MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-β and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

Background: Intramuscular fat(IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs(miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.Results: By porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4(SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6(TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.Conclusions: MiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling

Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.

The Heat Shock Protein Story—From Taking mTORC1,2 and Heat Shock Protein Inhibitors as Therapeutic Measures for Treating Cancers to Development of Cancer Vaccines

Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.

miR-214对异丙酚麻醉大鼠术后神经元损伤的保护作用

目的 探讨miR-214对异丙酚麻醉诱导的神经元损伤的保护作用及生物学机制。方法 7日龄SD雄性大鼠随机分为4组(每组15只):生理盐水组(NS)、异丙酚麻醉开腹探查组(模型)、miR-NC组和miR-214组。除NS组外,其余组别建立异丙酚麻醉开腹探查模型。miR-NC组和miR-214组分别在麻醉前将miR-NC-agomir或miR-214-agomir注射到海马内。采用免疫荧光法检测海马mTORC1的表达,TUNEL染色检测海马组织细胞凋亡,RT-qPCR分析海马组织中miR-214表达。分别将miR-214抑制剂和mTORC1抑制剂转染入HT22海马神经元细胞,然后暴露于异丙酚。采用流式细胞术分析细胞的存活情况和Western blot分析TEFB、C-caspase3蛋白表达。结果 与NS组相比,模型组大鼠海马中miR-214明显下调(P<0.05),并且海马神经细胞凋亡显著增加(P<0.05)。与模型组相比,miR-214组海马神经元凋亡减弱(P<0.05)。生物信息学预测证明mTORC1和miR-214之间存在特异性结合位点。免疫荧光检测显示,与NS组比较,模型组诱导海马神经组织中mTORC1表达增加(P<0.05),miR-214治疗显著降低了mTORC1表达(P<0.05)。此外,与NC对照组相比,si-mTORC1转染导致暴露于异丙酚的HT22海马神经元细胞的凋亡率显著降低,并且TFEB表达显著增加(P<0.01),以及C-caspase3降低(P<0.05)。而miR-214抑制剂转染显著逆转了si-mTORC1的保护作用以及诱导的TFEB、C-caspase3蛋白表达的变化(P<0.05)。结论 miR-214可能通过mTORC1-TFEB通路减轻异丙酚神经毒性,提高神经元的存活率。

Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway

CD8＋ cytotoxic T lymphocyte （CTL） exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin （OVA）-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 （PD-1） and lymphocyte-activation gene-3 （LAG-3） and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-7 production and cytolytic effects. Naive CD8＋ T cells upregulated inhibitory PD-ligand 1 （PD-L1）, B- and T-lymphocyte attenuator and T-cell anergy-associated molecules （Grail and Itch） while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with （i） the upregulation of a marker for CTL functionality, diacetylated histone-H3 （diAcH3）, （ii） a fourfold increase in CTLs, occurring independent of host DCs or CD4＋ T cells, and （iii） the restoration of CTL IFN-7 production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, elF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo- induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus （HIV-1） infection.

L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase(GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver(P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4(SIRT4)gene expression(P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1(m TORC1) signaling(P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1(S6 K1) and ribosomal protein S6(S6). Interestingly, the leucine-induced m TORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4.Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes(P > 0.05), while m TORC1 signaling was activated.Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes(P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating m TORC1/SIRT4 pathway in the liver of pigs.

Extracellular vesicles released by transforming growth factor-beta 1-preconditional mesenchymal stem cells promote recovery in mice with spinal cord injury

Spinal cord injury(SCI)causes neuroinflammation,neuronal death,and severe axonal connections.Alleviating neuroinflammation,protecting residual cells and promoting neuronal regeneration via endogenous neural stem cells(eNSCs)represent potential strategies for SCI treatment.Extracellular vesicles(EVs)released by mesenchymal stem cells have emerged as pathological mediators and alternatives to cell-based therapies following SCI.In the present study,EVs isolated from untreated(control,C-EVs)and TGF-β1-treated(T-EVs)mesenchymal stem cells were injected into SCI mice to compare the therapeutic effects and explore the underlying mechanisms.Our study demonstrated for the first time that the application of T-EVs markedly enhanced the proliferation and antiapoptotic ability of NSCs in vitro.The infusion of T-EVs into SCI mice increased the shift from the M1 to M2 polarization of reactive microglia,alleviated neuroinflammation,and enhanced the neuroprotection of residual cells during the acute phase.Moreover,T-EVs increased the number of eNSCs around the epicenter.Consequently,T-EVs further promoted neurite outgrowth,increased axonal regrowth and remyelination,and facilitated locomotor recovery in the chronic stage.Furthermore,the use of T-EVs in Rictor/SCI mice(conditional knockout of Rictor in NSCs)showed that T-EVs failed to increase the activation of eNSCs and improve neurogenesis sufficiently,which suggested that T-EVs might induce the activation of eNSCs by targeting the mTORC2/Rictor pathway.Taken together,our findings indicate the prominent role of T-EVs in the treatment of SCI,and the therapeutic efficacy of T-EVs for SCI treatment might be optimized by enhancing the activation of eNSCs via the mTORC2/Rictor signaling pathway.

