Background: Acute lymphoblastic leukemia (ALL) is the main type of cancer in children. In Mexico and other Hispanic populations, the incidence of this neoplasm is one of the highest reported worldwide. Functional polymorphisms of various enzymes involved in the metabolism of xenobiotics have been associated with an increased risk of developing ALL, and the risk is different by ethnicity. The aims of the present study were to identify whether NQO1, CYP2E1, and NAT2 polymorphisms or some genotype-environmental interactions were associated with ALL risk in Mexican children.Methods: We conducted a case-control study including 478 pediatric patients diagnosed with ALL and 284 controls (children without leukemia). Ancestry composition of a subset of cases and controls was assessed using 32 ancestry informative markers. Genetic-environmental interactions for the exposure to hydrocarbons were assessed by logistic regression analysis.Results: The polymorphisms rs1801280 , rs1799929 , and rs1208  were found to increase the risk of ALL; being the risks higher under a recessive model . Gene-environment interaction analysis showed that NAT2 rs1799929 TT genotype confers high risk to ALL under exposure to fertilizers, insecticides, hydrocarbon derivatives, and parental tobacco smoking. No associations among NQO1, CYP2E1, and ALL were observed.Conclusion: Our study provides evidence for the association between NAT2 polymorphisms/gene-environment interactions, and the risk of childhood ALL in Mexican children. Acute lymphoblastic leukemia (ALL) represents the most frequent type of cancer in pediatric population worldwide. Additionally, Mexico and other Hispanic populations have one of the highest incidence and mortality rates for this cancer around the globe , 8. The Hydrocarbons, parental smoking, alcohol drinking, pesticides, and air pollution have been reported as the main type of xenobiotics related with an increased risk of developing ALL in children –16. AfteCytochrome P450 Family 2 Subfamily E Member 1 (CYP2E1) are phase I enzymes, whereas, NAD(P)H quinone oxidoreductase 1 (NQO1) and Arylamine N-acetyltransferase 2 (NAT2) are phase II enzymes, which are commonly implicated in xenobiotics metabolism . In addiNQO1, CYP2E1, and NAT2 polymorphisms, or some genotype-environmental interactions, were associated with ALL risk in Mexican children. As well, to provide a picture of the genetic ancestry and admixture patterns of the patients with ALL from central Mexico.The aims of the present study were to identify whether A case-control study was conducted by the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia (MIGICCL), from August 1, 2014, to July 31, 2016. Cases and controls were younger than 17 years, residents of the Metropolitan Area of Mexico City.Cases were recruited from eight public hospitals of Mexico City in which an estimated 69.9% of children with leukemia who reside in Mexico City are attended. Acute lymphoblastic leukemia diagnosis was established according to clinical features, and bone marrow aspirate findings: cell morphology, immunophenotype, and genetics, as defined by the 2008 WHO classification of lymphoid neoplasms Children with Down syndrome were excluded from the analysis. Case registration required that trained personnel were assigned to each participating hospital to identify incident cases of leukemia through reviews of clinical charts. Afterwards, parents were approached and invited to participate.NQO1, CYP2E1, and NAT2 polymorphisms studied here on ALL survival. Those cases were also included in the present work for increasing the precision of the assessed interactions.The participating public hospitals and health institutions were: (1) Hospital de Pediatría, “Dr. Silvestre Frenk Freund” Centro Médico Nacional (CMN) “Siglo XXI”, Instituto Mexicano del Seguro Social (IMSS); (2) Hospital Infantil de México Federico Gómez, Secretaría de Salud (SS); (3) Hospital General “Dr. Gaudencio González Garza”, CMN “La Raza”, IMSS; (4) Hospital General Regional “Dr. Carlos McGregor Sánchez Navarro”, IMSS; (5) Hospital Juárez de México, SS; (6) Hospital Pediátrico de Moctezuma, Secretaria de Salud de la Ciudad de México (SSCDMX); (7) Hospital General de México, SS; and (8) Hospital CMN “20 de Noviembre”, Instituto de Seguridad Social al Servicio de los Trabajadores del Estado (ISSSTE). For this study the sample size was calculated with 250 cases and 250 controls, this being sufficient for obtaining a power of 80% to identify the associations. In parallel to this study the inclusion of cases continued for assessing the prognostic value of the The controls were selected from second-level hospitals of the same health institution  that referred the children with ALL to the third-level care hospitals. The controls were children without leukemia who were treated at different hospital departments, such as ambulatory surgery, pediatrics, ophthalmology and orthopedic outpatient clinics, and the emergency room. Children with diagnoses of neoplasms, hematological diseases, allergies, infections, and congenital malformations were not selected as controls. The interviewers were provided with a list of characteristics of individual leukemia cases, and charged with identifying one control per case according to sex, age (±18 months), and health institution. When more than one control per case met the matching criteria, the control that was closest in age to the case was selected. If the controls were of the same age, one control was selected randomly (by tossing a coin). Moreover, if no control of the same sex was found after three visits to the same hospital, a control matched by age (±18 months) was selected.Trained personnel were assigned to each of the second-level hospitals to search for controls who fulfilled the selection criteria. When a control was identified, parents were invited to participate.The Ethics and National Committee of Scientific Research approved this study with number R-2013-785-062. Additionally, we obtained approval by the Institutional Scientific Review Committees of each health institution to carry out the study.The interview was carried out using the same questionnaire for cases and controls. This questionnaire was previously standardized and adapted from the questionnaire module of the National Cancer Institute. General information obtained from the interviews included the child's history, socioeconomic status (SES), demographic characteristics, and the parents information .Study variables were child's sex, age, and birthweight , family history of cancer, maternal years of education, after birth home exposure to fertilizers, insecticides, and other hydrocarbon derivatives , and parental tobacco smoking.For the exposure of the parents to tobacco smoking, the information was obtained from the in-person interview and it was assessed in the four exposure periods: (1) pre-conceptionally, (2) during pregnancy, (3) during lactation, and (4) the last year before leukemia diagnosis or interview in cases and controls. The number of cigarettes smoked during each exposure period was calculated. Parental smoking was classified into three groups: (1) when both parents were lifelong non-smokers or ex-smokers, who stopped smoking more than 1 year before the birth of the index child, (2) when either parent smoked <5 cigarettes a day, and (3) when at least one parent smoked 6 or more cigarettes daily. Passive smoking in the child was considered when at least one of the parents smoked in the presence of the child for at least three times a week during the year before diagnosis/interview in cases and controls, respectively.Parental years of education were used as an indicator of SES as it has been previously used by the Childhood Leukemia International Consortium (CLIC) .Genomic DNA from saliva or peripheral blood was obtained according to the ORAGENE Purification Kit  and the Gentra Kit  manufacturer's instructions, respectively. DNA purity and concentration were determined by sypectrofotometry (Nanodrop-1000).NQ01), rs3813867 (CYPE2) and rs1041983 (C282T), rs1801280 (T341C), rs1799929 (C481T), rs1799930 (G590A), rs1208 (A803G), and rs1799931 (G857A) from NAT2 SNPS was performed using the 5′exonuclease technique and TaqMan MGB chemistry in a QuantStudio 5 system according to the manufacturer's instructions . TaqMan probes used were C___2091255_30 (rs1800566), C___2431875_10 (rs3813867), C___8684085_20 (rs3813867), C___1204093_20 (rs1801280), C___1204092_20 (rs1799929), C___1204091_10 (rs1799930), C____572769_20 (rs1208). Each polymerase chain reaction (PCR) reaction contained 25 ng of genomic DNA, 2.5 ul of TaqMan master mix, 0.0625 ul of 40X assay mix, and ddH2O up to a final volume of 5 ul. The PCR protocol included denaturing at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s and annealing and extension at 60°C for 1 min. The genotype of each sample was assigned automatically by measuring the allele-specific fluorescence using QuantStudio Desing and Analysis software 5 for allelic discrimination . The overall genotype call rate was 99.8%, and a subset of randomized samples were genotyped in duplicate and showed 100% reproducibility.Genotyping of rs1800566  as ancestry informative markers (AIMs). The SNPs chosen for inclusion were based on AIMs sets' that has been validated in a large group of Mexican subjects. This panel included 1 to 2 SNPs in all chromosomes, with the exception of chromosome 18 . ALL casNQO1 (rs1800566), CYP2E1 (rs3813867), and NAT2  were selected based on previous results in leukemia where they were the most consistently associated with leukemia risk (http://ihg.gsf.de/cgicbin/hw/hwa1.pl). Linkage disequilibrium (LD) and haplotyping of NAT2 SNPs were assessed by