The differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging in clinical practice. We aimed to evaluate the diagnostic accuracy of the IFN-γ/TNF-α FluoroSpot assay for differentiating ATB from LTBI.We conducted a pilot study of case-control design, using the FluoroSpot assay to simultaneously detect IFN-γ and TNF-α secretion at the single-cell level. The frequencies of antigen-specific single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells were detected. The optimal cutoffs value of frequencies for differentiating ATB from LTBI were determined according to receiver operating characteristic curve analysis. The sensitivity, specificity, predictive values (PV) and likelihood ratios (LR) of the FluoroSpot assay were calculated.P < 0.01). The frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay correlated significantly with those of T-SPOT.TB . After stimulated by ESAT-6 peptides, with total TNF-α-secreting T cells frequencies at a cut off value of 21 iSFCs/250,000 PBMCs, the sensitivity, specificity, PLR, NLR, PPV, NPV of IFN-γ/TNF-α FluoroSpot assay in differentiating ATB from LTBI were 96.7% , 94.3% , 16.92 , 0.04 , 93.6%  and 97.1% , respectively. With the frequencies of total TNF-α- and total IFN-γ-secreting T cells stimulated by ESAT-6 peptides combined, the specificity was increased to 97.1%, and the positive likelihood ratio to 31.5. The combination with CFP-10 might not improve the diagnostic accuracy of the ESAT-6 for differentiating ATB from LTBI.Thirty patients diagnosed microbiologically with ATB, 36 healthcare workers with LTBI and 36 healthy controls were enrolled. After stimulated by ESAT-6 or CFP-10 peptides, the median frequencies of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells in ATB patients were all significantly higher than those in LTBI and HC groups (IFN-γ/TNF-α FluoroSpot assay might have potential to help differentiate ATB from LTBI, but the findings need to be further verified by cross-sectional or prospective cohort studies.The online version contains supplementary material available at 10.1186/s12879-021-06351-w. Tuberculosis (TB) is known to be the leading cause of death from a single infectious pathogen. Worldwide 10.0 million people were newly diagnosed with TB disease, and TB caused 1.2 million deaths among HIV-negative people in 2019 [Mycobacterium. tuberculosis (MTB) antigens with no evidence of clinically manifest ATB [WHO defined LTBI as a state of persistent immune response to stimulation by fest ATB . It is efest ATB . Tubercufest ATB . IGRA difest ATB . Hence, Tumor necrosis factor (TNF) participates in MTB immune response by activating macrophages, inducing chemokines, participating in TB granuloma formation and maintaining granuloma integrity. In addition, animal experiments showed that blocking the TNF signaling pathway accelerated intracellular bacterial growth and necrotic death of overladen macrophages, implicating that TNF restricts mycobacterial growth within macrophages and prevents their necrosis . Human sFluoroSpot is a highly sensitive and practical technique based on ELISPOT, which simultaneously detect two or more cytokine secretions at single-cell level. The interpretation of the results is based on the visualization of the cells with distinct fluorescence, thus avoiding color confusion , 9. FluoThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of PUMCH (No: S-715). Informed written consent was obtained from all patients prior to their enrollment in this study.From March 2018 to September 2018, ATB patients and healthcare workers were enrolled from Peking Union Medical College Hospital and Beijing Chest Hospital. The inclusion criteria for the active TB group were: 1) aged from 18 to 75 years old, AND 2) had typical clinical manifestations of ATB such as cough, chest pain, night sweats, weight loss, etc. AND 3) ATB diagnosis made based on microbiological evidence  testing or Xpert MTB/RIF assay), AND 4) had not received any anti-TB therapy by the time of recruitment. The inclusion criteria for healthcare workers were: 1) aged from 18 to 75 years old, AND 2) without clinical manifestations suspected of ATB, AND 3) without evidence of previous TB such as self-reported TB history or positive X-ray findings, AND 4) without autoimmune diseases, diabetes mellitus, chronic hepatitis or malignancy. Healthcare workers with positive and negative T-SPOT.TB testing results were regarded as LTBI and healthy controls (HC), respectively. Persons who were pregnant, receiving anti-TNF-α therapy, or infected with HIV were excluded. This study was a case-control designed diagnostic accuracy study.6 PBMCs/ml with serum free culture medium . IFN-γ/TNF-α FluoroSpot  assays were performed according to the manufacturer. 96-well plates pre-coated with monoclonal antibodies against IFN-γ and TNF-α were seeded with 2.5 × 105 PBMCs and anti-CD28 . 50 μl of AIM-V  as nil control, 5 μg/ml PHA as positive control, and 6-kDa early secreted antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10) peptides  as MTB-specific antigens were added in duplicate wells. All peptides were 15mers overlapping their adjacent peptides by 10 amino acids, spanning the length of ESAT-6 (17 peptides) or CFP-10 (18 peptides). The purity of each peptide exceeded 80%, and identity was confirmed by mass spectrometry. Plates were incubated at 37 °C in 5% carbon dioxide for 16–20 h and then washed with washing buffer. After incubation, the detection antibodies (anti-TNF-α biotin and anti-IFN-γ-FITC) were added to each well and incubated for two hours at room temperature under dark condition. Then streptavidine-550 red-conjugate and anti-FITC-490 green were added to the reaction system. The reaction system was incubated for another one hour under dark and dry condition. Finally, fluorescence enhancer was loaded to make spots visible under the automated ELISPOT reader . When counting the frequencies of ESAT-6 or CFP-10 antigen-specific immunofluorescence spot forming cells (iSFCs), including frequencies of total TNF-α- or total IFN-γ- or dual IFN-γ/TNF-α-secreting T cells, the background spots in the nil control well were subtracted. If the number of spots was over 50 iSFCs/2.5 × 105 PBMC for TNF-α or over 10 iSFCs/2.5 × 105 PBMC for IFN-γ in nil control, or less than 200 iSFCs/2.5 × 105 PBMC in positive control, the result was considered to be indeterminate. Images of the results are presented in Fig. Four milliliters of peripheral blood were obtained from each participant by venipuncture. Within 4 h of sample collection, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood by density gradient centrifugation. Cell suspension was prepared at 2.5 × 10Laboratory staff who conducted the assays and researchers who interpreted the results were all blinded to patients’ clinical data.We used the histogram to check the distribution of the variables. For normally distributed variables, results were presented as mean ± standard deviation (SD), while variables with an abnormal distribution were denoted as median and interquartile range (IQR). Categorical data were presented as percentages and 95% confidence intervals (CIs). We used Mann-Whitney U test to compare the frequencies of iSFCs and Chi-square test to compare response proportions between different groups, respectively. We used Wilcoxon signed-rank test to compare the frequency of iSFCs between the two antigens in the same group. We used Spearman’s rank correlation to measure correlation between the frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay and T-SPOT.TB.Receiver operating characteristic (ROC) curves were used to show the overall accuracy of IFN-γ/TNF-α FluoroSpot assay for differentiating ATB and LTBI in frequencies of T cells. The areas under the ROC curves (AUROC) of each group of T cells were calculated and compared. The cutoff value with the largest Youden’s index on the ROC curve was regarded as the optimal cutoff of the frequency. The sensitivity, specificity, predictive value (PV) and likelihood ratio (LR) were calculated at the optimal cutoff.P < 0.05 (two-sided) were considered as statistically significant.Statistical analyses were performed using SPSS 24.0  and MedCalc . A total of 101 participants, including 30 patients with microbiologically confirmed ATB, 35 participants with LTBI and 36 healthy controls were enrolled. Of the 30 ATB patients, 23 (76.7%) were positive for MTB culture, 16 (53.3%) were positive for sputum smear acid-fast staining, 24 (80.0%) were positive for MTB-NAA testing, and 20 (66.7%) were positive for Xpert MTB/RIF test. 27 (90.0%) ATB patients were diagnosed with pulmonary tuberculosis, while tuberculous lymphadenitis and tuberculous pleuritis were diagnosed in 2(6.7%) and 1 (3.3%) patient, respectively. Detailed characteristics of the participants are shown in Table P < 0.01). The frequencies of total TNF-α-, single IFN-γ-, total IFN-γ-, dual IFN-γ/TNF-α-secreting T cell in LTBI group were significantly higher than those in HC group under single ESAT-6 stimulation . In consistency with the result, the median frequencies of single TNF-α-secreting T cells stimulated by ESAT-6 were significantly higher than those stimulated by CFP-10 (P < 0.001)  displayed over 10 iSFCs/2.5 × 10ctively. .P < 0.001, r = 0.845 for CFP-10, P < 0.001) . In active TB group, the sensitivity of Fluorospot assay detecting total IFN-γ-secreting T cell was 93.3%, and there was no significant difference compared with T-SPOT.TB .After stimulated by ESAT-6 or CFP-10 peptides, the frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay strongly and significantly correlated with those of T-SPOT.TB  Fig. . The agr5 PBMC, with a sensitivity of 96.7%  and a specificity of 94.3% , respectively. The combination of ESAT-6 and CFP-10 did not improve the diagnostic accuracy of differential diagnosis of ATB and LTBI   improved the differential diagnostic accuracy  smear lacks sensitivity and mycobacterial culture is time consuming . AlthougT-cell immunity plays a critical role in controlling MTB infection . IGRA woOur study showed that the frequencies of MTB specific IFN-γ-secreting T cells in the ATB group were significantly higher than those in the LTBI and HC groups. These findings were consistent with our previous research . The freInterestingly, ESAT-6 seemed to activate T cells more effectively than CFP-10. The frequencies of TNF-α-secreting T cells stimulated by ESAT-6 were significantly higher than those stimulated by CFP-10, though no difference was detected in the IFN-γ secretion under the stimulation of these two antigens. The immune mechanism is still unclear and remains to be further studied.In this study, the frequencies of ESAT-6-specific total TNF-α-secreting T cells might be a better potential marker for IFN-γ/TNF-α FluoroSpot assay to differentiate ATB and LTBI, compared with IFN-γ-secreting T cells. Wang’s study showed that the detection of. of MTB-specific TNF-α secretion by ELISA assay could distinguish ATB from LTBI, with a sensitivity of 72% and a specificity of 90.91% . AnotherFurthermore, this study showed that double-testing the ESAT-6 with the CFP-10 might not increase the differential diagnostic accuracy. The result was in consistency with a study of North India population which also observed significantly higher IFN-γ and TNF-α response to ESAT-6 stimulation in ATB group compared to healthy household contacts (considered as LTBI), while no significant difference was observed with CFP-10 stimulation . ClifforMedication may disturb the cytokine secretion. Several studies of IGRAs indicated that anti-tuberculosis drugs, corticosteroids and immunosuppressants may interfere T-cell responses towards MTB antigens , 32–34. Both our study and previous research found that a number of iSFCs secreting TNF-α in the nil control of some samples were detected, but no iSFCs secreting IFN-γ were found . A possiHowever, this study has some limitations. First, we excluded patients with malignancy or undergoing TNF-α antagonist therapy, whose TNF-α secretion may be disturbed. Thus, whether IFN-γ/TNF-α FluoroSpot can be applied to these population required further research. Second, the use of T-SPOT.TB as a diagnostic criterion for LTBI may lead to selection bias . HoweverIFN-γ/TNF-α FluoroSpot assay might be helpful for the differential diagnosis of ATB and LTBI. The frequencies of ESAT-6-specific total TNF-α-secreting T cells might be a better potential marker for IFN-γ/TNF-α FluoroSpot assay, compared with IFN-γ-secreting T cells. The results need to be further verified by cross-sectional or prospective cohort studies.Additional file 1 Supplementary Table 1. Frequencies of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ-, dual IFN-γ/TNF-α-secreting T cells following ESAT-6 or/and CFP-10 stimulation in ATB, LTBI and HC groups. Supplementary Table 2. Frequencies of spot-forming T cell in nil control in ATB, LTBI and HC groups. Supplementary Table 3. Diagnostic accuracy of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ-, dual IFN-γ/TNF-α-secreting T cells following ESAT-6 or/and CFP-10 stimulation in differentiating ATB and LTBI.