Regulation of mTORC1 by amino acids in mammalian cells: A general picture of recent advances

The mechanistic target of rapamycin complex 1(mTORC1)integrates various types of signal inputs,such as energy,growth factors,and amino acids to regulate cell growth and proliferation mainly through the 2 direct downstream targets,eukaryotic translation initiation factor 4 E-binding protein 1(4 EBP1)and ribosomal protein S6 kinase 1(S6 K1).Most of the signal arms upstream of mTORCl including energy status,stress signals,and growth factors converge on the tuberous sclerosis complex(TSC)-Ras homologue enriched in brain(Rheb)axis.Amino acids,however,are distinct from other signals and modulate mTORCl using a unique pathway.In recent years,the transmission mechanism of amino acid signals upstream of mTORCl has been gradually elucidated,and some sensors or signal transmission pathways for individual amino acids have also been discovered.With the help of these findings,we propose a general picture of recent advances,which demonstrates that various amino acids from lysosomes,cytoplasm,and Golgi are sensed by their respective sensors.These signals converge on mTORCl and form a huge and complicated signal network with multiple synergies,antagonisms,and feedback mechanisms.

Protein Supplementation for the Prevention and Management of Sarcopenia in the Elderly

Sarcopenia is common in patients with many physiological or pathological conditions, especially in aging people. Nutrition plays an important role in the prevention and treatment of sarcopenia. Sarcopenia is often related to insufficient protein intake in the elderly. Muscle protein synthesis occurs mainly through mTORC1 pathway, and degradation occurs by ubiquitination-mediated pathways. This review summarizes the growing body of evidence, including substantial clinical trials, which increasing the protein intake can serve as the basis for preventing and managing muscle loss in patients with sarcopenia. Supplementation of essential amino acids (EAA), branched chain amino acids (BCAA), and especially leucine-rich whey protein may promote muscle protein synthesis by activating the mTORC1 signaling pathway, and may inhibit protein degradation by decreasing ubiquitin-mediated degradation. Taking in sufficient energy and protein and engaging in active exercise are the main methods of stimulating muscle protein synthesis and preventing or managing sarcopenia. Therefore, it is necessary to strengthen research on the use of protein supplements for not only elderly patients, but also those with tumor cachexia and other diseases related to sarcopenia.

Cu2+抑制小胶质细胞内自噬-溶酶体途径介导的Aβ寡聚体清除

目的探讨Cu2+对小胶质细胞(MG)内自噬-溶酶体途径介导的Aβ寡聚体(Aβo)清除功能的调控作用及其机制。方法应用ELISA检测MG对Aβo的摄取和细胞内降解;应用Western蛋白印迹法及免疫荧光技术检测Cu2+刺激下MG内自噬相关蛋白及溶酶体相关蛋白的表达变化;应用单荧光和双荧光质粒转染实验检测自噬体和自噬流变化;应用Lyso Tracker red荧光探针检测溶酶体体积改变。结果 (1) Cu2+处理BV-2细胞后可导致Aβo清除(包括摄取和细胞内降解)障碍。(2)并引起自噬标志性蛋白LC3-Ⅱ和自噬底物P62蛋白水平升高;GFP-LC3转染结果显示,Cu2+刺激后自噬体数量增加。(3)溶酶体酸化抑制剂bafilomycin A1处理后,LC3-Ⅱ蛋白水平较赋形剂处理组明显升高,但bafilomycin A1+Cu2+刺激未导致LC3-Ⅱ蛋白水平的进一步升高;自噬体形成抑制剂3-MA预处理后,Cu2+仍可以增强LC3-II水平;提示Cu2+可能是通过抑制溶酶体降解过程导致LC3-Ⅱ蛋白水平升高,即抑制自噬降解过程。(4) RFP-GFP-LC3质粒转染结果显示,Cu2+可导致自噬流障碍。(5) Cu2+可导致溶酶体LAMP1和总Cat B(包括pro Cat B和mature Cat B)表达下降,并降低溶酶体体积。(6) Cu2+处理后,转录因子TFEB蛋白水平及其溶酶体相关靶基因的表达均有下调;(7) Cu2+可增强m TOR及其底物蛋白p70S6k磷酸化水平,提示m TORC1可能参与了Cu2+调控自噬及溶酶体功能的作用;(8)应用m TORC1抑制剂PP242预处理,可显著改善Cu2+导致的LC3-Ⅱ和P62蛋白水平升高及自噬流障碍,TFEB及其靶基因表达下调,溶酶体生物合成及体积下降,以及Aβo清除障碍。结论Cu2+可能通过m TORC1-TFEB通路抑制性调控MG自噬-溶酶体途径介导的Aβo清除;m TORC1抑制剂PP242可改善Cu2+作用。