using the haploview software , rs1801280 (T341C), rs1799929 (C481T), rs1799930 (G590A), rs1208 (A803G), and rs1799931], named NAT2*4, were classified as a rapid acetylator phenotype. Haplotypes containing more than one mutant allele were identified as slow acetylators and the remainder as an intermediate acetylator phenotype, using the NAT2PRED web-server   loci and environmental factors to identify combinations of both factors that are associated with the risk of a disease . Descriptive analyses were conducted. Odds ratio (OR) and 95% confidence intervals (95% CI) were estimated by unconditional logistic regression. The logistic regression model was constructed as follows: (1) the overall interaction in the model was evaluated, (2) it was assessed whether study variables had a global confounding effect, those variables that, in the stratified analysis the ORs prior to (crude OR) and after stratifying (aOR) showed a difference between the two ORs >10%. These variables alongside with the matching variables , were included in the multivariate analyses. As a result, the most parsimonious model included: child's sex, age, paternal education level, maternal education level, maternal age at pregnancy, active smoking of the mother after birth, alcoholism of the mother before pregnancy, family cancer history, health institution, birthweight, maternal exposure to hydrocarbons at home during pregnancy, X-rays exposure during pregnancy, alcoholism of the father before pregnancy, and active smoking of the father after birth. Accordingly, the adjusted ORs (aOR) were calculated.http://ihg.gsf.de/cgicbin/hw/hwa1.pl). Alleles and genotypes frequencies were compared among groups by using chi-square and Fisher's exact test, when appropriate, which are implemented in the STATCALC program . The level of significance was set at 5%. To evaluate ancestry composition of our sample, STRUCTURE software was used assuming two populations (European and NA), and each analysis was performed at least three times using >100,000 replicates and 20,000 burn-in cycles under admixed model. To determine the statistical power of our study, we used Quanto software (http://hydra.usc.edu/gxe) accounting minor allele frequency (MAF) of all SNPs in the control the group; likewise, considering a recessive genetic model, and odd ratio (OR) of 2.0, the prevalence of ALL in Mexican children, and the sample size.Hardy-Weinberg Equilibrium (HWE) test was performed using the FINETTI program  and rs1799931 (G857A), the remaining SNPs were found in HWE in the control population. The association analysis between individual SNPs and ALL are described in CYP2E1 (rs3813867) and NQO1 (rs1800566) SNPs were not associated with ALL   and rs1789929 (C481T) ; as well, under recessive model for the rs1208 (A803G) . with ALL .NQO1 (rs1800566), CYP2E1 (rs3813867), and NAT2  and the exposure variables were noted (NAT2 SNPs rs1041983 (C282T) and rs1799931 (G857A), respectively, were associated with a reduced risk of ALL under exposure to hydrocarbons by the mother at home before and during pregnancy and after birth (HEMAB), drug consumption, active smoking, alcohol consumption and insecticide exposure by the mother before pregnancy, whilst being pregnant and while child feeding. As well as active smoking and alcohol drinking by father before and during pregnancy. NAT2 variants rs1799929 and rs1208 variants, which were associated with ALL risk, exhibited an increased ORs under maternal hydrocarbons exposure and drug consumption. In the same way, active smoking, and alcohol consumption by the parents during pregnancy incremented the risk to ALL in homozygotes cases for mutant alleles to both SNPs. In fact, the rs1799929 (C481T) and active smoking by the father after birth displayed the highest OR  to ALL observed in the present analysis. The rs1799930 (NAT2), rs1800566 (NQO1), and rs3813867 (CYP2E1) were not associated neither after adjusting by these variables  was selected  (To investigate for interactions among selected . The rs1selected  and cont5–10.71) , Table 4NAT2-rs1799929 had the larger effect on the susceptibility to develop ALL (3.0%). A synergistic interaction (read lines) between NAT2-rs104183-rs1799931 and NAT2-rs1799929-rs1801280 SNPs, which reveals an epistasis effect between them, was observed. Redundancy (green lines) was observed between several NAT2 and CYP2E1 SNPs which means that jointly those SNPs provide less information than studying one SNP. Gold lines showed independence among SNPs  and controls (n = 167) who met the selection criteria during the study period was evaluated. A total of 32 AIMs, previously validated in a Mexican population were used -medicine consumption by the mother before pregnancy (MCMBP)-PSFBC suggesting that these factors jointly contributed to the etiology of ALL  and during pregnancy (PSFDP) was the best factor with statistical significance . The multifactor model with a minimum prediction error (TBA 0.7648) and CVC (10/10) was y of ALL , Table 5NAT2_rs1799929 as the best one factor (TBA 6021 CVC 10/10) and the six-factor interaction model NAT2_rs1041983, NAT2_rs1799929, NAT2_rs1799931, NQ01_rs1800566-insecticide exposition after child's birth (IEAB)-active smoking by the father after child's birth (ASFAB) as the best model for ALL with a TBA of 0.6062 and CVC 9/10  and after child's birth (4.21%) , ACMBP (9.88), and MCMBP (9.65%) have the larger effect on susceptibility to ALL before pregnancy , whilst  (4.21%) .Acute lymphoblastic leukemia, the most prevalent cancer in children, like other hematologic malignancies is likely to develop from a complex interaction between genetic and environmental factors. Hydrocarbons, such as benzene, pesticides, and air pollutants, are among the common xenobiotics that have been implicated in ALL etiology , 15. DetNQO1, CYP2E1A, and NAT2 polymorphisms and their interaction with environmental factors, such as home exposure to fertilizers, insecticides, hydrocarbon derivatives , and parental tobacco smoking and wood smoking, among other variables in the risk of developing childhood ALL. In the present study, we found that NAT2 polymorphisms, but not NQO1 and CYP2E1 polymorphisms were associated with ALL risk. NQ01 modifies internal exposure to bioactivated carcinogens. NQ01 has been previously described as an anticancer enzyme. There is a documented a relation between NQ01 (rs1800566) with several types of cancer, including infant leukemia , rs1799929 (C481T), rs1799930, rs1208 (A803G), and rs1799931 (G857A) defined the low acetylator phenotype  and rapid CTTGAG  and CTTGGG  haplotypes, which were rare in the healthy subjects (<1%), were associated with higher risk of ALL. These results seem paradoxical; however, we predicted the acetylation phenotype according to the results of the NAT2 haplotypes and potential haplotype misclassification could exist  and rs1799931 (G857A) confer protection to ALL under parents exposition to diverse xenobiotics . To our knowledge, no previous studies have explored the relationship between controls .NQO1, CYP2E1, and NAT2 are enzymes involved in the metabolism of xenobiotics , which increase the risk to develop diverse human diseases , CYP2E1 (rs3813867), and NAT2  polymorphisms and exposure to common environmental hydrocarbons. In This is a study that included patients from 8 public hospitals in Mexico City, what represents slightly <70% of all cases with leukemia in this city . It is aOn the other hand, with respect to the variables included in this study to assess the exposure to hydrocarbons, we used a similar strategy reproduced in previous studies where the association between the exposure to hydrocarbons and the risk of developing childhood leukemia has been analyzed –65. ThisNQ01 and CYP2E1 genes, thus we cannot discard the association among these genes and ALL or the interaction among SNPs in these genes and environmental xenobiotics. On the other hand, subjects were grouped into three different NAT2 acetylator phenotypes based only on six NAT2 SNPs . Considering that we did not study the rs1801279 (191G>A), the assessment of only these six SNPs could result in a misclassification of some NAT2 alleles (NAT2 have been identified and there is there is phenotype heterogeneity within the slow and intermediate acetylator genotype groups, due to variation in enzyme activity conferred by different alleles (NAT2 association findings among studies could be explained by factors, such as sample size, age group, genotyping method, and the time of exposure to risk agents (There were some limitations in the present study. On one hand, we tested only one SNP of  alleles . In addi alleles . Low gen alleles , 67. Othk agents , 69.NAT2, NQO1, and CYP2E1 on the risk of childhood ALL in the Mexican population, and the first report of ancestry background in Mexican children with ALL. Our study provides evidence that polymorphisms of NAT2 might be genetic factors involved in childhood ALL. These results shed light on the contribution of NAT2 polymorphisms to increase the risk of developing ALL in children.To the best of our knowledge, this is the first assessment of the interaction between hydrocarbon exposure and genetic polymorphisms of The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics and National Committee of Scientific Research of the Instituto Mexicano del Seguro Social approved this study with number R-2013-785-062. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.AM-S, JM-A, and SJ-M: conceptualization. AM-S, JN-E, JCF-L, JM-A, and SJ-M: methodology. JN-E, JCF-L, JM-A, and SJ-M: formal analysis. AM-S, JN-E, and SJ-M: investigation. AM-S, EH-C, MP-S, AM-G, EJ-H, JM-T, HP-L, JF-L, RA-S, FM-R, JGP-G, DD-R, JT-N, JF-B, RE-E, PR-Z, LF-V, JG-U, SM-S, GE-A, CA-H, RR-C, LH-M, LG-L, GC-O, AG-E, IC-H, AM-H, ML-C, NH-P, JG-K, MR-V, DT-V, CC-R, FM-L, JAP-G, AM-R, AA-D, BS-D, VB-M, MM-R, OS-R, JR-B, HR-V, and AH-M: resources. AM-S and SJ-M: writing—original draft preparation. SJ-M and JM-A: writing—review and editing, supervision, and funding acquisition. All authors reviewed the final manuscript, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated primarily from endogenous biochemical reactions in mitochondria, endoplasmic reticulum (ER), and peroxisomes. Typically, ROS/RNS correlate with oxidative damage and cell death; however, free radicals are also crucial for normal cellular functions, including supporting neuronal homeostasis. ROS/RNS levels influence and are influenced by antioxidant systems, including the catabolic autophagy pathways. Autophagy is an intracellular lysosomal degradation process by which invasive, damaged, or redundant cytoplasmic components, including microorganisms and defunct organelles, are removed to maintain cellular homeostasis. This process is particularly important in neurons that are required to cope with prolonged and sustained operational stress. Consequently, autophagy is a primary line of protection against neurodegenerative diseases. Parkinson's is caused by the loss of midbrain dopaminergic neurons (mDANs), resulting in progressive disruption of the nigrostriatal pathway, leading to motor, behavioural, and cognitive impairments. Mitochondrial dysfunction, with associated increases in oxidative stress, and declining proteostasis control, are key contributors during mDAN demise in Parkinson's. In this review, we analyse the crosstalk between autophagy and redoxtasis, including the molecular mechanisms involved and the detrimental effect of an imbalance in the pathogenesis of Parkinson's. Eukaryotic cells employ a variety of catabolic pathways to degrade altered/damaged proteins and redundant macromolecular components . These pathways are critical for cellular homeostasis, and alterations in any have been linked to diverse human diseases –4. Autopα-synuclein (α-syn) and tau are hallmarks, respectively, of Parkinson's and tauopathies [There are three types of autophagy, each with a distinct mechanism for delivery of substrates to the lysosome. These are microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, the cargo is directly engulfed into lysosomes through lysosomal invaginations or protrusions . CMA is opathies .Macroautophagy is the best understood of the three autophagy forms. It is commonly referred to simply as “autophagy,” and we will adopt the same nomenclature herein. Defects in autophagy are common hallmarks of human diseases, including neurodegenerative diseases . During β [α-syn [Although autophagy occurs in cells under basal conditions, it is dramatically upregulated in response to stresses including starvation, oxidative stress, and pathogen infection . Crucialβ , 40, thaβ , the cauβ [α-syn , a majorβ [α-syn –23, 43.The process of autophagy consists of several sequential steps: (i) initiation and nucleation; (ii) elongation; (iii) maturation; and (iv) fusion with the endolysosomal compartment. In mammalian cells, autophagy initiation involves the recruitment of several complexes to the autophagy initiation sites and the formation of the phagophore . Upon autophagy induction , the Unc51-like kinase 1 (ULK1) complex—formed by the catalytic subunit ULK1, the regulatory subunit ATG13, ATG101, and focal adhesion kinase family interacting protein of 200 kDa (FIP200)—is activated . ULK1 ac113 versus Y113 [At the omegasome, PI3P effector proteins are recruited, including the zinc-finger FYVE domain-containing protein (DFCP1), the autophagy-linked FYVE protein (ALFY), and WD repeat domain phosphoinositide-interacting proteins  . DFCP1 rsus Y113 ), LC3C, sus Y113 . Prior tsus Y113 , 22, 69.sus Y113 , 71, a sAutophagosome maturation and fusion with lysosomes involves (i) membrane fission for autophagosome closure; (ii) trafficking of the autophagosome along the cytoskeleton, typically in the retrograde direction ; and finally (iii), fusion with the lysosome to form a degradative autolysosome . Here, cFully formed autophagosomes go through a maturation process during which ATG8 proteins link autophagosomes to motor proteins and the microtubule cytoskeleton. For example, RAB7 is recruited to mature autophagosomes and further recruits the FYVE and coiled-coil domain-containing 1 protein (FYCO1), which in turn binds to LC3 (via LIR- (LC3-interacting region-) type interaction) and PI3P to mediate anterograde kinesin-driven transport . Alternaα- (PKCα-) mediated ULK1 phosphorylation reducing this interaction via ULK1 degradation by the CMA pathway [For membrane fusion and formation of the autolysosome, the SNARE  fusion machinery is required. On the autophagosome membrane resides STX17 and synaptosomal-associated protein 29 (SNAP29), whereas on the lysosome, vesicle-associated membrane protein 7 or 8 (VAMP7 or VAMP8) mediates membrane fusion supported by the homotypic fusion and protein sorting complex (HOPS) which interacts with STX17 , 85. STX pathway .α-syn) and damaged organelles that are selected by ubiquitylation and recognised by the autophagy machinery [Atg7 knockout mice, α-syn and p62/SQSTM1 both accumulate in inclusions within neurites in an ageing-dependent fashion that is ultimately linked to mDAN loss and motor dysfunction [Autophagy pathways in general are especially important in neurons, as these are postmitotic cells that cannot dilute cytoplasmic damage through proliferation/division, and thus autophagy is required to maintain long-term neuronal functionality. Although this review focuses on (macro)autophagy, it is important to mention that CMA and microautophagy are also present in neurons . In neurachinery , 93. Misachinery , otherwiachinery , 96. Theachinery . In autoAtg5 knockout mice is rescued after restoration of neuronal-specific expression of ATG5 [Autophagy also contributes to axonal regeneration, presynaptic modelling, dendritic spine pruning, and synaptic plasticity , 97–100. of ATG5 ; meanwhi of ATG5 , and aut of ATG5 . Consist of ATG5 , 107–109Previous work in primary rodent neurons points to unique characteristics of autophagosome assembly, maturation, and trafficking in these specialised cells , 110. ImSYNJ1 are associated with Parkinson's [Autophagosome biogenesis is a constitutive process that can be triggered in the soma or distal axon where elements of the core autophagy machinery are actively recruited . Supplekinson's ), are prkinson's , but havkinson's  and to pkinson's , respectkinson's . Autophakinson's , 121. Fukinson's , 122.Neuronal autophagy properties also appear to vary as a function of ageing, with accumulation of neuromelanin and lipofuscin progressively observed in autophagosomes in aging brain tissues . OverallAs a key component of cellular and tissue homeostasis, with protective roles in human neurodegenerative diseases, a full appreciation of mitophagy regulatory control in neurons is desirable. In particular, mitophagy dysfunction is a hallmark of Parkinson's, implicated in a number of early onset genetic forms , and is PINK1 and PRKN mutations are linked to familial Parkinson's [The best characterised route for mitochondrial degradation is via the PINK1/Parkin (PRKN) pathway, although several Parkin-independent pathways have been described –135. Crukinson's . When mikinson's , 136. Hokinson's . Parkin kinson's . For Parkinson's . These ukinson's .There are several cargo receptor proteins involved in mitophagy, including p62/SQSTM1, NIX (or BNIPL3), Neurabin-1 (NRB1), FUNDC1 (FUN14-domain-containing 1), NDP52, Optineurin (OPTN), and Tax1 binding protein 1 (TAX1BP1) . RecruitSome cargo receptors involved in mitophagy—including NDP52, OPTN, NRB1, and p62/SQSTM1—also facilitate the degradation of protein aggregates (aggrephagy), or other organelles such as peroxisomes (pexophagy) , 148. Peα (IRE1α), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These cascades trigger the translational and transcriptional regulation of redox enzymes, chaperones, foldases, lipid synthesis proteins, autophagy-related proteins , FAM134B (family with sequence similarity 134 member B); ATL3 (atlastin 3), SEC62 (secretory 62 homolog), CALCOCO1 , RTN3 (reticulon 3), and TEX264 (testis-expressed protein 264); they are found in different regions of the ER, and they might have different roles and be tissue-specific –160. The., CCPG1 ), and ER., CCPG1 .