The proteolytical cleavage of transmembrane proteins with subsequent release of their extracellular domain, so-called ectodomain shedding, is a post-translational modification that plays an essential role in several biological processes, such as cell communication, adhesion and migration. Metalloproteases are major proteases in ectodomain shedding, especially the disintegrin metalloproteases (ADAMs) and the membrane-type matrix metalloproteases (MT-MMPs), which are considered to be canonical sheddases for their membrane-anchored topology and for the large number of proteins that they can release. The unique ability of TIMP-3 to inhibit different families of metalloproteases, including the canonical sheddases (ADAMs and MT-MMPs), renders it a master regulator of ectodomain shedding. This review provides an overview of the different functions of TIMP-3 in health and disease, with a major focus on the functional consequences in vivo related to its ability to control ectodomain shedding. Furthermore, herein we describe a collection of mass spectrometry-based approaches that have been used in recent years to identify new functions of sheddases and TIMP-3. These methods may be used in the future to elucidate the pathological mechanisms triggered by the Sorsby’s fundus dystrophy variants of TIMP-3 or to identify proteins released by less well characterized TIMP-3 target sheddases whose substrate repertoire is still limited, thus providing novel insights into the physiological and pathological functions of the inhibitor. The human genome encodes for about 15,000 transmembrane proteins, encompassing 19% of the total genome. Ectodomain shedding is a post-translational modification by which membrane proteins are proteolytically cleaved and their ectodomain released. This biochemical mechanism is essential in a number of cellular processes, including cell communication, adhesion and migration. For instance, tissue bioavailability of certain cytokines and growth factors is controlled by ectodomain shedding, as it depends on membrane-level signalling receptors and therefore the ability of cells to respond to specific cues. Moreover, shedding controls cell-to-cell adhesion or adhesion of cells to the ECM, and it modulates cellular transport by controlling levels of endocytic receptors on the cell surface . MetalloGiven the large number of substrates and biological processes in which they play a role, the activity of metalloprotease sheddases has to be finely modulated. When dysregulated, aberrant metalloprotease-dependent ectodomain shedding can lead to pathological conditions, such as cancer, arthritis, inflammatory diseases and Alzheimer’s ,8. Its rThis review provides an overview of different functions of TIMP-3 in health and disease related to its ability to control ectodomain shedding. TIMP-3-unique structural features and regulatory mechanisms that enable it to modulate ectodomain shedding, together with in vivo functional consequences of enhanced TIMP-3 expression or ablation, will be discussed. Finally, we describe a collection of mass spectrometry-based approaches that have been used in recent years to systematically identify substrates of TIMP-3 target proteases. These methods have led to the identification of new proteins regulated by the inhibitor. High-resolution proteomics approaches represent a powerful tool to study proteolysis and have provided novel insights into TIMP-3 biology and the functions of its target sheddases, which may be applied in future to other TIMPs or sheddases that have been less well characterized.2+ ion of metalloproteases by their conserved Cys1. This displaces the water molecule needed by metalloproteases for peptide bond hydrolysis [n-terminal domain of about 125 amino acid residues, which retains the full inhibitory capability, and a smaller C-terminal domain whose biological function is not fully elucidated yet, but it has been reported to affect the affinity of TIMPs against ADAMs and therefore ectodomain shedding [n-terminal five residues  that are highly conserved among all TIMPs and residues of the metalloprotease active site cleft [All mammalian TIMPs appear as “wedge-shaped” with the inhibitory ridge that perfectly complements the active site cleft of target metalloproteases. The TIMP mechanism of inhibition comprises the coordination with the proteolytic Zndrolysis . TIMPs cshedding ,11. Despshedding ,12. The te cleft . The abite cleft . Lee andte cleft .Given its role in modulating ECM turnover and ectodomain shedding, the bioavailability of TIMP-3 must be finely regulated. This occurs at several levels, including transcriptional regulation, by growth factors and cytokines, and epigenetically by promoter methylation, post-transcriptional regulation by specific miRNAs and by receptor-mediated endocytosis .TIMP3 promoter. Qureshi and colleagues also reported that TIMP-3 expression could be induced by TGF-β1 through different mechanisms involving Smad-2, -3 and -4 and the phosphatidylinositol 3-kinase/Akt signaling pathway [TGF-β1 is a multifunctional growth factor produced by different cell types, such as monocytes, platelets and chondrocytes, that promotes ECM deposition. It has been shown to induce the expression of TIMP-3 in articular chondrocytes . This oc pathway ,15. Onco pathway . Epstein pathway . Similar pathway . Missens pathway . Loss of pathway .TIMP3 gene has been reported to be a key mechanism in cancer progression [The epigenetic regulation of TIMP-3 involves a number of mechanisms that are not dependent on its gene sequence. For example, aberrant methylation of the promoter region of TIMP-3 gene was found to be associated with primary cancer of the brain, kidney, colon, breast, etc. . TIMP-3 gression ,22,23. Lgression .Post-transcriptional regulation of TIMP-3 expression has been investigated in both physiological processes and pathological conditions. Several miRNAs have been found to bind the 3′-UTR of the TIMP-3 mRNA, resulting in the degradation of TIMP-3 transcripts and its gene silencing ,26. miR-Exosomes can be used as means for transferring miRNAs to target recipient cells. Few examples of exosomal miRNAs have been reported to regulate the expression of TIMP-3 and, in turn, cell migration. Melanoma-derived exosomes carried miR21, which promoted invasion of fibroblasts by downregulation of TIMP3 expression . SimilarEndocytosis has emerged as a major mechanism to regulate bioavailability of TIMP-3 in the tissues, with the low-density lipoprotein receptor-related protein-1 (LRP-1) playing a key role in this process ,35. TIMPTimp3-null cortical neurons subjected to oxygen–glucose deprivation (OGD) showed delayed cell death. Loss of TIMP-3 enhanced shedding of FAS ligand and subsequent lower FAS activation and neuronal cell death [Timp3-null mice were resistant to middle cerebral artery occlusion (MCAO), indicating that TIMP-3 promotes neuronal cell death in vivo [Timp3-null mice undergoing BCAO had significantly higher levels of shed TNFR1 and ADAM17 activity [TIMP-3 has a number of unique features among TIMPs, including the ability to promote apoptosis, as was shown in a number of cancer cells ,42. TIMPll death . As a co in vivo . Additio in vivo . Furtheractivity . Intriguactivity .Timp3-null mice, and the process is reversed by addition of recombinant TIMP-3, but not TIMP-1. Thus, although the molecular mechanism has not been fully elucidated yet, these data suggest that epithelial apoptosis during mammary gland involution in Timp3-null mice is due not to a general lack of MMP inhibition but more likely to one of the unique features of TIMP-3, including its ability to regulate shedding or binding to the ECM [The effects of TIMP-3 on apoptosis in vivo were also investigated by Fata and colleagues using mammary gland involution as a model for physiological apoptosis . In resp the ECM .TIMP3 gene [n-terminus (S38C) and six at the C-terminus  [Sorsby’s fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the MP3 gene ,52. SFD MP3 gene . All TIMd S181C) ,52. An ad S181C) . SFD mutd S181C) . Howeverd S181C) . Thus, td S181C) ,54.CADASIL  is an inherited form of cerebrovascular disease that is associated with changes in the number of cysteines causing an odd number of cysteines in the mutant NOTCH3. Such mutations lead to the extracellular deposition of NOTCH3 ectodomain on the vessels. Shed NOTCH3 interacts with TIMP-3 and promotes its accumulation in the vascular extracellular matrix. TIMP-3 plays a crucial role in the pathology of CADASIL. Its haploinsufficiency in a mouse model of CADASIL rescues reduced cerebral blood flow and impaired functional hyperemia, two CADASIL-related phenotypic features . MechaniTimp3-null mice display increased susceptibility to LPS-induced septic shock. This phenotype is rescued by either ablation of the TNF receptor 1 (TNFR1) or by ectopic administration of a metalloprotease inhibitor. This study clearly shows that TIMP-3 is a master regulator of inflammation in vivo by controlling the ADAM17/TNF/TNFR1 axis [Timp3-null mice failed to resolve the inflammatory process. When subjected to bleomycin-induced lung injury, Timp3-null mice showed higher and persistent neutrophilia and macrophage infiltration compared to wild-type mice, mainly due to increased expression of genes associated with proinflammatory (M1) polarization, including Il6, Il12, Nos2 and Ccl2 [Tumour necrosis factor alpha (TNF) is an essential pro-inflammatory cytokine in immune responses to invading pathogens. TNF is synthesized as a transmembrane protein that needs to be shed in order to elicit its pro-inflammatory potential. This process occurs by the action of ADAM17 and it is finely regulated by TIMP-3. TIMP-3 deficient macrophages release higher levels of soluble TNF in response to LPS. As a consequence, FR1 axis . Other tand Ccl2 .Leukocyte migration is a key component of the inflammatory process. Upon pathogen invasion, leukocytes move to sites of infection. This process is mediated by an array of cell adhesion molecules that regulate rolling, adherence of leukocytes to the vascular endothelium and their extravasation out of the circulatory system. Shedding of adhesion molecules is a key mechanism to control leukocyte migration and, as a consequence, TIMP-3 is a major regulator of the process. In