triphenyl phosphate (TPP) a carboxylesterase inhibitor, lowered the LC50/LD50 values of chlorantraniliprole in L. decemlineata [P. xylostella [C. suppressalis [S. frugiperda [P. xylostella revealed that most of the metabolic detoxification enzyme genes were slightly up-regulated [C. suppressalis [S. exigua [RyR might also be a possible resistance mechanism to diamide insecticides, which was demonstrated via RNAi in S. furcifera [L. decemlineata [RyR led to a decrease in the diamide efficacy. In another study, RyR was found to be slightly down-regulated in P. xylostella populations with lower to moderate levels of resistance (RR: 6–35 fold) against chlorantraniliprole, while the gene was significantly down-regulated in a population with high levels of resistance (RR:1750-fold) [RyR was down-regulated in C. suppressalis upon treatment with chlorantraniliprole [RyR might slow the release and depletion of intracellular Ca2+ stores from the SR in muscles and the ER of many cell types when induced by RyR activators, and consequently enhances resistance to diamide insecticides [Other mechanisms might also confer to diamide resistance; this includes metabolic resistance and down-regulation of mlineata , P. xylolostella , C. suppressalis  and S. fugiperda . Additiougiperda ,408,409.egulated . Up-reguegulated ,412,413.ressalis  and S. e. exigua . It is n. exigua ,414,415.urcifera  and L. dmlineata . Down-re50-fold) . Similariliprole . Down-recticides . It is ncticides ,64,416. 3Rs as targets in pest control are limited due to their higher similarity with their mammalian counterparts. Nevertheless, a single study has examined the role of IP3R in diamide resistance. Interestingly, silencing 3RIP in B. tabaci adults dramatically decreased susceptibility to cyantraniliprole [RyR silencing in S. furcifera [L. decemlineata [3RIP expression during the entire period of the treatment in B. tabaci, which might be a strategy to adjust the RyR-linked increase in intracellular Ca2+ and decreased ER Ca2+ levels [As mentioned before, studies on IPiliprole , similarurcifera  and L. dmlineata . It is i+ levels . However2+ homeostasis and interfering with IP3R and RyR. Botanicals, entomopathogens, repellents, toxins, Ca2+ inhibitors or biomolecules such as dsRNA or miRNAs or peptide agonists or antagonists are promising in this regard. For example, Ma et al. [Tripterygium wilfordii (Celastraceae) [Mythimna separate (Lepidoptera: Noctuidae). This investigation revealed that wilforine acts on myocytes leading to an increase in cytosolic Ca2+ levels when applied at nanomolar levels and activates both RyR and IP3R based on use of specific inhibitors of both channel proteins [3R and RyR in neurons are activated by the botanical insecticide Celangulin I, extracted from Chinese bittersweet Celastrus angulatus, another species from Celastraceae [Bacillus thuringiensis Cry toxins might also interfere directly or indirectly with Ca2+ signaling and intracellular Ca2+ levels [2+ homeostasis also has great potential in this manner [There might be other pest control tools targeting cellular Caa et al.  examinedtraceae)  on Mythiproteins . Similarstraceae . Other b+ levels ,428,429.s manner ,431,432.2+ homeostasis is vital for insects, and the ER is one of the major intracellular sources for Ca2+. The RyR and IP3R are the two channel proteins associated with the ER and are involved in the intracellular Ca2+ supply. Insects possess a single RyR and IP3R gene, in contrast to mammalians which possess three for each. Both RyR and IP3Rs cluster separately in phylogenetic analyses; however, they share common domains, such as the MIR, RIH, RIH-associated regulatory domains at the amino-terminus, and transmembrane helices at the carboxy-terminus. Alternative splicing, which is regulated in a tissue-specific and developmental manner, occurs for both genes and each receptor has its own, distinct, regulatory mechanism. IP3R genes are expressed in most cells, in particular in the ER of neurons, adipocytes, and oocytes, while RyR gene expression has a more restricted distribution and is predominantly found in the SR of muscle cells and the ER