α indirectly activates BECLIN1, thus promoting autophagy initiation, while PERK and ATF6, respectively, activate two autophagy transcription factors, ATF4 (activating transcription factor 4) and CHOP (C/EBP homologous protein) [Under nutrient starvation or ER stress , ER-phagy is induced via different pathways after UPR activation. Calcium released via the inositol trisphosphate receptor IP3R and other calcium channels activates calcium-dependent proteins, namely, calmodulin-dependent protein kinase (CAMKK), which inhibits mTORC1 , 165; deprotein) , 171 (foprotein) ). ER-phaprotein) , 174). Rprotein) , and recprotein) ).RTN3 is linked to AD [In neurons, the ER extends from the cell body and along the axon to the axonal distal tip. It is crucial for neuronal function , and the ER tubular network is disrupted in several neurodegenerative diseases –178. Coned to AD ; and mosed to AD . However2O2) and superoxide (O2•-)) and reactive nitrogen species (RNS) ) are highly reactive molecules generated under both basal and pathological or stress conditions  . They acals see . These rcals see , 188. Incals see . For thi2) generated from succinate in the citric acid cycle and consequently delivers electrons to the ETC (ubiquinone); (iii) Complex III (cytochrome c oxidoreductase), where electrons (from ubiquinone) are transferred to cytochrome c; (iv) Complex IV (cytochrome c oxidase), where electrons are removed from cytochrome c to generate H2O with energy released used to translocate protons to the intermembrane space; and (v) Complex V (ATP synthase), for the generation of ATP via proton flow to the matrix (at a ratio of 4H+ : 1ATP) [2•- due to electron leakage [2 while transferring electrons to ubiquinone in the ETC, generates additional O2•-. Similarly, also in the inner mitochondrial membrane, the electron transfer to flavoprotein, ubiquinone oxidoreductase, and dihydroorotate dehydrogenase, that, respectively, links fatty acid β-oxidation and pyrimidine biosynthesis to electron transfer to the ETC, also generates O2•- [2 to be used in the ETC. Particularly, dihydrolipoamide dehydrogenase (DLD), an E3 component of pyruvate dehydrogenase (for the production of acetyl-CoA from pyruvate), and α-ketoglutarate dehydrogenase , generates unwanted O2•- via the flavin cofactor of this enzyme. Finally, superoxide dismutase 2 (SOD2) in the mitochondrial matrix, and SOD1 in the intermembrane space, convert O2•- into H2O2, which can potentially be turned into •OH radicals via the Fenton reaction. Mitochondria are also sources of RNS, including •NO that is produced by nitric oxide synthases (NOS) in the oxidation of L-arginine [β-oxidation, lipid synthesis, purine catabolism, and amino acid and glyoxylate metabolism. Importantly, most enzymes involved in these processes produce ROS. For example, acyl-coA oxidase(s), which catalyse the first step in peroxisomal fatty acid β-oxidation, generates H2O2 [2•- and H2O2 [2O2. Other peroxisomal enzymes producing ROS/RNS include L-α-hydroxyacid oxidase (involved in oxidation of glycolic acid), polyamine oxidase (involved in polyamine degradation), sarcosine oxidase , D-aspartate oxidase , SOD1, and NOS2 [Mitochondria are the major source of cellular ATP, generated via the electron transport chain (ETC), comprising (i) Complex I (NADH dehydrogenase), which uses NADPH generated in the citric acid cycle for proton translocation from the mitochondrial matrix to the intermembrane space, with electrons being transferred to ubiquinone; (ii) Complex II (succinate dehydrogenase), which uses flavin adenine dinucleotide (FADH : 1ATP) . Complex leakage . In addites O2•- . The otharginine , 193. Petes H2O2 . Similarand H2O2 , 196. Duand NOS2 , 198.2O2. Similarly, quiescin sulfhydryl oxidase, also present in the Golgi, generates H2O2 for the introduction of disulfide bonds into unfolded reduced proteins and can compensate for the loss of ERO1 [2O2 in the ER is the NADPH oxidase 4 (NOX4). Proteins that belong to the NOX family are the only cellular enzymes exclusively involved in the production in ROS by using NAD(P)H for oxygen reduction to produce a superoxide anion [The ER is involved in diverse functions including protein folding, processing and vesicular transport, calcium storage, lipid synthesis, cell signalling, and xenobiotic toxicity. Particularly, during protein folding, ER oxidoreductin (ERO1) catalyses oxidation of protein disulfide isomerase, involved in disulfide bond formation, generating H of ERO1 , 200. Thde anion . Finallyde anion .Other sources of cellular ROS include the plasma membrane and lysosomes, as well as cytosolic reactions . In addi2+/calmodulin independent protein kinase II (CaMKII). Other pathways that display crosstalk with ROS/RNS include the NF-κB pathway, the MAPK pathway, the PI3K/AKT pathway, ATM signalling, the insulin pathway ), iron metabolism , calcium signalling , the ubiquitin system , the UPS ), and the autophagy pathway, as will be described later in detail.Crucially, ROS and RNS are not only detrimental to cells, but they are also important for cellular homeostasis, regulating numerous important cellular activities, also known as physiological oxidative stress or oxidative eustress. ROS and RNS act as second messengers in signal transduction pathways involved in cell survival, cell to cell communication, and cell growth and proliferation –208. TheIn neurons in particular, physiological levels of ROS are important for (i) axonal growth via cytoskeletal regulation ; (ii) pr2O2 release in mitochondria) and other specific unidentified channels [α-syn in Parkinson's, tau in Alzheimer's, and HTT in Huntington's) [ROS/RNS generated inside organelles can be readily released into the cytoplasm. They diffuse across membranes through aquaporins  . Lipid pngton's) .The nucleus is highly susceptible to oxidative stress. Diffusion of ROS/RNS into the nucleus influences diverse pathways/components, including chromatin organisation, DNA methylation, histone function , nucleobases, interactions between DNA and DNA-binding proteins, mutagenesis, transcription via targeting of purines and pyrimidines, single- and double-strand breaks, and abasic site formation . Indeed,Yoboue et al. proposed a “redox triangle” formed by ER-mitochondria-peroxisome structures, generating a multiorganellar protein complex called the “redoxosome,” where ROS and RNS accumulate to impact organelle function , an idea that awaits mechanistic validation , 226. Inc release into the cytosol; ATP depletion; and the induction of mitochondrial membrane depolarization. Low levels of oxidative stress in the ER activate the unfolded protein response (UPR) to inhibit protein translation, and to induce chaperone expression and protein degradation, as will be described later; whereas high levels of ROS trigger the activation of ER stress-mediated apoptosis via different pathways , some of which are interconnected with mitochondrial pathways, leading to caspase activation and apoptosis [Ultimately, high levels of oxidative stress can induce cell death via apoptosis, necroptosis, and autophagy-associated programmed cell death. Indeed, ROS/RNS activate the extrinsic death receptor pathways , leading to the activation of caspases, as well as the internal mitochondrial and ER cell death pathways. In mitochondria, ROS can induce apoptosis through diverse pathways, including activation of p53 and JNK, which in turn activate proapoptotic Bcl-2 proteins; oxidation of cardiolipin, leading to cytochrome poptosis , 230.2O2 by monoamine oxidase (MOA), whose activity is disrupted in Parkinson's); (vii) neurotransmitter oxidation (formation of toxic intermediates); (viii) lower antioxidant response in comparison to other tissues; (ix) microglia activation and astrogliosis (as a big source for ROS/RNS); (x) presence of redox active transition metals ; (xi) lipid peroxidation (high levels of fatty acids in the brain); (xii) NOS and NOX for neuronal signalling; and (xiii) RNA oxidation [Crucially, the brain is particularly susceptible to oxidative stress damage. Cobley et al. defined 13 reasons why the brain is predisposed to oxidative stress and consequent neurodegeneration: (i) redox signalling (high levels of ROS/RNS can induce proapoptotic pathways via redox modifications); (ii) calcium homeostasis ; (iii) excessive glutamate uptake ; (iv) glucose metabolism necessary to support neuronal activity (oxidative stress can affect this pathway via the formation of advanced end glycation products (AGE)); (v) mitochondria ; (vi) neurotransmitter metabolism  endogenous antioxidant mechanisms , (ii) a metabolic switch to the pentose phosphate pathway , (iii) t2O2 into H2O, and by a defence system for the conversion of O2•- into the less harmful radical, H2O2, comprising superoxide dismutase 2 (SOD2) in the mitochondrial matrix, and SOD1 in the intermembrane space. Glutathione peroxidases (GPX1 and GPX4) in the outer mitochondrial membrane, reduce H2O2 into H2O, using reduced glutathione (GSH) as cofactor. Other mitochondrial antioxidant enzymes include the peroxiredoxins (PRX3 and PRX5), which also catalyse the reduction of H2O2 into H2O [2O2 into H2O, and indeed deficiencies in this system are linked to cancer and diabetes. In addition, in this organelle, SOD1 and PRX5 are also involved in the formation and reduction of H2O2, respectively [Mitochondria are protected from ROS by the presence of antioxidant enzymes that contain cysteine catalytic residues for the reduction of Hinto H2O , 236. Peectively , 237. Thectively . Other aectively . An addiectively , 239.Transcriptionally, NRF2 is considered to be a master regulator of redoxtasis, controlling around 1% of human genes that share in common the Antioxidant Response Element (ARE) in their promoters –243. CruFinally, the two major quality control mechanisms in the cell have antioxidant roles in preventing the aggregation of oxidized proteins and/or the persistence of damaged organelles  and autophagy , 2, 5–7)Under stress conditions , ROS/RNS are induced, and have the potential to influence autophagy via core autophagy protein oxidation, or by altering the activities of transcription factors –250. In The activity of several proteins upstream of the autophagy pathway is affected by ROS/RNS. These proteins are typically also involved in the regulation of several pathways; thus, their redox modifications influence diverse cellular activities.Reversible oxidative and nitrosative modifications include sulfenylation, glutathionylation, disulfide bonds, acylation, and nitrosylation. These affect RTK receptors , affecting their activation, localisation, or trafficking, depending on the modification and residues involved . In add2O2 oxidation inactivates PTEN catalytic activity by the formation of disulfide bonds (C124-C71), leading to autophagy activation in a noncanonical pathway induced my mTOR activation; and (ii) peroxynitrite inhibits PTEN activity and induces neuronal