fact, TIMP-3 has been reported to reduce shedding of L-selectin, a cell adhesion molecule that is expressed on most circulating leukocytes and plays a major role in directing leukocytes towards the inflammation sites . Its sheTimp3-null mice subjected to antigen-induced arthritis display a dramatic increase in inflammation and higher levels of circulating TNF compared to wild-type controls [Timp3-null mice showed signs of spontaneous cartilage degradation after 6 months of age [While the ADAM17/TNF/TNFR1 axis plays a key role in defending the organism against invading pathogens, when deregulated, this signalling pathway can lead to autoimmune and inflammatory diseases, including arthritis ,72,73,74s of age . In agres of age .Timp3-null mice challenged with intravenous administration of concavalin A to develop autoimmune hepatitis displayed enhanced activation of hepatic T-cells, cytokine release and, ultimately, increased mortality. Mechanistically, loss of TIMP-3 exacerbates the pathology in a TNF-dependent manner, as Tnf ablation rescued the increased susceptibility to concavalin A-induced hepatitis. Nevertheless, congenic chimeras generated by transplanting wild-type bone-marrow into Timp3-null recipient mice displayed similar pathological features to Timp3-null mice, suggesting that TIMP-3 produced by hepatic stromal cells, rather than TIMP-3 released by immune cells, has a prominent role in the pathology of autoimmune hepatitis [Inflammation is an orchestrated process that involves a heterogenous population of immune and non-immune cells. Murthy and colleagues found that TIMP-3 has a broader role in immunity using a murine model of autoimmune hepatitis in which inflammation is not only driven by canonical immune cells but also epithelial and stromal cells of the liver, such as hepatocytes, endothelial cells and stellate cells . Timp3-nepatitis .Fulminant hepatitis is a rare syndrome leading to rapid liver failure, mostly caused by drug overdose. TIMP-3 was reported to be a major regulator of this process, not only by modulating TNF release and its hepatotoxic consequences but also hepatoprotective signals . ApoptosTimp3-null mice showed exacerbated organ damage and further impaired liver function when challenged with IRI. Intriguingly, additional to an enhanced inflammatory response, loss of TIMP-3 increased ADAM10-dependent shedding of E-cadherin during hepatic IRI. Moreover, a specific ADAM10 inhibitor partly rescued this effect after IRI. Altogether these results indicated that in contrast with the majority of the phenotypes displayed by Timp3-null mice, which are related to aberrant activity of ADAM17, some effects of Timp3 loss in vivo are related to its target protease ADAM10 [Another model of liver damage, hepatic ischemia/reperfusion injury (IRI), is characterized by elevated ADAM17 activity and TNF levels . Timp3-ne ADAM10 .Timp3-null and Timp3-overexpressing mice with 2, 4, 6-trinitrobenzenesulfonic- acid (TNBS) to induce chronic colitis. They found that Timp3-null mice developed severe colitis after administration of TNBS, while mice overexpressing TIMP-3 were resistant to the disease [Both ADAM17 and TIMP-3 are constitutively expressed by epithelial cells in the human intestinal barrier, but levels of the inhibitor were found low in patients affected by Crohn’s disease . In orde disease . Althoug-/- mouse model of the disease. TIMP-3 overexpression decreased macrophage infiltration and the size of atherosclerotic plaques, indicating a protective role for the inhibitor in the development of atherosclerosis [ApoE-null mouse model of the disease [Atherosclerosis is characterized by chronic inflammation, with macrophages playing a role in all stages of the disease. In order to investigate the role of TIMP-3 in the progression of atherosclerosis, Casagrande and colleagues generated a new mouse model in which TIMP-3 was specifically overexpressed in atherosclerotic plaques via a macrophage-specific promoter (MacT3) and crossed it with an LDLRclerosis . Convers disease . Since n disease ,85.Timp3-null mice display higher body temperature, oxygen consumption and carbon dioxide production than wild-type mice, regardless of food intake and locomotor activity [Insr+/− mice) exhibit metabolic dysfunctions of human diabetes, such as glucose intolerance and insulin resistance. Insr+/− mice exhibit low levels of TIMP-3. In addition, TIMP-3 expression inversely correlates with the susceptibility of mice to develop diabetes and vascular inflammation [Insr+/− mouse. When exposed to a high-fat diet, the resulting Timp3-/-; Insr+/− mouse developed hepatic defects that are features of non-alcoholic fatty liver disease as a consequence of uncontrolled ADAM17 activity [The role of TIMP-3 in controlling metabolism and in the pathogenesis of metabolic disorders has been well documented. activity . This efammation . Pharmacammation . A similammation . TIMP-3 activity .Timp3-null mice are highly resistant to developing HCC in response to a hepatocarcinogen agent (n-diethylnitrosamine) [Timp3-null and wild-type mice. Although the mechanism is not yet fully elucidated, Timp3 ablation led to activation of a number of factors promoting senescence, including p53, p38 and Notch, and to decreased expression of the senescence repressor FoxM1 [Another pathological consequence of obesity is an increased risk of hepatocellular carcinoma (HCC). As will be discussed in more detail in osamine) ,91. The or FoxM1 ,91. ThusTimp3-null mice exposed to a high-fat diet show reduced diversity in gut microbiota, liver steatosis and an increase in circulating soluble IL-6 receptors, which, in turn, can activate inflammatory cells to drive metabolic inflammation [It has emerged that TIMP-3 can affect gut microbiota and, subsequently, obesity and insulin resistance. ammation .Timp3-null mice subjected to unilateral ureteral obstruction (UUO), a common experimental model of renal injury, developed a more severe hydronephrosis (kidney swelling) compared to wild-type mice [Timp3-null mice displayed higher interstitial fibrosis due to increased fibroblast activation and deposition of type I collagen [Timp3-null mice developed an age-dependent chronic tubulointerstitial fibrosis, even in the absence of renal injury, further underlining a crucial role of TIMP-3 in maintaining kidney function. Renal fibrosis in Timp3-null mice is associated with aberrant ADAM17-mediated shedding of TNF, as TNF ablation rescued tissue thinning and the other lesions displayed by Timp3-null mice.TIMP-3 is the most expressed TIMP in the kidneys, and its levels are increased in patients with kidney disease . Timp3-nype mice . This wacollagen . IntriguTimp3-null mice challenged with streptozocin exhibited increased signs of fibrosis, thicker glomerular basement membrane and albuminuria compared to wild-type mice [Timp3 was genetically ablated in the Akita mouse, which carries an autosomal dominant allele consisting of a missense mutation in the Ins2 gene and recapitulates aspects of human diabetes [Timp3 ablation in this mouse exacerbated the diabetic nephrology-like pathology, characterized by increased albuminuria, mesangial matrix expansion and kidney hypertrophy [Timp3-null mice was accompanied by increased ADAM17 and NADPH oxidase activity, it is conceivable that TIMP-3 mainly exerts its function in the kidney by regulating ADAM17-mediated shedding. In agreement, Casagrande and colleagues demonstrated that Timp3-overexpression in myeloid cells protected mice from streptozotocin-induced diabetic nephropathy at the same extent as ablation of ADAM17 in podocytes [Diabetic nephropathy, one of the major complications of diabetes, is characterized by a gradual decline in glomerular filtration rate and progressive albuminuria due to increased accumulation of extracellular matrix and podocyte dysfunction. Loss of TIMP-3 is a hallmark of diabetic nephropathy. Patients affected by the disease show decreased levels of the protein, and TIMP-3 polymorphisms are associated with the disease . Ablatioype mice . Secondldiabetes . Timp3 aertrophy . There iertrophy . Since todocytes . This evTnf in the same Timp3-null mouse ameliorates the pathology and prevents heart failure. TNF is a potent activator of MMP expression. Thus, excess TNF released in the absence of TIMP-3 leads to elevated production of MMPs, especially MMP-8, matrix turnover and ventricular dilation [Dilated cardiomyopathy is the most common cause of heart failure. Ectodomain shedding, as well as inflammation and extracellular matrix (ECM) remodelling, play a pivotal role in regulating the homeostasis of the cardiovascular system. When dysregulated, these processes can lead to dilated cardiomyopathy and heart failure. Patients with dilated cardiomyopathy express high levels of the sheddases ADAM10 and ADAM15, which may promote integrin shedding and cause a reduction of cell–matrix interactions, thereby contributing to cardiac dilatation. On the other hand, TIMP-3 levels are reduced in patients with dilated cardiomyopathy, suggesting a protective role for the inhibitor . In agredilation .TIMP-3 plays a key role in another cardiovascular condition characterized by an excess of ECM turnover, the aortic aneurysm. TIMP-3 loss caused augmented remodelling of the abdominal aorta, thereby leading to exacerbated aortic aneurysm, in the angiotensin II-induced murine model of hypertension . In addiTimp3-null mice to Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. When challenged with L-NAME, Timp3-null mice showed lower blood pressure, less thickened vascular walls, reduced fibrosis and production of reactive oxygen species in cardiac microvessels [Hypertension, a major risk factor for cardiovascular disease, is finely regulated by ectodomain shedding ), ECM remodelling and inflammation, suggesting a critical role for TIMP-3 in the process. A protective role for TIMP-3 in hypertension and myocardial vascular remodeling in vivo was demonstrated by exposing ovessels .Timp3-null mice displayed higher pulmonary microvascular leakage after sepsis, leading to tissue edema and multiple organ dysfunction. These defects are associated with dysfunction of microvascular endothelial cells caused by aberrant shedding of cadherins and other proteins that contribute to cell-to-cell