of neurons. Both receptors have essential roles in insect physiology and development. RyRs mediate many cellular and physiological activities related to muscle contraction and hormone secretion, while IP3Rs are involved in key events related to learning, memory, neuronal signaling, lipid metabolism, and sensory transduction. Efforts have concentrated on the development of pest control strategies targeting the operation of RyRs and IP3Rs; however, RyRs appear to be safer targets due to their lower similarity with mammalian counterparts compared to IP3Rs. Diamides are the best examples of a pest control chemistry targeting RyRs, although resistance developed by pests against diamides has become an increasing issue. Various pest control tactics based on use of botanicals, microbials and toxins, as well as biomolecules such as dsRNA and miRNAs, targeting cellular Ca2+ homeostasis and affecting the operation of RyRs and/or IP3Rs directly or indirectly might be also promising.In conclusion, Ca


POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations.Mutations in the POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0–6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations , which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations , patients with both MSI and extensive methylation in MLH1 , patients with MSI but no extensive methylation in MLH1 , and patients without MSI . The POLE group was younger at diagnosis , with frequent right-sided tumour localisation , and was diagnosed at an earlier stage . The mean methylation score in POLE was not different from that in MSI-U and non-MSI, but the methylation signature was distinct from that of the other subgroups. Additionally, although the examined number of POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene.Tumour tissues  obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.CRC patients with The online version contains supplementary material available at 10.1186/s13148-021-01104-7. POLE gene are found in 3–7% of CRCs, mutations within the proofreading (exonuclease) domain of POLE are present in only 1–2% of CRCs [POLE is essential for ensuring replication fidelity, and its disruption by the pathogenic heterozygous mutations found in cancers leads to an ultra-hypermutated phenotype of tumours, with the highest burden of single-nucleotide variants among human cancers [POLE exonuclease domain mutations have excellent prognosis [Most colorectal cancers (CRCs) occur sporadically and progress through sequential accumulation of multiple genetic and epigenetic alterations by influencing the expression and behaviour of genes that regulate cell growth and differentiation –3. Sever of CRCs –9. The p cancers , 10, 11.rognosis , possiblrognosis , 14.POLE-mutant CRCs with their corresponding MSI status, genetic mutations, tumour lymphocyte infiltration, and clinicopathological findings, including clinical outcomes [POLE-mutant CRCs in the context of epigenetic alterations. The present study aimed to evaluate the epigenetic profiles of POLE-mutant CRC and elucidate the clinicopathological features associated with key genetic and epigenetic alterations in this malignancy.To date, several pivotal studies have characterised outcomes –8, 15. HPOLE mutations. None of the patients with synchronous multiple cancers displayed POLE mutations in their cancer tissues; thus, patients with synchronous multiple cancers were analysed by the most advanced tumour lesion for further molecular studies . Therefore, a total of 1,070 CRC tissues were analysed for POLE mutations displayed MSI features in the four mononucleotide repeat markers [The MSI status of the 1,052 CRC tissues was evaluated. None of the four tumours harbouring pathogenic  markers , 16. AmoThe expression status of the four DNA mismatch repair (MMR) proteins  was confirmed in all 67 MSI CRC tissues. By IHC analysis, 11.9% (8/67) were classified as MMR-proficient (pMMR), and the remaining 86.8% of MSI CRCs (59/67) were confirmed to be MMR-deficient (dMMR). Of the 59 dMMR tumours, 79.7% (47/59) exhibited both MLH1 and PMS2 deficiency (dMLH1), 15.3% (9/59) exhibited both MSH2 and