survival and can be oxidized by a lipid peroxide which is prevented by PRX3 [PTEN opposes PI3K activity by dephosphorylating PIP3 and inhibiting AKT signalling. Redox modifications affect PTEN activity; for example, (i) H by PRX3 –259.PDK1 is a Ser/Thr kinase that activates AKT. Redox modifications of this protein include nitrosylation in different residues leading to inhibition of its kinase activity .Several cysteines within the pleckstrin homology domain of AKT have been identified as being reversibly oxidised, forming new disulfide bonds. These modifications affect protein function, including the stabilisation of the PI3P pocket, or its inhibition, depending on the modification. In addition, AKT can be inactivated via glutathionylation, which is reversed by glutaredoxin 1 , 262.Nitrosylation of TSC2 impairs its dimerization with TSC1, leading to mTOR activation .Generation of disulfide bonds affects mTOR stability and activity depending on the residues involved. Oxidised mTOR can be rescued by Thioredoxin 1 . Relatedα and β AMPK subunits, affecting AMPK activity depending on the modified residues . In addition, free radicals induce calcium release  leading indirectly to the activation of the AMPK via CaMKII activation. Similarly, it has been reported that the induction of autophagy, as a consequence of ROS production in starvation conditions and ATP depletion, is via activation of the AMPK pathway [Disulfide bonds, sulfenylation, and glutathionylations have been described to be present in both  pathway –270.2O2 treatment induces nuclear ATM redox disulfide bond formation, indirectly promoting the downstream expression of proteins involved in the pentose phosphate pathway, and an activator effect via nytrosilation has also been suggested [ATM is a threonine/serine kinase involved in the DNA damage/repair response. Crucially, ATM is also involved in the induction of pexophagy to maintain redox balance. In response to ROS, ATM activates MAPK, and ATM is transported into peroxisomes via the PEX5 import receptor. Here, it phosphorylates PEX5 triggering ubiquitylation via the E3-like ubiquitin ligase complex, PEX2-PEX10-PEX12, and later recognition by cargo receptors , 272. H2uggested –275.α, NF-κB, PPARs, and FXR [A class III histone deacetylase regulating numerous cell activities  , SIRT1 i and FXR , 279. Co and FXR –282.α, PERK, and ATF6 . For ex pathway . Other m pathway , and dis pathway .It is also important to mention that redox modifications can also affect cytoskeletal dynamics , and thuProteins involved in the autophagosome assembly pathway can also be targeted by ROS/RNS. These redox modifications affect the efficiency and productivity of autophagosome biogenesis .ATG8 processing mediated by ATG4 proteins needs to be spatiotemporally regulated to support both autophagy initiation and the availability of a pool of primed ATG8 . Scherz-Frudd et al. described that these proteins can be modified by oxidation, including glutathionylation and disulfide bond formation, affecting their function, and leading to autophagy inhibition. Conversely, while inactive, ATG3 and ATG7 form covalent thioester complexes with LC3, preventing their oxidation. However, after autophagy induction, their interactions become more transient, thus increasing susceptibility to redox modifications .Redox modifications affect BECLIN1 function indirectly: under normal conditions, BECLIN1 forms a complex with proapoptotic BCL-2, which inhibits BECLIN1 activity; however, under autophagy-inducing conditions, BECLIN1 dissociates to establish the PI3KC3 complex. Kitada et al. described that BCL-2 is a target of redox modification, particularly nitrosylation, stabilising the interaction with BECLIN1, and thus preventing autophagy induction .Carroll et al. identified two cysteine residues in p62/SQSTM1 that can be redox modified, forming disulfide bonds that promote its oligomerisation to enable autophagy induction . They fuNumerous redox modifications have been described for Parkin, including nitrosylation, sulfonation, and methionine oxidation. Particularly, Chung et al. described that S-nitrosylation reduces Parkin E3 ligase activity, thus affecting its protective function , althougOh et al. described that PINK1 can be nitrosylated, inhibiting its kinase activity, and this posttranslational modification is present in Parkinson's mice models where Parkin recruitment is reduced, restricting mitophagy .α-synuclein fibrillation [DJ-1 overexpression induces mitophagy via the activation of ERK in mDANs, and this protects against rotenone-induced cell death . Indeed,illation . Conversillation , 310. Inillation .ATG4, ATG9B, BECLIN1, LC3B, GABARAPL1, ATG16, WIPI, UVRAG, and p62/SQSTM1) by binding to CLEAR  sequences in their promoters. These genes are involved in autophagosome biogenesis, autophagosome-lysosome fusion, and lysosomal biogenesis [α-syn in Parkinson's, and HTT in Huntington's [Autophagy gene expression is influenced in different tissues by diverse transcription factors, microRNAs (miRNAs), and by epigenetic modifications . In the ogenesis , 314. TFogenesis , 315, viogenesis , 317. Phogenesis . Calcineogenesis –321. Cytogenesis . An incrogenesis , 324. Inington's –327).In this review, we focus on transcription factors whose activity is regulated by redox modifications, thus affecting autophagy transcriptional control.In addition to the well-characterised control of TFEB activities via phosphorylation (above), Wang et al. described the regulation of TFEB (and other members of the MiT family) via ROS-mediated cysteine oxidation (C212). This inhibits the interaction of TFEB with Rag GTPases, and induces its nuclear translocation, thus inducing the expression of autophagy/lysosomal genes independently of mTORC1  .SOD1, SOD2, and GPX1 [2O2 treatment in human mesenchymal stem cells, thus leading to autophagy induction that is crucial for osteogenic differentiation; however, a direct redox modification has not yet been described [In particular, FOXO1 and FOXO3 have been identified as autophagy transcription factors, regulating the expression of numerous autophagy-related genes –331. In and GPX1 . Under band GPX1 . Under sand GPX1 , 335. Crand GPX1 . Mainly,and GPX1  and disuand GPX1 , 339). Gescribed . Other eescribed .p62/SQSTM1, NDP52, ULK1, ATG2B, ATG4, ATG5, and GABARAPL1) [LAMP2A expression [HIF-1α expression, and interacts with ATF4 [2 homeostasis and contributors to autophagy-gene expression ; whereas in severe hypoxia, ATF4 regulates the expression of autophagy genes  [The master regulator of oxidative stress, NRF2 contributes to the regulation of autophagy-gene expression under these conditions  . CrucialBARAPL1) . In addiBARAPL1) , 344 to BARAPL1) . Converspression . In addiith ATF4 , 348, mand LC3B) , 350). Bnd LC3B) –354.GPX1) [ULK1, ATG4, ATG7, and ATG10), and to induce TFEB nuclear translocation [As one of the best characterised transcription factors, p53 has been reported to regulate antioxidant genes  , autophalocation . It alsolocation . Howeverlocation . Severallocation . Indeed,location ; howeverlocation .κB, is inactivated in the cytosol where it interacts with IκB (inhibitor of κB) preventing its nuclear translocation. Previous data suggest that oxidative stress can induce or inhibit the NF-κB pathway, depending on conditions [κB is phosphorylated under oxidative stress, leading to its polyubiquitination and consequent degradation [κB upregulates the expression of several anti-inflammatory and antioxidant genes  [κB mainly inhibits autophagy, but it can also activate the expression of the autophagy genes including BECN1 and p62/SQSTM1 [κB activity. Disulfide bonds in cysteine in the DNA-binding domain inhibits DNA binding, and this can be rescued by Thioredoxin 1 [κB including glutathionylation and nitrosylation also inhibit DNA binding [Under basal conditions, the proinflammatory transcription factor, NF-nditions . As one radation . The connd SOD2) , and can2/SQSTM1 –367). ROedoxin 1 . Similar binding , 370.ULK1, ATG2, ATG5, ATG7, WIPI, GABARAP, and TFEB) [Examples of other transcription factors involved in both the regulation of autophagy-related and antioxidant genes include (i) the peroxisome proliferator-activated receptors (PPARs) that upregulate autophagy and can be directly regulated by redox modifications  , 372 andnd TFEB) .Parkinson's is one of the most common neurodegenerative disorders, second only in prominence to Alzheimer's, and it affects 1-3% of the population aged over 60 , 378. Liα-syn-rich Lewy's bodies; (ii) increased oxidative stress accompanied by a reduction in antioxidants; (iii) neuroinflammation; (iv) mitochondrial dysfunction; (v) ER stress; (vi) and disruption in protein quality control, including autophagy dysregulation [At the cellular level, Parkinson's is characterised by the loss of mDANs, initially in the substantia nigra pars compacta (SNc). As the condition progresses, defects in serotonergic, noradrenergic, cholinergic, GABAergic, and glutamatergic neuronal pathways can also be observed . Althouggulation . Crucialgulation , 393–396mDANs are localized in the mesencephalon, and are characterised by the production of the catecholaminergic neurotransmitter, dopamine . DopaminThere are three different clusters of mDANs, designated as follows: (i) A8, those originating from the retrorubral field (RRF); (ii) A10, those found in the ventral tegmental area (VTA) forming the mesolimbic (to the nucleus accumbens) and mesocortic  dopaminergic pathways; and (iii) A9, the cells located in the SNc that project to the striatum. Functionally, mDANs involved in emotion-based behavior are found in the A8 and A10 groups, whereas those responsible for voluntary movement control are specified as A9 . As A9 S2+ channels, to provide tonic dopamine release to the striatum [2+. Unfortunately, SNc mDANs have intrinsically low Ca2+-buffering capacity, unlike their counterparts in the neighbouring VTA, thus placing an extra reliance on energy-dependent Ca2+ efflux and Ca2+ sequestration in mitochondria [2+ supports enhanced oxidative phosphorylation (OXPHOS) rates [Perhaps the most compelling explanation for the vulnerability of mDANs is that their atypical morphology and physiology require that these cells operate close to their energy demand/supply threshold . A9 mDANstriatum . This buchondria –415. AltS) rates , this coS) rates . In addiS) rates –421. IndS) rates , 414, anS) rates , 422.