adhesion [TIMP-3 controls the microvascular endothelial cell barrier function, as was shown in a model of sepsis induced by caecal-ligation and perforation . This meadhesion .Timp3-null mice exhibited abnormalities in retinal vasculature, comprising dilated capillaries throughout the choroid [Timp3-null mouse displayed an increase of VEGF signalling and pathologic angiogenesis in two different models of corneal neovascularization [Timp3-null mice showed greater revascularization compared to littermate controls in a mouse model of oxygen-induced retinopathy. Conversely, pharmacological inhibition of ADAM17 decreased pathological neovascularization [TIMP-3 is a potent inhibitor of angiogenesis. This activity is partly separated from metalloprotease inhibition and due to its direct binding to VEGF receptor 2. In turn, the interaction of TIMP-3 with VEGFR2 prevents the binding of VEGF to the receptor and its downstream signaling and angiogenesis . Further choroid . In addirization . On the rization .TIMP-3 is downregulated in several human cancers, and its loss correlates with worse prognoses . The proWhile properties of TIMP-3 in cancer have been extensively reviewed elsewhere ,121, thiA crucial event in the innate immunity to tumours is the engagement of the NK group 2D (NKG2D) receptor on natural killers (NKs) and NKG2D ligands on tumour cells. Such an engagement overcomes inhibitory signals on NK cells, activates NK cells to release cytotoxic molecules, such as perforin and granzyme, and triggers apoptosis of tumour cells. Thus, proteolytic shedding of ligands for (NKG2D) receptors is a strategy used by tumours to modulate immune recognition by NK cells and cytotoxic T cells. Shedding of NKG2D ligands, including MICA, MICB and ULBP2, is inhibited by TIMP-3 in acute myeloid leukemia (AML) cells, and this process enhances their sensitivity to lytic activity of NK .Some effects promoted by TIMP-3 loss are not evident due to redundant functions of the other TIMPs. This is the case in cancer-associated fibroblast (CAF)-like state regulation. CAFs play a crucial role in cancer progression for their ability to secrete stromal factors that promote cell migration and invasion. Ablation of all four TIMPs induces fibroblasts to acquire a CAF-like state and to secrete exosomes enriched in ADAM10 . This, iTimp3-null mice and mice overexpressing Timp3 in chondrocytes. Loss of TIMP-3 compromises tibial bone mass and structure in both cortical and trabecular compartments, with a corresponding increase in the number of osteoclasts [Timp3-null mice had reduced load and stress capacity [Timp3-overexpression also led to bone abnormalities, including defects in tibial structure [Timp3 allele [Timp1-/-, Timp2-/- or Timp4-/- did not show trabecular loss, which is evident in the Timp3-/- mouse. QT mice are clearly smaller in size than wild-type mice and display a dramatic reduction of trabeculae in long bones. The authors showed that this phenotype is due to an imbalance between bone deposition and resorption, which is a consequence of unleashed MMP activity and enhanced osteoclast differentiation. Intriguingly, the latter process is mainly driven by aberrant shedding activity of ADAMs in the absence of TIMPs, especially TIMP-3. Unleashed ADAM activity triggers bone resorption by a dual mechanism. First, excess TNF release drives osteoclast differentiation of osteoclast precursors. Secondly, TNF is responsible for Dkk1 overexpression by osteoblasts, which, in turn, negatively regulates Wnt signaling and therefore osteoblast differentiation [The function of TIMP-3 in bone and cartilage has been characterized in vivo by analysing the skeletal architecture of eoclasts . Such decapacity . Intrigutructure . These s3 allele . Timp1-/ntiation .Timp3-null mice which are not direct consequences of shedding regulation but mainly consequences of an excess of ECM turnover.In addition to functions that TIMP-3 exerts in vivo by regulating ectodomain shedding, there are a number of phenotypes developed by the Timp3-null animals develop spontaneous air space enlargement in the lungs as a consequence of enhanced degradation of collagen in the peribronchiolar space and disorganization of collagen fibrils in the alveolar interstitium, while no increase in inflammation or fibrosis is displayed [Timp3-null mice showed decreased bronchiole branching, enhanced activity of MMPs and fibronectin degradation throughout lung development compared to controls [isplayed . In addicontrols .Timp3-null mice exhibited deterioration in cognitive function and decreased habituation in the open-field test, as a consequence of enhanced hippocampal MMP activity [Extracellular matrix (ECM) turnover, especially degradation of proteoglycans, plays a crucial role in regulating synaptic activity, nerve recovery and cognition. activity . Other tactivity . Neuroglactivity . IgLONs activity .In conclusion, TIMP-3 regulates shedding of numerous proteins, spanning from pro-inflammatory cytokines, such as TNFα, to growth factors and adhesion molecules, thereby modulating a number of biological processes in vivo .The function of a protease strictly depends on the collection of substrates that it cleaves. Some proteases cleave a large number of substrates, while others have a very restricted activity towards a few proteins. Furthermore, the activity of proteases can be spatially and/or temporally confined, so that even proteases with a large spectrum of substrates may only access a limited number of them at specific locations and at specific times. In the last years, high-resolution mass spectrometry techniques have been developed to investigate the function of specific proteases by the systematic identification of their substrates in a specific cellular context ,142. TheHigh-resolution secretome analysis allows the determination of alterations of protein levels in the secretome of cultured cells. This includes the possibility to evaluate levels of proteins that are released by ectodomain shedding, whose levels are reduced when their sheddases are inhibited or genetically ablated . ConditiTimp3-null mice, whose most prominent phenotypes are related to a deregulated ADAM17 activation [Secretome of TIMP-3-overexpressing HEK293 cells were analysed using a similar approach and revealed that TIMP-3 mainly inhibited shedding of ADAM10 substrates in these cells and under steady-state conditions . A similtivation . AltogetAs extensively discussed within this review, shedding regulates levels of transmembrane proteins and therefore biological responses. In turn, TIMP-3 has been reported to stabilize membrane proteins by inhibiting ectodomain shedding, such as death receptors and adhesion molecules, thereby modulating cellular processes. However, shedding can be a means to control the homeostatic turnover of transmembrane proteins. The effects of TIMP-3 on proteins that are shed at a low rate, as a consequence of their constitutive turnover at the cell surface, may be less pronounced and not lead to an evident increase of their levels at the cell surface and clear functional consequences on cell function. In order to discriminate which proteins are effectively stabilized on the cell surface by TIMP-3 , quantitative proteomics can be used. Different approaches can be used to isolate cell surface proteins prior to being analysed by mass spectrometry, including enrichment of glycosylated membrane proteins by SPECS (discussed in the next section) and biotinylation of membrane proteins followed by streptavidin pulldown. The latter approach was used to identify proteins stabilized by TIMP-3 at the surface of HEK293 cells . In thisSecretome protein enrichment with click sugars (SPECS) is a proteomic method that has been proven useful to investigate shedding, especially in those cells that require serum and other additives to grow, such as primary neurons ,144. SPEn-termini and lysine residues of proteins are chemically labeled with formaldehyde and isotopes are labeled for the following mass spectrometry analysis. Then, labeled proteins are digested by trypsin, which will cleave after arginines, but it will not be able to cleave after lysines, as they will be blocked after formaldehyde conjugation. Then, TAILS peptides are enriched and subjected to liquid chromatography and high-resolution mass spectrometry. The neo-n-termini peptides arising from cleavages of the protease of interest will appear with higher relative abundance, and from this the substrates of specific proteases can be deduced.Additional to ADAM-mediated ectodomain shedding, TIMP-3 modulates the activity of secreted metalloproteases, such as MMPs and ADAMTSs. These proteases are majorly involved in processing components of the ECM. The previously described proteomics methods, which have been used to investigate ectodomain shedding, are not suitable to identify soluble substrates of TIMP-3 target metalloproteases and therefore they are not appropriate to study the role of TIMP-3 in ECM turnover. In fact, these methods comprise a tryptic digestion of protein samples and evaluation of relative protein abundance. Relative abundance of an ECM component would not change upon cleavage by a protease, and therefore methods that are specifically targeted to identifying cleavages should be used. For instance, the terminal amine-based isotope labeling of substrates (TAILS) has been extensively characterized to investigate MMPs and their inhibitors , a degenerative disease of the macula. Despite scientific efforts that have been made to understand how SFD mutations affect TIMP-3 function and cell behavior, the molecular mechanisms linking SFD mutations to the onset of the disease are still unknown [Mutations in the  unknown . The pro unknown . Neverth unknown ,149. Met unknown . The funIn conclusion, TIMP-3 is a major regulator of ectodomain shedding in vivo. Its ablation or overexpression in mice has led to the identification of several biological processes in which TIMP-3 plays a key role. Nevertheless, many aspects of TIMP-3 biology are not fully characterized yet. Proteomics represents a promising tool to uncover such TIMP-3 functions that have not been elucidated by in vivo analysis and targeted biochemical approaches, thus providing new insights into the biology of the inhibitor.


CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-A. Here, we focused on RTN3 as its contribution to CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a well-characterised model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin, however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury. Reticulons (RTNs) are membrane-bound proteins of which four have been identified in mammals and termed RTN1, 2, 3 and 4 (Nogo). RTNs are characterised by highly conserved reticulon homology domains (RHD); approximately 150–200 amino acids are located in the RTN C-terminal, which are involved in the improvement of the localisation and function of these proteins. RTNs are found in a variety of tissues including the brain, kidney, spleen and liver, playing diverse roles such as ER shaping and morphology, membrane trafficking and apoptosis. Moreover, RTNs are found in neurodegenerative diseases such as Alzheimer’s, amyotrophic lateral sclerosis and multiple sclerosis [In neurons, a specific transport system for membrane-bound vesicles that contain lipids and proteins is required for the extension and maintenance of neurites ,6. KinesProtrudin also interacts with RTN3 and promotes its binding to KIF5 allowing the protrudin-KIF5 complex to cargo RTN3 from the cell soma to neurites . More reAll experiments were licensed by the UK Home Office (PDA74B4CC6) and experimental protocols were approved by the University of Birmingham’s Animal Welfare and Ethical Review Board . All animal surgeries were carried out in strict accordance with the guidelines of the UK Animals Scientific Procedures Act, 1986 and the Revised European Directive 1010/63/EU. The ARRIVE guidelines were also followed in the reporting of in vivo studies. Surgical procedures conformed to the guidelines and recommendation of the use of animals by the Federation of the European Laboratory Animal Science Associations (FELASA). Adult 6–8-week-old female Sprague-Dawley rats weighing 170–220 g  were housed in dedicated facilities at the University of Birmingham. Up to four rats per cage were maintained in a temperature-controlled environment on a 12 h light/dark cycle with free access to food and water ad libitum. All in vitro and in vivo experiments were performed with the experimenter blinded to the treatment conditions and unblinded after data analysis.g for 60 min. The white material at the interface was collected and purified in 0.32 M sucrose solution and the white pellet was collected and freeze-dried overnight. The protein content of the CME was determined using the Pierce BCA assay  and the content of myelin-derived axon growth inhibitory molecules such as myelin basic protein , Nogo-A , myelin-associated glycoprotein  and the CSPG brevican  was determined by Western blot, as described below.CME was prepared from adult rat brains as described by us previously . Briefly2 overexposure before harvesting L4/L5 DRG pairs. Cells were dissociated into single cells using collagenase  and DRGN were grown in Neurobasal-A  at a plating density of 350 cells/well in 8-well chamber slides precoated with 100 μg/mL poly-D-lysine . CNS myelin extracts (CME) were prepared and added to cultures at 100µg/mL to simulate the post-injury environment of the injured spinal cord and incubated with or without appropriate treatments for 3 days at 37 °C and 5% CO2, as described previously [n = 9 wells/condition). Rat primary DRGN were prepared according to our previously published method . Brieflyeviously . ExperimSince preconditioning SN (pSN) lesioned DRGN contain significantly higher levels of RTN3 mRNA/protein, we exposed the left SN at mid-thigh level and crushed the nerve using fine forceps at the level of the sacrotuberous ligament. We then harvested the ipsilateral L4/L5 DRGs at 7 days after injury and dissociated them into single cells and cultured them as described above. 3 cells were plated in 8-well chamber slides in NBA pre-coated with 100 μg/mL poly-D-lysine  and CME and incubated with or without appropriate treatments for 3 days at 37 °C and 5% CO2 [n = 9 wells/condition). Retinal cells were dissociated into single cells using a Papain dissociation kit , as described by us previously . Brieflyd 5% CO2 . ExperimDRGN or retinal cells were fixed in 4% paraformaldehyde (PFA) , washed in several changes of PBS and immunocytochemistry performed as described previously ,21. BrieTo overexpress RTN3 in DRGN and retinal cells in vitro, a CMV promoter-driven rat RTN3 plasmid was purchased from Origene, Rockville, MD, USA (#RR200211L3). Transfection controls were carried out using a CMV driven, non-targeting, EGFP plasmid . In preliminary experiments, optimal concentrations of RTN3 plasmids were determined as 2 µg. In vivo-jetPEI  was prepared according to the manufacturer’s instructions and used to deliver 2 µg of RTN3 (PEI-RTN3) or EGFP (PEI-EGFP) plasmids were used to transfects DRGN and retinal cells. Additional controls included untreated DRGN/retinal cells or PEI alone. DRGN and retinal cells were allowed to incubate for 3 days prior to harvesting of cells and extraction of total protein/RNA for validation of RTN3 protein/mRNA overexpression.n = 9 wells/condition). For siRNA transfection, a preliminary dose determination experiment was run and 5 nM siRTN3 delivered using Lipofectamine  was used to determine optimal knockdown of RTN3 mRNA/protein in DRGN/RGC cultures, as described previously . The effn = 6–9 wells/treatment) was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen). Appropriate primers were used to validate each gene of interest using the specific primers , as described previously [Total protein was extracted from L4/L5 DRG, DRGN cultures, retinal cells and retinae after lysis in ice-cold lysis buffer as appropriate and subjected to Western blot as described previously ,21. A DCn = 4 rats/group/test and each experiment repeated on at least 2–3 independent occasions , except for the electrophysiology, ladder crossing and tape sensing/removal tests where n = 6 rats/group were used and repeated on 3 independent occasions . The same rats were used for electrophysiology, ladder crossing and tape sensing/removal tests. Animals were randomly assigned to treatment groups and experimenters were masked to the treatment conditions. Sample sizes were not computed using power calculations, instead, these were based on previous similar experiments published by us ,19. Unlen = 16 DRG/group ) and immunohistochemistry studies ). For the in vivo experiments, 5 groups of animals each comprised of 4 adult female Sprague-Dawley rats were designated as: (1), uninjured intact controls (IC); (2) sham-treated controls  (3), non-regenerating DC model (DC) that received a DC lesion alone; (4), regenerating SN model (SN) that received an SN lesion alone; and (5), regenerating pre-conditioning (p) SN lesion (pSN) + DC injury model (pSN + DC-pSN lesion performed 7 days before the DC lesion). The ipsilateral L4/L5 DRG were harvested from each animal at 10 days after DC, SN and pSN + DC lesions, pooled together and RNA and protein extracted for qRT-PCR and Western blot, respectively (To stimulate the intrinsic capacity of DRGN to grow neurites in vivo (‘regeneration readiness’), rats received a unilateral pSN lesion 7 days prior to harvesting of affected L4/L5 DRG ,26. In vTo overexpress RTN3 in DRGN or retina in vivo, a CMV promoter-driven rat RTN3 plasmid was purchased from Origene, Rockville, MD, USA (Cat no. RR200211L3). A CMV driven, non-targeting, EGFP plasmid (Cat No. PS100065) was used as control. Plasmid DNA for either RTN3 or the control EGFP vector were delivered by intra-DRG injection into L4/5 DRG or intravitreal injection into the eye, respectively, condensed in in vivo JetPEI , a non-viral vector that we have shown to be as efficient at transducing neurons as efficiently as AAV8 ,27. PEI n = 18 rats/group from 3 independent repeats) received pSN lesions 7 day before DC lesion to stimulate regeneration ’readiness’. Immediately after DC injury at day 0, animals received an intra-DRG injection of in vivo-jetPEI (referred to as PEI from herein), PEI-GFP, or PEI-RTN3 and were allowed to recover for 6 weeks during which behavioural tests were performed at baseline, 2 days and then weekly at 1–6 weeks after injury [To test the potential of RTN3 to promote DC axon regeneration and improve functional recovery, groups of 6 adult Sprague-Dawley rats  also received 0.5 µL of the bi-directional tracer, FluoroRuby  #D1820; MW 10,000; ThermoFisher Scientific), injected slowly using a micropipette with a tip diameter between 30–40 µm, at a depth of 0.5 mm and 10 mm caudal to the lesion site 1 day before animal sacrifice. Rats were then intracardially perfused with 4% paraformaldehyde, cryoprotected in sucrose, embedded in optimal cutting temperature  embedding medium and longitudinal sections of the spinal cord cut at a thickness of 15 µm using a cryostat, as described in the immunohistochemistry section below. Some animals (n = 12 rats/group) received intravitreal injection of RTN3 or the control EGFP