MSH6-deficiency (dMSH2), 5.1% (3/59) exhibited MSH6-deficiency alone (dMSH6), and none of the tumours displayed PMS2-deficiency on their own (dPMS2). The precise status of IHC staining and MSI markers in MSI tumours is shown in Additional file MLH1 gene, which has a large CpG island within its promoter region that divides it into at least two discrete regions of methylation  of MSI CRCs, and 85.1% (803/944) of non-MSI CRCs. Partial methylation in MLH1 (i.e. affecting the AB region only) was observed in 50.0% (2/4) of POLE-mutant CRCs, 16.9% (11/65) of MSI CRCs, and 14.9% (141/944) of non-MSI CRCs. Extensive methylation (i.e. affecting both the AB and C regions) was observed in 44.6% (29/65) of MSI CRCs  tumours with extensive MLH1 methylation were categorised as MSI with MLH1 methylation (MSI-M) and 3.6%  tumours without extensive MLH1 methylation as MSI with unmethylated MLH1 (MSI-U).In this cohort, no methylation in the AB or C region (unmethylated) was observed in 50.0% (2/4) of POLE mutations , MSI CRC patients with extensive MLH1 methylation , MSI CRC patients with unmethylated MLH1 , and non-MSI CRC patients , similar to 72.4% (21/29) of MSI-M and 77.8% (28/36) MSI-U, compared to 46.6% (440/944) of non-MSI (P < 0.0001).Table BRAF and KRAS mutations were not observed in POLE-mutant tumours. In contrast, BRAF mutations were observed in 69.0% (20/29) of MSI-M, 11.1% (4/36) of MSI-U, and 3.7% (35/944) of non-MSI, whereas KRAS mutations were observed in 22.2% (8/36) of MSI-U and 32.5% (307/944) of non-MSI (P < 0.0001).POLE mutations and the other subtypes, we evaluated the methylation status of discrete regions within the promoter of the MGMT and SFRP2 genes.To better understand the differences between tumours with MGMT methylation status, lack of methylation in the minimal promoter (Mp) and enhancer (Eh) regions (defined as unmethylated) was observed in 100% (4/4) of POLE, 37.9% (11/29) of MSI-M, 75.0% (27/36) of MSI-U, and 76.1% (718/944) of non-MSI. Partial methylation in MGMT (i.e. affecting either MP or Eh) was observed in none of the POLE cases, but in 3.5% (1/29) of MSI-M, 13.9% (5/36) of MSI-U, and 6.4% (60/944) of patients without MSI. Extensive methylation of MGMT (i.e. affecting both Mp and Eh) was observed in none of the POLE, 58.6% (17/29) of MSI-M, 11.1% (4/36) MSI-U, and 17.6% (166/944) non-MSI  and region-2 (R2) in SFRP2 (defined as unmethylated) was not observed in POLE or MSI-M, but in 13.9% (5/36) of MSI-U and 15.7% (148/944) of non-MSI. Partial methylation in SFRP2 (i.e. affecting either R1 or R2) was observed in none of the POLE, but in 3.5% (1/29) of MSI-M, 22.2% (8/36) of MSI-U, and 20.6% (194 of 944) of non-MSI patients. Extensive methylation of SFRP2 (i.e. affecting both R1 and R2) was observed in all of the POLE, 96.6% (28/29) of MSI-M, 63.9% (23/36) of MSI-U, and 63.8% (602/944) of non-MSI . Although tumour-infiltrating lymphocytes were likely to be more common in older POLE-mutant patients and/or with methylation in the MLH1-AB region, the analysed number of patients was too small to find associations.Finally, Table ltration , 14, 21.ues Fig. b. The nuHPF Fig. c. The mePOLE mutations, especially those with simultaneous epigenetic alterations. As previously reported, pathogenic somatic POLE mutations are usually found in the early stages of CRC in the right side of the colon and relatively younger patients with CRC.In the present study, we report a biologically distinct subset of CRCs with pathogenic somatic BRAF V600E mutations [BRAF mutations can directly induce CIMP [BRAF V600E mutation does not directly cause CIMP, and thus, aging-like acquisition of DNA methylation may favour the survival of cells with such mutations in the BRAF gene through suppression of senescence and activation of stem cell pathways.CIN, MSI, and CIMP constitute the three major mechanisms of genomic or epigenetic instability in CRC , 2, 5. Eutations , 22, 23.uce CIMP , 25. Recuce CIMP  reportedMLH1 gene, which justifies our reason for selecting methylation status of the MLH1 gene at the beginning as a critical biomarker for classifying CRCs [BRAF V600E mutations, but rarely KRAS mutations [KRAS gene [BRAF and KRAS gene products function in the same MAP-kinase signalling pathway, and activating mutations in these genes occur mutually exclusive [BRAF mutations, KRAS mutations have been proposed as a possible cause of aberrant methylation. Fibroblasts transformed by fos or ras show upregulated DNA methyltransferase expression and consequent global hypermethylation [KRAS mutations [BRAF or KRAS. From this point of view, it might be sufficient to summarise the features of methylation by using the MLH1, MGMT, and SFRP2 genes.Clinically, most sporadic MSI CRCs arise from a CIMP background caused by the epigenetic silencing of the ing CRCs . CIN andutations , 28. HowRAS gene , 29. BRAxclusive , 31. Inthylation . Indeed,utations . TherefoKRAS mutations, promoter methylation within the MGMT gene, which encodes a DNA repair gene that removes pro-mutagenic O6-methylguanine residues from DNA, is associated with KRAS-mutant CRC [MGMT methylation status to clarify the epigenetic features of POLE-mutant CRCs. Interestingly, although half of the POLE-mutant CRCs showed partial methylation in the MLH1 promoter, none of the POLE-mutant CRCs displayed any methylation in the discrete promoter regions of the MGMT gene.In the case of tant CRC , 26, 33.MLH1 and MGMT, we evaluated SFRP2 methylation  because most CRCs possess extensive methylation in their promoter regions [SFRP2 methylation is that extensive methylation is rare in adenomatous polyps, while it is common in CRCs [SFRP2 methylation is more frequently observed in CRCs with KRAS mutations than in those with BRAF V600E mutations and those with wild-type KRAS or BRAF mutations [MGMT and SFRP2 has a similar feature in terms of association with CRC with KRAS mutations, all POLE-mutant tumours demonstrated extensive methylation in SFRP2, but no methylation in MGMT.In addition to  regions –36. An i in CRCs , 36. Addutations , 35. ThuPOLE mutations induces a distinct pattern of mutations in cancer driver genes, a substantially increased mutation burden, and an enhanced immune response that is detectable even in precancerous lesions [POLE-mutant tumours was similar to that in the MSI-U and non-MSI groups, POLE mutations may also cause a distinct pattern of epigenetic alterations in cancer-associated genes.Temko and colleagues demonstrated that acquisition of  lesions . SimilarPOLE-mutant CRCs have been reported to have a favourable prognosis, as noted for early-stage dMMR tumours [POLE-mutant CRC patients in this study were diagnosed at stages I to II, and there was no recurrence within five years after surgical resection. IHC analysis showed that the number of CD8-positive cytotoxic tumour-infiltrating lymphocytes in POLE-mutant CRCs significantly increased in POLE-wild-type CRCs, especially in mismatch repair protein-proficient CRCs [MLH1-AB region methylation status and age at diagnosis. In this study, CD8-positive cells were likely to be more common in older POLE-mutant patients and/or with methylation in the MLH1-AB region, but the number of analysed POLE-mutant patients was too small to reach statistical significance. tumours . The fouent CRCs , 14. We POLE proofreading mutations were found in only four tumours. Therefore, this rarity suggests that our results should be interpreted with caution. We conclude that CRC patients with POLE mutations are rare, such mutations are observed in younger individuals, lesions are often located within the right colon, diagnosis occurs at an earlier stage, and distinct epigenetic alterations might be associated with CD8 cell infiltration.Although we analysed over 1,000 CRC samples, CRCs with POLE proofreading mutations in a large cohort of CRC patients in Japan and to evaluate the epigenetic profiles of POLE-mutant CRC and elucidate the clinicopathological features associated with key genetic and epigenetic alterations in this malignancy.This study aimed to clarify the incidence of A cohort of 1,052 CRC patients underwent surgical resection between 1998 and 2017 at the Okayama University Hospital, Japan. Tumour specimens and corresponding normal mucosa samples were collected according to institutional