+ and transported by DAT into mDANs [Perhaps most tellingly, neurotoxins that target mitochondria can induce selective mDAN cell death. A neurotoxin used to generate Parkinson's mouse models, 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibits mitochondrial Complex I in the ETC, causing ROS damage and mitochondrial dysfunction . MPTP cato mDANs . Anotherto mDANs –429. Rotto mDANs , 430–433to mDANs , 434. Inα-syn Parkinson's model, Nrf2 deficiency increased dopaminergic cell death, neuroinflammation, and protein aggregation [α-syn Parkinson's mice models [Drosophila Parkinson's model [Elevated levels of oxidative stress in Parkinson's correlate with lipid peroxidation, nucleic acid oxidation, elevation of intracellular calcium and increased iron content, and protein oxidation and nitration , 435. Inregation ),while ue models , 441 and's model ). On the's model , and ind's model , while i's model .α-syn aggregates [α, impairs autophagy flux in microglia via mTORC1 [One of the main features of oxidative stress in mDANs is the oxidation of dopamine. In dopaminergic neurons, cytosolic excess dopamine is oxidised to the metabolite aminochrome or other toxic dopamine quinones. These are intermediates in the normal process of dopamine oxidation to neuromelanin, a dark polymer pigment that accumulates with age in the SNc and has neuroprotective roles as a metal chelator  , 447. Amgregates ; (ii) migregates ; (iii) cgregates , 451; angregates . Consequgregates , and nona mTORC1 ).Atg7 knockout mice), inclusions containing α-syn and p62/SQSTM1 are observed predominantly in neurites, and these increase with age, preempting neurodegeneration and motor dysfunction [BECN1, TFEB, and LAMP2A overexpression) in α-syn (SNCA) overexpression mouse models ameliorates synaptic and dendritic pathology [α-syn aggregates and reduces oxidative stress levels in a paraquat-induced Parkinson's mice model [Atg7 conditional knockout) in a SNCA mouse model led to dopamine release and improved motor movement, while aggravating pathology as reported by increased p62/SQSTM1 inclusions and neuronal death [PINK1, PRKN/Parkin, and FBXO7—three proteins involved in the recognition of damaged mitochondria—are linked to familial Parkinson's  [Nipsnap1—a mitochondrial matrix protein involved in PINK1/Parkin-independent mitophagy and highly expressed in mDANs—caused Parkinson's hallmarks [Over the previous decade, numerous lines of evidence have highlighted the importance of autophagy in neuronal homeostasis. In the absence of autophagy in dopaminergic neurons  . In addictively) . Similarctively) , 300. Inallmarks . In the allmarks , 465.UCHL1, DJ-1, LRRK2, ATP13A2, USP24, HTRA2, VPS35, SYNJ1, VPS13C, and GBA) [Apart from the mutations found in mitophagy genes, several genes linked to familial Parkinson's either directly or indirectly modulate autophagy (including and GBA) , 466, anUCHL1 I93M mutant overriding this suppression [α-syn aggregation [UCHL1 is a deubiquitylating enzyme . It can pression . In respregation , 470.α-syn; and (iv) the regulation of transcriptional activity via activation of NF-κB, p53, and NRF2 pathways [κB regulation, DJ-1 controls the expression of mitochondrial uncoupling proteins, UCP4 and UCP5, that decrease mitochondrial membrane potential, thereby suppressing ROS production; meanwhile, DJ-1 binds to Complex I via NDUFA4 to maintain its activity, and is thus crucial for mDAN survival [DJ-1 acts in parallel with the PINK1/Parkin pathway, playing an important antioxidant role to protect mDANs against oxidative damage . Howeverpathways , 472. Crsurvival , and DJ-survival , 473, ansurvival .2O2 treatments downregulate LRRK2 phosphorylation, preventing binding to 14-3-3 in vitro [LRRK2 is degraded by CMA, and its most common mutation (G2019S) increases its kinase activity, restricting its degradation –477. G20in vitro , 487.GBA)—in which homozygous mutations lead to Gaucher's disease—are one of the most common risk factors for Parkinson's, and GBA deficiency is associated with mitochondrial dysfunction and oxidative stress [Mutations in the lysosomal enzyme, glucocerebrosidase  mouse model [α-syn aggregates, and this was rescued with a CMA activator [C. elegans, neurons release vesicles called exophers in a process that is enhanced during stress or when autophagy is inhibited, and these contain protein aggregates and organelles that are subsequently engulfed by adjacent cells [Finally, as we previously mentioned, glial cells—i.e., astrocytes, microglia, and oligodendrocytes—represent about 90% of all cells in the brain, and are critical for maintaining neuronal homeostasis  and alterations of neuron-glia signalling pathways are associated with neurodegenerative diseases, including Parkinson's –493. A pse model , 495. Sictivator . Howeverctivator . In thisctivator . In addint cells .Certain hallmarks of Parkinson's are thought to exacerbate pathology through the disruption of autophagic flux. Here, we will describe how different Parkinson's features are linked to autophagy disruption and oxidative stress damage.α-Syn is a major component of this fibrillar aggregate; however, more than 70 additional molecules have been identified as coconstituents , posttranslational modifications , and autophagy dysfunction increase the rate of oligomerisation, and thus the formation of inclusions [α-syn. For example, Giasson et al. reported that α-syn can be nitrated at specific tyrosine residues, and these modifications are found in Lewy's bodies [α-syn that enhance its propensity to aggregate [α-syn methionine oxidation inhibits secondary structure formation [α-syn, and mutations or overexpression of α-syn enhances the extent of contact sites and affect mitochondrial function [The best characterised feature of Parkinson's is the presence of Lewy's bodies; however, these are not observed in all Parkinson's cases, and are also found in healthy patients where they are referred as incidental LB disease . α-Syn iormation ), and LRormation . Mutatioclusions , 502. Ims bodies . Jiang aggregate . Ponziniormation . In addifunction –509.α-syn in oligomers affects neurotransmitter release, synaptic vesicle recycling and trafficking, and autophagy (both CMA and macroautophagy); meanwhile, it increases ROS/RNS levels , triggers microglial activation, impacts on mitochondrial homeostasis, and induces ER stress and calcium homeostatic imbalance [α-syn can block CMA, thereby preventing the degradation of itself and other CMA substrates [α-syn also stimulate cell death via oxidative-nitrosative stress, and this [α-syn persistence leading to a compensatory response of increased macroautophagy and an accumulation of aggregates in autophagosomes [α-syn aggregates can also impair macroautophagy  [519, 5 ZKSCAN3 . In addihe brain , and indpairment , 523.α-syn [α impairs autophagy flux in microglia, and autophagy induction promotes microglia polarisation towards a M2 neuroprotective phenotype [β signalling [Another Parkinson's hallmark that may impact on or be affected by redox imbalance is neuroinflammation . Neuroinα-syn . In turnα-syn , 527. Inhenotype . Similarhenotype , and mithenotype . In addignalling –532. Indgnalling . Similargnalling , 535.α-Syn aggregates, mitochondrial dysfunction, oxidative stress, familial Parkinson's , and other conditions impair UPS function, which in turn, leads to an increase in oxidised and damaged protein levels, increased toxic iron, and increasing vulnerability of mDANs [Disrupted protein quality control in Parkinson's is linked to impairment of the UPS, involved in the selective degradation of the majority of abnormal proteins in the cell . Oxidiseof mDANs –539. Indof mDANs . UPS andof mDANs –543. In of mDANs .ER stress/dysfunction and chronic UPR activation are further Parkinson's hallmarks . Disruptα-syn aggregation [Finally, it is also important to mention that the other major source of ROS/RNS in the cell, peroxisomes, are also affected in Parkinson's. Indeed, peroxisomes are required for neuronal homeostasis and function, and peroxisomal dysfunction has been suggested to contribute to regation –555. In regation —acting aregation . Howeverα-syn aggregation.Overall, autophagy dysfunction and increased oxidative stress are two closely related hallmarks present in dopaminergic neurons in Parkinson's, aggravated by other Parkinson's features including mitochondrial dysfunction, elevated iron and calcium levels, increase in dopamine oxidation, UPS dysfunction, ER stress, neuroinflammation, and Autophagy is an intracellular process required for the maintenance of cellular homeostasis, being particularly crucial in neurons as they are postmitotic cells highly vulnerable to stress. Dysfunctional autophagy typically correlates with neurodegenerative diseases, with mitophagy being a particularly important link due to the increased vulnerability of mDANs to autophagy deficits and mitochondrial dysfunction , 417. Thα-synuclein oxidation); (vi) UPS dysfunction; (vii) disruption in the cytoskeletal