vector and were allowed to recover for 28 days after ONC. Optic nerve regeneration was then assessed using the gold standard method in the rat of GAP43 immunohistochemistry, as described later. For RGC axon regeneration, groups of 4 adult Sprague-Dawley rats , 3 independent repeats  or 6 retinae/timepoint), by qRT-PCR analysis, with cells expressing control non-targeting EGFP shRNA. DNA fragments encoding stem-loop–type shRNAs specific for human protrudin mRNA (5′-GCTGAGGTGAAGAGCTTCTTG-3′) or GFP (non-specific control) mRNA (5′ GCTGACCCTGAAGTTCATC-3′) were synthesised, attached to the U6 promoter and subcloned into pBluescriptII SK(+) vector , as described previously . The eff2). After a partial laminectomy, DC were crushed bilaterally at the level of T8 using calibrated watchmaker’s forceps [For DC and SN crush lesions, rats received subcutaneous Buprenorphine injections before anaesthesia , we used FluoroRuby (FR) bidirectional tracer to confirm regenerating axons in the DC. A micropipette with a tip diameter between 30–40 µm was used to inject 0.5 µL of 5% FR  dissolved in 0.9% saline at a depth of 0.5 mm, 10 mm caudal to the lesion site 1 day before animal sacrifice. Rats were then intracardially perfused with 4% paraformaldehyde, cryoprotected in a graded series of sucrose, embedded in optimal cutting temperature  embedding medium and longitudinal sections of the spinal cord cut at a thickness of 15 µm using a cryostat, as described in the immunohistochemistry section below. The same sections were stained for GAP43 and FR to demonstrate co-staining, as described below. 2 with constant body temperature and heart rate monitoring. Optic nerves were unilaterally crushed 2 mm from the lamina cribrosa using a calibrated watchmaker’s forceps, as described by us previously [n = 4 rats/group were used and each experiment was repeated on 3 independent occasions .After injection of rats with Buprenorphine to provide analgesia, rats were anaesthetised using 5% isoflurane in 1.8 L/min of Oeviously . In gene2 overexposure and animals were intracardially perfused with 4% formaldehyde, retinae dissected out, immersion fixed and wholemounted onto glass slides. Images were captured from three different areas of each retinal quadrant  and expressed as the number of RGC per mm2 ± S.E.M.FluorGold (FG) retrograde tracer was used to count the number of surviving RGC in retinal wholemounts as described by us previously . Briefly2 overexposure and freshly isolated L4/L5 DRG, spinal cord, retinae or optic nerves from appropriate experiments were harvested and snap-frozen in liquid N2 before RNA extraction for quantitative RT-PCR (qRT-PCR) or protein extraction for Western blot, as described later.For the microarray and Western blot studies, rats were killed at 10 days after DC lesion by COFor immunohistochemistry, animals were intracardially perfused with 4% PFA (TAAB Laboratories) in phosphate-buffered saline  and both ipsilateral and contralateral L4/L5 DRG and lesion site plus 5 mm either side of the spinal cord and retinae/optic nerves were dissected out and post-fixed for 2 h at 4 °C. All tissues were then cryoprotected in a graded series of sucrose, blocked up in optimal cutting temperature embedding compound  and sectioned through a para-sagittal plane on a cryostat set at 15 µm-thick. Sections from the entire DRG, spinal cord, retinae or optic nerves were collected onto charged slides and stored at −20 °C until required, noting the middle sections of each sample. The levels of RTN3 mRNA were validated by qRT-PCR using a pre-validated primer sequence . Levels of protrudin mRNA were analysed using pre-validated primer sequences . Q-PCR was performed on a LighCycler Q-PCR machine  following published methods . Fold ch+ staining) were diluted 1:400 in PBS and incubated for 1 hr at room temperature in a humidified chamber. Sections were then washed in several changes of PBS and coverslips mounted using Vectashield containing DAPI . Negative controls, including omission of primary antibody, were included in each run and were used to set the background threshold level for each antibody before image capture. Slides were viewed using an Axioplan 200 epi-fluorescent microscope equipped with an Axiocam HRc and run using Axiovision Software  The middle sections of each DRG, spinal cord, optic nerve or retinae were chosen for immunohistochemistry as described by us previously ,31,32,33The following antibodies were used for immunoassays: primary antibodies-rabbit polyclonal anti-RTN3 , monoclonal anti-NF200 , monoclonal anti-βIII-tubulin , mouse anti-GAP43 (#33-5000 (clone 7B10); Invitrogen; 1:400); secondary antibodies-Alexa488 anti-rabbit , Texas Red anti-mouse , Alexa488 anti-mouse .+ axons in the spinal cord were quantified by collecting all serial parasagittal 50 µm-thick sections  and counting the number of GAP43+ fibres through a dorsoventral oriented line from 4 mm rostral to 4 mm caudal to the lesion site, as described previously [+ fibres seen 4 mm above the lesion site where the DC was intact. Regenerating GAP43eviously ,34. Axon+ axons in the optic nerve were quantified from 3 sections/optic nerve  at 0.2, 0.5, 1, 1.5, 2, 2.5, 3 and 4 mm from the lesion epicenter. GAP43 in the optic nerve correlates with the number of axons detected by the anterograde tracer, Rhodamine B and thus accurately demonstrates regenerating RGC axons [Regenerating GAP43GC axons . The cro−5.5 log cd·s/m2 to 1.0 log cd·s/m2 in 0.5 log unit increments. Traces were analysed using the built-in Espion software and traces at a light intensity of −5.5 log cd·s/m2 were chosen for analysis as they produced clean, unambiguous positive scotopic thresholds (pSTR) at 100 ms after stimulus. The peak amplitude of the pSTR was recorded by an experimenter masked to the treatment conditions.Electroretinography (ERG) was performed as described by us previously . Brieflyn = 6 rats/group, 3 independent repeats ) as previously described [At 6 weeks after surgery or treatment, compound action potentials (CAP) were recorded after vehicle, RTN3 overexpression or RTN3 overexpression/Protrudin suppression ) received training to master traversing the horizontal ladder functional testing. Baseline parameters for all functional tests were established 2–3 days before injury. Animals were then randomly assigned and treatment status masked from the investigators and tested at 2 days after DC lesion + treatment and then weekly for 6 weeks. Experiments were performed by 2 observers masked to the treatment status, with animals being tested in the same order and at the same time of day. Three individual trials were performed each time for each animal for each test.Functional testing was carried out as described by us previously ,19,28,38Horizontal ladder crossing test: This tests the animals’ locomotor function and is performed on a 0.9-m-long horizontal ladder with a diameter of 15.5 cm and randomly adjusted rungs with variable gaps of 3.5–5.0 cm. The total number of steps taken to cross the ladder and the number of left rear paw slips were recorded and the mean error rate was then calculated by dividing the number of slips by the total number of steps taken.Tape removal test (sensory function): The tape removal test determines touch perception from the left hind paw. Animals were held with both hind-paws extended and the time it took for the animal to detect and remove a 15 × 15 mm piece of tape  was recorded and used to calculate the mean sensing time.p < 0.05 in SPSS . All p and n values are specified within each figure legend. No statistical tests were used to predetermine sample sizes, but our sample sizes were similar to those reported in previous publications [Data are presented as mean ± SEM. Normally distributed data were interrogated by one-way analysis of variance (ANOVA), with Dunnett’s post hoc tests, set at ications ,17,18,19glmer function and p values calculated using parametric bootstrap with pkbrtest in R package , set to between 1000 and 20,000 simulations, as described previously [lme4 and glmer functions in the R package. p values were then calculated in R using pkbrtest, with the Kenward-Roger method [For the horizontal ladder test, data were analysed by comparing lesion versus sham-treated rats, using binomial generalised linear mixed models (GLMM), with lesioned/sham  and operated/unoperated  as fixed factors, animals as a random factor and time as a continuous covariate ,19,38. Deviously ,28. For r method ,28.To examine changes in RTN3 mRNA levels, we harvested dorsal root ganglion (DRG) containing the DRG neurons (DRGN) at 10 days after (1) DC injury (non-regenerating model); (2) sciatic nerve (SN) injury (regenerating SN model); (3) preconditioning (p) SN + DC lesion  A. PreconTo understand the contribution of RTN3 to DRGN neurite outgrowth, we overexpressed RTN3 in freshly isolated primary adult DRGN using a non-viral vector, in vivo-JetPEI , which we have shown produces similar efficiencies of gene transduction as adeno-associated vectors (AAV8) in DRGN in vitro and in vivo . DissociSince preconditioning lesions (pSN + DC) stimulate significantly higher levels of RTN3 mRNA and protein in DRGN, we dissociated DRGN from animals that had received a preconditioning lesion at −7 days followed by a DC crush at day 0 and harvested DRGN 10 days later A. We theWe then determined that overexpression of RTN3 using PEI in preconditioned DRGN further upregulated RTN3 levels above that induced by preconditioning lesions alone A–D. OverRTN3 overexpression has previously been shown to result in neurite abnormalities and reduction in synaptic plasticity in transgenic mice by the aggregation of RTN3 in RTN3 immunoreactive dystrophic neurites (RINDs) ,40. RINDTaken together, these results demonstrate that RTN3 overexpression promotes DRGN neurite outgrowth in naïve DRGN cultures. DRGN exposed to preconditioning lesions grow significantly longer neurites than naïve cultures and overexpression of RTN3 in preconditioned DRGN further promotes disinhibited DRGN neurite outgrowth. Since knockdown of RTN3 suppressed DRGN neurite outgrowth, this suggests that DRGN require RTN3 for neurite outgrowth.We next sought to understand how RTN3 may signal to enhance DRGN neurite outgrowth. Since the interaction of RTN3 with protrudin and protrudin to RAB7, FYCO1 and SYST7 is important in regulating membrane protrusions resembling neurites, we overexpressed RTN3 in preconditioned (pSN + DC injured) DRGN and treated cultures with appropriate siRNAs to determine the contribution of protrudin, FYCO1, RAB7 and SYST7 to disinhibited DRGN neurite outgrowth A. OptimaAs off-target effects of siRNA transfection such as activation of interferon responses have been shown by us and others and appear to be siRNA sequence-dependent, we checked for a panel of interferon-mediated cytokines and innate immunity-related related genes in our cultures after knockdown of protrudin, FYCO1, RAB7 and SYT7 by qRT-PCR. We did not observe any activation of TNFα, IFNγ, IL-6, IL-12, IL1β, MX1, IFIT, OAS1 or Casp7 mRNA in RTN3-overexpressed DRGN. In contrast, transfection of RTN3-ver expressed DRGN cultures with a positive control sip75NTR, which we know invokes a significant interferon response , induced+ axons were seen crossing the lesion site (*) and reaching at least 6 mm (>20%) into the distal segment of the spinal cords of pSN + DC + PEI-RTN3-treated rats compared to little or no GAP43+ staining in pSN + DC + PEI-EGFP-treated control spinal cords  ablated GAP43+ regenerating axons in spinal cords, normally observed after RTN3 overexpression (pSN + DC + PEI-RTN3 + shEGFP) (To determine if RTN3 overexpression promotes DC axon regeneration in vivo and whether knockdown of Protrudin with shRNA (shProtrudin) ablates RTN3 overexpression-mediated axon regeneration over 6 weeks , we staial cords A,B. Fluo shEGFP) A,B.To determine if overexpression of RTN3 led to functional benefits, we used electrophysiology, tape sensing and removal (sensory function) and ladder crossing tests (locomotor function) ,18,19. IThese results demonstrate that overexpression of RTN3 promotes DC axon regeneration after injury with a return of significant beneficial sensory and locomotor function.+ DRGN . We observed that overexpression of RTN3 led to increased numbers of RTN3+ DRGN , whilst  galanin . This su+ RGC demonstrated that overexpression of RTN3 was significantly more neuroprotective than the control shEGFP plasmids, and as neuroprotective as the positive control, CNTF  injury leads to rapid death of retinal ganglion cells (RGCs) and axon regeneration can be promoted and easily visualised ,41,42,43ol, CNTF . These r+ RGC  in the GCL in both RTN3 and shEGFP vector control eyes  injury model, to show that RTN3 is also required for enhanced RGC neurite outgrowth in vitro and RGC axon regeneration in vivo, effects that were also dependent on protrudin. Moreover, overexpression of RTN3 was also RGC neuroprotective in vitro and after ONC in vivo.It is increasingly clear that axon regeneration fails after CNS injury including SCI and ONC due to a multitude of factors, including the low intrinsic capacity of CNS neurons to regenerate their axons, the presence of a glial scar containing axon growth inhibitory molecules and the presence of axon growth inhibitory molecules in degenerating myelin ,45,46,47However, we report here that upregulation of RTN3 correlated with both SN and pSN + DC axon regeneration and its overexpression enhanced DRGN neurite outgrowth/axon regeneration, leading to significant improvements in electrophysiological, sensory and locomotor function after DC injury in rats. We backed up these findings using a second CNS population of neurons, RGCs, which also normally fail to regenerate their axons due to the same problems as is apparent after SCI. However, significant RGC neurite outgrowth and axon regeneration were promoted by overexpression of RTN3 in vitro and in vivo, effects that were also protrudin dependent, suggesting a commonality between DRGN and RGC in their response to RTN3. In contrast to the DC injury model, RTN3 overexpression was neuroprotective for RGC both in vitro and after ONC in vivo, which was not the case for DRGN where no effects of DRGN survival were observed. This is probably explained by our observations that after ONC, significant RGC death occurs within 3 weeks after injury ,22,32,55RTN3 overexpression and subsequent improvements in DRGN neurite outgrowth were dependent on protrudin and its interacting molecules since knockdown of either protrudin, RAB7, FYCO1 or SYT7 ablated RTN3-induced DRGN neurite outgrowth. Moreover, RTN3-induced improvements in DC axon regeneration and electrophysiological, sensory and locomotor function were also ablated in vivo by suppression of protrudin. These results suggest that RTN3 works through protrudin and its interacting molecules and that suppression of any of these ablates RTN3-induced axon regeneration and subsequent functional improvements. It is possible that protrudin facilitates increased translocation of LEs after RTN3 overexpression and hence mediates enhanced axon regeneration and sprouting, bringing about enhanced functional recovery. In addition, the sprouting of preserved fibres around the lesion site could also account for some of the observed benefits of RTN3 to functional recovery.Whether this is a direct or indirect effect needs to be elucidated. However, protrudin normally forms contact sites with late endosomes (LEs) via RAB7, mediating the transfer of kinesin-1 from protrudin to the motor adaptor FYCO1 on LEs ,12,13,14The improvements in sensory and locomotor recovery after RTN3 overexpression in the spinal cord was remarkable since injured animals were indistinguishable from sham-treated animals by 4 weeks after DC injury, suggesting significant recovery. Even at 2 days after injury, there were significant improvements in sensory and locomotor function, which we are unable to explain at present since it is likely to be too soon for axonal regeneration/plasticity to account for these improvements. The improvements at 2 days after RTN3 overexpression may be due to enhanced survival of injured cells in the spinal cord or greater preservation of cord tissue architecture. This remains to be investigated. Moreover, we have not analysed any effects of RTN3 overexpression on astrocyte responses, microglia/macrophage activation status nor scarring and scar-related inhibitory molecules. It is possible that RTN3 causes changes in these parameters which then facilitates the enhanced axon regeneration and functional recovery. This is important to investigate in future experiments.In addition, the promotion of axon regeneration and functional benefits in the DC injury, we have shown that overexpression of RTN3 also promotes RGC survival, neurite outgrowth, axon regeneration and restoration of significant RGC function by electroretinography in the ONC injury model. The ONC injury model is an excellent model to evaluate neuroprotective and axon regenerative therapies since RGC soma are easily accessible after intravitreal injection. Like our observations in the DC injury model, RGC axon regeneration promoted by RTN3-overexpression was dependent on protrudin function since knockdown of protrudin ablated RTN3-induced RGC neurite outgrowth/axon regeneration and functional benefits. Moreover, RTN3 had an RGC neuroprotective effect after ONC compared to DRGN after DC injury. This is probably because RGC death occurs rapidly after ONC whilst DRGN death rarely occurs after DC injury. Hence, RTN3 probably exerts a neuroprotective effect on both systems but is only evident after ONC due to the overt death of RGC that normally occurs.This study supports the notion that overexpression of RTN3 enhances neuroprotection and CNS axon regeneration, and in particular DC and RGC axon regeneration and thus leading to improved sensory, locomotor and visual function. Our study, therefore, implies that overexpression of RTN3 may be a possible treatment for SCI and ONC injury and may restore lost function in affected patients.