review board (IRB)-approved protocols .Genomic DNA was extracted from fresh-frozen samples using a QIAamp DNA Mini Kit . The extracted DNA was quantified using a Qubit 4 fluorometer with a Qubit dsDNA BR assay kit . After determining the DNA quality, an EZ DNA methylation kit  was used for bisulfite conversion of the normalised samples.POLE hotspot mutations in the proofreading domain  were evaluated by Sanger sequencing. The primer sequences used are listed in Additional file Pathogenic BAT26, NR21, NR27, and CAT25), as described previously [MSI status was analysed in all CRC tissues using four mononucleotide repeat markers , MSH2 , PMS2 , and MSH6 protein . Further incubation was performed with a secondary antibody and the avidin–biotin–peroxidase complex , followed by incubation with biotinyl tyramide and streptavidin–peroxidase. Diaminobenzidine was used as a chromogen, and haematoxylin was used as a nuclear counterstain. Tumour cells were scored negative for MMR protein expression only if the epithelial cells within the tumour tissue lacked nuclear staining, while the surrounding stromal cells showed positive staining. Samples showing proficiency in all MMR protein expressions were defined as pMMR, and samples showing deficiency in at least one of the four MMR proteins were defined as dMMR. When a tumour showed neither MLH1 nor PMS2 with staining, the tumour was classified as MLH1-deficient (dMLH1); when a tumour showed neither MSH2 nor MSH6 with staining, the tumour was classified as MSH2-deficient (dMSH2); when a tumour showed negative staining only for PMS2 but was positive for MLH1, the tumour was classified as PMS2-deficient (dPMS2), and when a tumour showed negative staining only for MSH6 but was positive for MSH2, the tumour was classified as MSH6-deficient (dMSH6).We employed immunohistochemistry to examine the MMR protein expression of MLH1, MSH2, PMS2, and MSH6 in primary tumour tissues of CRC specimens that showed the MSI phenotype. Staining was performed manually using FFPE specimens. Thin (5 µm) sections of representative blocks were deparaffinised and dehydrated using gradient solvents. Following antigen retrieval in citrate buffer (pH 6.0), endogenous peroxidase was blocked with 3% HPOLE-mutant tumours, as described previously [IHC analysis for CD8 was performed in the four eviously . The numMLH1, we examined the discrete regions of MGMT and SFRP2 promoters that affect these expressions or other critical features in tumorigenesis to clarify the epigenetic features in tumours with POLE mutations. Like MLH1, the MGMT gene is inactivated as a consequence of extensive methylation in its promoter region  [MGMT is significantly associated with MGMT protein downregulation and an increased burden of KRAS mutations in CRC patients [SFRP2 gene promoter is a definitive feature of advanced CRCs [In addition to  region) , 38, 39.patients , 33, 38.ced CRCs , 34–36.MLH1, MGMT, and SFRP2 promoters in each sample, a modified combined bisulfite restriction analysis (COBRA) with fluorescence dyes was performed to quantitatively measure the methylation density [To quantify the population of methylated alleles of the  density , 36, 38.MLH1, MGMT, or SFRP2 genes, two tumours that were otherwise characterised as dMLH1 by IHC analysis and 37 tumours without MSI were excluded from further analysis , as described previously [Sanger sequencing was performed to confirm mutations in eviously , 26. PCRMLH1, MGMT, and SFRP2 promoters were analysed as both continuous and categorical variables .All statistical analyses were performed using JMP Pro software . Methylation levels in the MLH1-AB region methylation status was evaluated by the Kruskal-Wallis test. All reported P values were two-sided, and statistical significance was set at P < 0.05.Categorical variables were compared using the Chi-squared test. The pair-wise comparisons for each subgroup were performed using a nonparametric multiple comparison method using the Steel–Dwass test. The association between age at dignosis and Additional file 1. Table 1. Detailed information of 65 MSI CRC patients.Additional file 2. Table 2. Primer sequences.