transport; (viii) neuroinflammation; (ix) ER stress and chronic activation of the UPR; and possibly (x) peroxisomal dysfunction.However, when the balance of antioxidant mechanisms and ROS/RNS generation is disrupted because the antioxidant defence is reduced , or because ROS generation is increased, oxidative stress is initiated. This can damage the cell, including triggering neuronal cell death programmes, the primary driver in Parkinson's. In addition, dopaminergic neurons are particularly vulnerable to autophagy deficits and high levels of oxidative stress. Consistent with this, different factors contribute to the disruption of the autophagy pathway linked to an increase in oxidative stress, including (i) dopamine oxidation leading to the formation of the toxic molecules like aminochromone; (ii) familial Parkinson's-associated genes involved in autophagy and oxidative stress regulation; (iii) mitochondrial dysfunction, including an increase in calcium and iron levels; (iv) neurotoxins affecting almost exclusively dopaminergic neurons ; (v) oxidation of biomolecules  [α-synuclein aggregates and cell death in Parkinson's models [Currently, there are only symptomatic treatments for Parkinson's, and no disease-modifying therapies have been described. The most commonly used approaches to treat motor deficiencies are based on pharmacological stimulation of the dopaminergic pathway—e.g., levodopa , dopamine agonists , and nonregates) –567. In s models , 569. Hos models . For this models , 571–573s models –576.Overall, there is a strong evidence for the interplay of autophagy and redox homeostasis and how it plays a crucial role in Parkinson's. However, there is still a lot to explore and future research would contribute to a better understanding of this tight relationship and potential target for selective therapies.


Thymic stromal lymphopoietin (TSLP) is released by epithelial cells following disturbed homeostasis to act as “alarmin” and driver of Th2-immunity. Aberrant TSLP expression is a hallmark of atopic diseases, including atopic dermatitis (AD). Mast cells (MCs) are overabundant in AD lesions and show signs of degranulation, but it remains unknown whether TSLP contributes to granule discharge. Degranulation of skin MCs proceeds via two major routes, i.e., FcεRI-dependent  and MRGPRX2-mediated . Evidence is accumulating that MRGPRX2 may be crucial in the context of skin diseases, including eczema. The current study reveals TSLP as a novel priming factor of human skin MCs. Interestingly, TSLP selectively cooperates with MRGPRX2 to support granule discharge, while it does not impact spontaneous or FcεRI-driven exocytosis. TSLP-assisted histamine liberation triggered by compound 48/80 or Substance P, two canonical MRGPRX2 agonists, was accompanied by an increase in CD107a+ cells (a MC activation marker). The latter process was less potent, however, and detectable only at the later of two time points, suggesting TSLP may prolong opening of the granules. Mechanistically, TSLP elicited phosphorylation of STAT5 and JNK in skin MCs and the reinforced degranulation critically depended on STAT5 activity, while JNK had a contributory role. Results from pharmacological inhibition were confirmed by RNA-interference, whereby silencing of STAT5 completely abolished the priming effect of TSLP on MRGPRX2-mediated degranulation. Collectively, TSLP is the first factor to favor MRGPRX2- over FcεRI-triggered MC activation. The relevance of TSLP, MCs and MRGPRX2 to pruritis and atopic skin pathology indicates broad repercussions of the identified connection. Acute mast cell (MC) activation leads to hypersensitivity reactions and is the root of disorders like rhinoconjunctivitis, asthma, urticaria, angioedema, food allergy, and anaphylaxis ,2. In thOnly certain MC-subtypes, especially those in the skin  express MRGPRX2 at high level ,18,19,20The allergic and pseudo-allergic pathways differ fundamentally in several aspects, including spatiotemporal patterns of granule discharge and dependence on IKK-β, the latter involved in SNAP23/STX4 complex formation, which is key to the so-called compound exocytosis observed following FcεRI aggregation, in which granules fuse together and are released as bigger, more heterogeneously shaped entities . InteresTogether with IL-33, thymic stromal lymphopoietin (TSLP) is widely recognized as a critical factor in allergic disorders, including rhinitis, asthma, food allergy, eosinophilic esophagitis, and atopic dermatitis (AD), i.e., conditions, in which MCs act as critical effector cells ,35,36. ABoth IL-33 and TSLP belong to the subgroup of epithelial-derived innate cytokines that support Th2 responses in part by re-programming of dendritic cells and support of innate lymphoid cells ,39,40,41TSLP, cutaneous MCs, FcεRI and, more recently, MRGPRX2 have all been linked to AD pathogenesis ,36,48, aThis was an unlinked anonymous study. Donor skins, which otherwise would be disposed of, were obtained from circumcisions (foreskin), with written informed consent of the patients or their legal guardians, as in previous studies from our group ,19,28,445/mL in Basal Iscove medium with 10% FCS  for around 3 weeks. SCF  (at 100 ng/mL) and IL-4 (Peprotech) (10 ng/mL) were provided twice a week.MCs were isolated from skin samples by a routine procedure, as described ,50. BrieThe HRA was performed according to a method routinely employed in our laboratory ,53. In bFor inhibitor studies in the absence of TSLP, cells were pretreated with SP600125 at 5 µM (JNK inhibitor) or Pimozide at 5 µM (STAT5 inhibitor) versus PAG-CM buffer (control) for 15 min and then stimulated with the above stimuli for 30 min.To study the involvement of selected kinases in TSLP’s potentiation of degranulation, cells were pretreated with the above inhibitors for 15 min, TSLP was added to selected vials, and finally the secretagogues (AER-37 or MRGPRX2 ligands) were added as above.® siRNA technology  [6/mL in Accell siRNA medium and treated with 1 µM STAT5-targeting siRNA (E-005169-00-0050), or JNK-targeting siRNA (E-003514-00-0050) or non-targeting siRNA as control (D-001910-10-50) for 48 h. After incubation, cells were primed with or without TSLP and degranulation was induced by MRGPRX2 ligands as above.RNA interference in MCs was performed according to an established protocol by using the AccellCO, USA) ,44,54. B5 cells/mL in PAG-CM buffer. Aliquots of 100 μL were stimulated by compound 48/80 (10 μg/mL), or SP (30 µM), or kept in buffer only. After incubation for 60 min, supernatants (SNs) were collected by centrifugation at 500× g, 4 °C for 3 min, and the pelleted MCs rapidly frozen at −80 °C. After thawing, aliquots of 50 μL of 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide  solution at 5 μM in citrate buffer (pH 4.5) were mixed with the same volume of supernatant or lysate and incubated for 60 min at 37 °C. The reaction was stopped by adding 100 mM sodium carbonate buffer (pH 10.7). Fluorescence intensity was measured at an emission wavelength of 460 nm after excitation at 355 nm. Percent β-hexosaminidase release was calculated as (fluorescence intensity SN/(fluorescence intensity SN + fluorescence intensity lysate)) × 100. Net release was calculated by subtracting spontaneous release, as in the histamine release assay above.β-hexosaminidase assays were run exactly as described ,50. BrieFlow-cytometric detection of CD107a cell surface expression was as described ,55. In bMRGPRX2 cell surface staining was performed according to established protocols ,28. In bIntracellular staining of signaling intermediates was performed exactly as described . In brieAll data were analyzed with the FlowJo 7.6.5 analysis software .Cell preparation and immunoblotting were performed as described . In briep < 0.05 was considered statistically significant.Statistical analyses were performed using PRISM 8 . Pairwise comparisons of TSLP priming versus control were conducted by Student’s t-test. One sample t-test (against 1) was used to calculate significance in normalized immunoblot data. For multi-group comparisons, RM one-way ANOVA with Sidak’s multiple comparisons test were applied. Fold changes (FC) of histamine and CD107a were calculated as net “parameter” after TSLP priming/net “parameter” without TSLP priming, whereby parameter refers to percent histamine release or the proportion of CD107a+ cells. The resulting FC (fold change) values were compared by Mann–Whitney test. So far, a positive impact of TSLP on MC cytokine production was reported ,57,58 anThe most selective MC function, i.e., degranulation with the release of prefabricated histamine and other mediators confined to these cells, has not been investigated or not found to be modulated by TSLP .In skin-derived MCs, TSLP alone had likewise no impact on histamine release, i.e., did neither degranulate MCs on its own nor inhibit spontaneous histamine liberation A. ConverCollectively, TSLP selectively promotes MRGPRX2-driven degranulation of human skin MCs.Degranulation is accompanied by transient detection of granule markers at the cell surface as a result of granule-plasma membrane fusion, whereby CD107a best distinguishes activated from non-activated MCs, which are visible as two clearly separated populations ,55 and iPrior treatment with TSLP led to slightly increased expression of CD107a following SP or c48/80 stimulation in a time-dependent fashion . The effSince the impact of TSLP priming seemed more robust at the level of histamine release compared to CD107a-positive cells, we quantified the respective effect of TSLP as a fold-change ratio and indeed found a significant difference between outcomes . This unCollectively, TSLP has a positive, yet modest impact on the