Recent studies suggest potential benefits of applying L-carnitine in the treatment of cancer cachexia, but the precise mechanisms underlying these benefits remain unknown. This study was conducted to determine the mechanism by which L-carnitine reduces cancer cachexia.C2C12 cells were differentiated into myotubes by growing them in DMEM for 24 h (hrs) and then changing the media to DMEM supplemented with 2% horse serum. Differentiated myotubes were treated for 2 h with TNF-α to establish a muscle atrophy cell model. After treated with L-carnitine, protein expression of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K was determined by Western blotting. Then siRNA-Akt was used to determine that L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx. In vivo, the cancer cachexia model was established by subcutaneously transplanting CT26 cells into the left flanks of the BALB/c nude mice. After treated with L-carnitine, serum levels of IL-1, IL-6 and TNF-α, and the skeletal muscle content of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K were measured.L-carnitine increased the gastrocnemius muscle (GM) weight in the CT26-bearing cachexia mouse model and the cross-sectional fiber area of the GM and myotube diameters of C2C12 cells treated with TNF-α. Additionally, L-carnitine reduced the protein expression of MuRF1, MaFbx and FOXO3a, and increased the p-FOXO3a level in vivo and in vitro. Inhibition of Akt, upstream of FOXO3a, reversed the effects of L-carnitine on the FOXO3a/MaFbx pathway and myotube diameters, without affecting FOXO3a/MuRF-1. In addition to regulating the ubiquitination of muscle proteins, L-carnitine also increased the levels of p-p70S6K and p70S6K, which are involved in protein synthesis. Akt inhibition did not reverse the effects of L-carnitine on p70S6K and p-p70S6K. Hence, L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx and p70S6K pathways. Moreover, L-carnitine reduced the serum levels of IL-1 and IL-6, factors known to induce cancer cachexia. However, there were minimal effects on TNF-α, another inducer of cachexia, in the in vivo model.These results revealed a novel mechanism by which L-carnitine protects muscle cells and reduces inflammation related to cancer cachexia. The incidence and mortality of cancer have been increasing for several decades . Based oMuscle wasting is the core feature of cancer cachexia , which iIn atrophying muscles, there is activation of the Forkhead box O transcription factor family (FOXO), as well as NF-κB, which can be activated by TNF-α, soluble TNF-like weak inducer of apoptosis (TWEAK), or IL-1 , 16. FOXAs the etiology of cancer cachexia is multifactorial and complex, the deterioration resulting in muscle wasting can not be fully reversed by conventional nutritional support . L-carniL-carnitine was obtained from Sigma Aldrich . The anti-AKT , anti-phospho-AKT1 (Ser 473) , anti-FOXO3a , and anti-phospho-FOXO3a (Ser 253)  antibodies were purchased from Abcam Corp. . Anti-p70S6K  and anti-phospho-p70S6K (Thr 389)  were purchased from Cell Signaling Technology . Anti-MAFbx  was purchased from Everest Biotech. . Anti-MuRF1  was purchased from GeneTex . The anti-GAPDH , anti-mouse and anti-rabbit secondary antibodies (1:5000–1:1000) were purchased from ZSGB-BIO . Goat anti-mouse/rabbit IgG horseradish peroxidase-conjugated antibodies were purchased from Bio-Rad . The trypsin and BCA assay kit were purchased from Beyotime . Lipofectamine™ 2000 was purchased from Invitrogen  and OPTI-MEM Reduced Serum Medium were purchased from Gibco . The ELISA kit was purchased from Abebio .The TNF-α-induced C2C12 model is a widely used muscle cell atrophy model that simulates the muscle wasting of cancer cachexia , 11, 25.The differentiated C2C12 cells were stained with Crystal Violet Staining Solution , and images were taken using an inverted microscope . The myotube diameters (μm) were measured at ¼, ½, and ¾ along the length of a selected myotube and the average diameters were calculated. A total of 30 myotubes were measured for each group in each single experiment. Three separate experiments were performed to determine the myotube diameters.C2C12 cells were treated with 100 ng/mL TNF-α for 2 h to establish the muscle atrophy cell model. The cells were transfected with 3 μg of AKT-siRNA  in 240 μL OPTI-MEM Reduced Serum Medium for 5–7 h using a previously reported procedure. The cells were then switched to DMEM differentiation medium and treated with 1000 μg/mL of L-carnitine for an additional 24 h. Thereafter, the cells were harvested and Western blotting was performed for p-p70S6K, p70S6K, p-FOXO3a, FOXO3a, MuRF-1, MAFbx and GAPDH. The siRNA sequences used were as follows: siRNA-AKT#1 (control): GCACCTTTATTGGCTACAA; siRNA-AKT#1:CCATGAACGAGTTTGAGTA;siRNA-AKT#2:GTGCCACTATGAAGACATT.For the in vitro studies, differentiated C2C12 cells were collected at the end of experiments by cell lysis in ice-cold RIPA buffer. The lysates were subjected to SDS-PAGE according to a previously reported procedure . The PVD2 in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin  and 100 mg/ml streptomycin  [6 CT26 cells into the left flanks of male mice as described in a previous study [The mouse colon cancer cell line, CT26, which shows high tumorigenicity and low metastatic potential , was impus study  (as day L-carnitine was dissolved in 0.9% sodium chloride (NaCl). A total of 30 male BALB/c nude mice were randomly divided into four groups: a CT26 + 0.9%NaCl group , a 0.9%NaCl group , a CT26 + LC1 group  and a CT26 + LC10 group (8 mice with CT26 tumors treated with10 mg/kg bodyweight L-carnitine). All of the treatments (0.9%NaCl or different doses of L-carnitine) were administered by oral gavage daily from Day 6 (D6) to Day 24 (D24)  was calculated using the formula: 0.5 × tumor width (cm) × length (cm)The gastrocnemius muscles were fixed with 4% paraformaldehyde in 0.2 mol/LPBS, washed with PBS, and stained with hematoxylin and eosin (HE) solution. Images of muscle sections were recorded using the Image J software.The serum IL-1, IL-6 and TNF-α levels were detected by ELISA  following the manufacturer’s instructions. The blood glucose, triglyceride and total cholesterol levels were measured using a biochemical analyzer  in the clinical laboratory of our hospital.P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) for post-hoc comparisons. All tests were two-sided and P ≤ 0.05 was considered statistically significant.All data were expressed as the means ± SD and were analyzed using an analysis of variance (ANOVA) followed by Tukey’s test  siRNA to test the effects of L-carnitine on TNF-α-induced FOXO3a, p70S6K, MAFbx and MuRF1 expression in the absence of AKT. Photomicrographs of myotube cultures showed the effective inhibition of AKT by transfection with the siRNA against AKT, which made the myotube diameters smaller  confirmed that the myofibers were thinner and less ordered (evidence of wasting) in the cachexia control (CT26 + 0.9% NaCl group) compared with the normal control group , as noted in the cross-sections of muscle fibers. L-carnitine treatment ameliorated the muscle wasting, with the 10 mg/kg/d L-carnitine treatment nearly reversing the muscle fiber changes Fig. B. The crth day, with slight fluctuations continuing in the normal control group. The increase in the total body weight of the cachexic mice and mice treated with L-carnitine  group began to decline on the 12th day, and that in the 10 mg/kg/d L-carnitine (CT26 + 10 mg/kg LC) group began to decline on the 14th day  cancer and non-gastrointestinal (non-GI) cancer compared with healthy subjects . The pat19 kg/m2 . These l19 kg/m2 . PatientAlthough providing supplemental L-carnitine or increasing the CPT levels is considered to be beneficial to ameliorate cancer cachexia, the precise mechanism is not fully understood. Our present study implies that L-carnitine directly ameliorates cancer-induced cachexic muscle atrophy in vivo and in vitro, and these effects result from the improved inflammatory status, inhibition of the muscle catabolic pathways and stimulation of the anabolic pathways.Anorexia is common in cachexic cancer patients . AnorexiNumerous cell signaling pathways are involved in the initiation and progression of cancer cachexia, including the TNF-ɑ pathway, TGFβ/p38/MAPK pathway, NFκB pathway, IL-6 pathway, IGF/Akt pathway and FOXO1/FOXO3a pathway, among others . Within An increasing number of studies are showing that TNF-α and IL-6 are the primary inflammatory cytokines implicated in cancer cachexia , 9, and Cancer cachexia induces major metabolic disruptions, including alterations in lipid metabolism and glycometabolism, that contribute to the aggravation of cachexia symptoms . Our datAlthough L-carnitine could attenuate the symptoms of cancer cachexia , the preOur data support a role for FOXO in atrophy, which is consistent with the studies mentioned above , 57. MyoPI3K/AKT signaling leads to the activation of mTOR, p70S6, and various other pathways, which also promote muscle synthesis . Our stuOur current studies showed that L-carnitine efficiently ameliorated ongoing muscle atrophy in a cell cachexia model and murine cancer cachexia model. It should be noted that L-carnitine enhanced p-AKT, p-P70S6, and p-FOXO3a expression and inhibited the expression of MuRF1 and MaFbx in the gastrocnemius muscle, with major decreases in inflammatory cytokines IL-6 and IL-1.Cachexic patients usually present with rapid weight loss, extreme muscle loss, anorexia, and ultimately, multiple organ dysfunction syndromes. Our data showed that L-carnitine could ameliorate muscle wasting in in vitro and in vivo models of cachexia. L-carnitine treatment also ameliorated inflammation in the tumor-bearing mouse model, which suggests that it could be beneficial for cancer patients. Further, L-carnitine decreased skeletal muscle wasting, possibly by blocking ubiquitin-mediated protein degradation via the Akt/FOXO3/MaFbx axis. Further studies will be needed to confirm whether L-carnitine is effective in the clinical setting.