percentage of responding MCs, while prolonged granule opening may be the reason for the more efficient histamine release.We recently elaborated that among several signaling cascades, TSLP selectively elicits STAT5 and JNK activation in skin MCs . This waThe focus of the current study was to dissect which of the cascades, if any, was required for TSLP-supported degranulation. Prior to examining this aspect, it was mandatory to realize whether the signaling components participate in granule discharge of skin MCs in the absence of TSLP.The STAT5-inhibitor Pimozide had no effect on either FcεRI- or MRGPRX2-triggered degranulation . ConversWe next explored whether JNK and STAT5 are implicated in TSLP’s promotion of exocytosis. Using a three-component setting , we found that this was indeed the case.In the overall view of all groups A,B, TSLP® mediated RNAi protocol [Exploiting our recently established Accellprotocol ,28,54, wprotocol , we finaWe conclude that STAT5 is absolutely essential for TSLP’s supportive effect on pseudo-allergic degranulation, while JNK has a further contributory role.MCs adjust their responsiveness to secretory stimuli in dependence of the tissue microenvironment, whereby modulating factors like cytokines can critically influence their secretory competence without acting as secretagogues on their own. In doing so, extracellular stimuli can modify the manifestation and course of MC-dependent diseases by diminishing or increasing the concentration thresholds of triggering factors like allergens or drugs ,62,63. HDegranulation can be efficiently elicited via two major routes in skin MCs, i.e., FcεRI  and MRGPRX2 . The former involves the high affinity IgE receptor on the MC surface, antigen-specific IgE bound to it, and the antigen  which cross-links FcεRI to start a cascade which culminates in exocytosis ,2,25,64.Notwithstanding, several themes have started to emerge. In particular, MRGPRX2-triggered degranulation can be negatively affected by cues typically supportive of the lineage. Specifically, the dominant MC growth factor SCF interferes with the pseudo-allergic route, while simultaneously strengthening allergic stimulation ,19. In aTSLP, mostly expressed by epithelial cells, is considered a Th2-promoting master switch implicated in Th2-dominated pathologies in the skin, lung and other organs, and is therefore a focus in allergy-related research ,70,71,72MC modulating activities on maturation markers, production of cytokines and survival have previously been ascribed to TSLP ,73,74,75The sensitivity of skin MCs towards TSLP can be linked to the abundant expression of the TSLP receptor in these cells ,42,44. TWe therefore asked whether STAT5, JNK, both or none form part of the apparatus, by which TSLP potentiates MRGPRX2-dependent degranulation. To be able to judge, we first needed to clarify whether JNK and STAT5 are involved in MRGPRX2-evoked degranulation in the absence of TSLP. STAT5 perturbation had no impact on histamine liberation whatsoever, and this applied not only to the MRGPRX2- but also to the canonical IgER-route studied in parallel for comparison . DependeJNK is a well-established event in the signaling cascade downstream of FcεRI that can be activated by multiple upstream kinases, including MKK7, PI3K or PKC ,85. So fHere we found a slight, yet significant effect of JNK perturbation on degranulation triggered by SP (as well as FcεRI), yet not by c48/80 . In factIn contrast to the standard situation, STAT5 activity was indispensable to the pro-secretory function of TSLP, which was nullified by Pimozide. JNK supported STAT5, although its inhibition had a less complete effect than that of STAT5. This was discernible in the ratio-centered view, whereby TSLP still had a supportive (yet reduced) effect under JNK inhibition, while this effect was virtually absent upon interference with STAT5. The indispensable nature of STAT5 was verified following its RNAi-mediated knockdown. In this setting TSLP was unable to increase degranulation in both independent experiments and to both stimuli (c48/80 and SP) . The impIn summary, while STAT5 is the dominant entity, joint action of STAT5 and JNK is likely required to optimally orchestrate the heightened secretory competence bestowed by TSLP. Synchronized activation of STAT5 and JNK may thus mediate different TSLP outcomes in skin MCs, including survival protection  and MRGPMRGPRX2 has moved to the center of attention not least because of the large and still increasing number of secretagogues activating the receptor, of which many can be linked to symptoms or diseases. These include injection-site hypersensitivity, red man syndrome, and anaphylactic reactions to an array of drugs  ,13,16,92As mentioned, aberrant TSLP activity is closely tied to AD, and tezepelumab (anti-TSLP antibody) is in clinical trials for this condition . Of impoThe relevance of overexpressed TSLP and deregulated MRGPRX2 activity in skin pathologies indicates that their synergy may have important ramifications for understanding and treatment of skin disorders, but also host-defenses organized by MRGPRX2 on the other end of the spectrum. In fact, evidence is accumulating that MRGPRX2/Mrgprb2 can mobilize anti-bacterial mechanisms, e.g., after exogenous activation in the context of Staphylococcus aureus induced dermonecrotic lesions  or by reIt remains to be elucidated whether newly synthesized mediators like cytokines can be elicited via MRGPRX2 in human skin MCs, i.e., whether this MC type behaves rather like cells of the leukemic LAD2 line (efficient production of cytokines with MRGPRX2 agonists) or rather like  primary MCs (cytokine production inefficient or undetectable) ,99. DiffAs of now, TSLP may be provisionally assumed to produce a hyperactive phenotype with selectivity for MRGPRX2, as has been suggested for aberrant KIT activity (due to SCF overexpression or the D816V mutation) in connection with allergen-dependent activation . In conc


The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth, proliferation, and metabolism by integrating various environmental inputs including growth factors, nutrients, and energy, among others. mTOR signaling has been demonstrated to control almost all fundamental cellular processes, such as nucleotide, protein and lipid synthesis, autophagy, and apoptosis. Over the past fifteen years, mapping the network of the mTOR pathway has dramatically advanced our understanding of its upstream and downstream signaling. Dysregulation of the mTOR pathway is frequently associated with a variety of human diseases, such as cancers, metabolic diseases, and cardiovascular and neurodegenerative disorders. Besides genetic alterations, aberrancies in post-translational modifications (PTMs) of the mTOR components are the major causes of the aberrant mTOR signaling in a number of pathologies. In this review, we summarize current understanding of PTMs-mediated regulation of mTOR signaling, and also update the progress on targeting the mTOR pathway and PTM-related enzymes for treatment of human diseases. The mechanistic target of the rapamycin (mTOR) pathway is a central sensor of environmental clues, such as growth factors, nutrients, and energy, through which it controls proper cell growth and metabolism. Imbalances in the mTOR pathway including full inhibition or constitutive activation may disrupt the cellular homeostasis, leading to human diseases including but not limited to cancers, metabolic diseases, and aging and neurodegenerative disorders ,2,3. In mTOR is an evolutionarily conversed protein kinase from yeast to human. In mammals, mTOR is the catalytic subunit of two different complexes , termed mTORC1 exerts its biological functions by phosphorylating a large number of downstream proteins. mTORC1 phosphorylates S6K and 4E-BP1 to promote protein synthesis . mTORC1 mTORC1 activation is spatially and temporally controlled by upstream inputs, notably amino acids and growth factors. In response to amino acids stimulation, Rag GTPases (Rags) are recruited to the lysosome by the Ragulator complex  ,20. SubsUnlike mTORC1, mTORC2 is insensitive to rapamycin and has distinct roles on metabolism, cytoskeleton regulation, cell proliferation, and survival ,11,37. THaving discovered dozens of proteins in the mTOR pathway, it is important to understand how these components are regulated, which may provide explanations for the aberrant mTOR signaling in human diseases. Besides genetic alterations and transcriptional regulations, post-translational modifications (PTMs) including phosphorylation, ubiquitination, acetylation, and glycosylation have been demonstrated to be key regulators of mTOR signaling. In this review, we will summarize PTMs-mediated regulation of the mTOR pathway  and discStudies have demonstrated that mTOR kinase activity can be regulated by several types of PTM, notably phosphorylation. Growth factors promote the phosphorylation of mTOR at S2448 by Akt or S6K1 ,44,45,46mLST8 knockout mice are defective in vascular development [As the shared subunit of the mTORC1 and mTORC2 complexes, mLST8 was initially identified to bind to the mTOR kinase domain and stabilize mTOR interaction with Raptor, leading to enhanced mTORC1 activity toward S6K1 and 4E-BP1 . Howeverelopment , indicatβ-TRCP, resulting in its ubiquitination and degradation [DEPTOR, also known as DEPDC6, is an mTOR-interacting protein that serves as an endogenous inhibitor of both mTORC1 and mTORC2. DEPTOR depletion enhances mTOR signaling, cell growth, and survival. In contrast, DEPTOR overexpression blocks mTORC1 activation but unexpectedly activates mTORC2 signaling through relieving the mTORC1-mediated inhibitory feedback signal to the PI3K/mTORC2/Akt pathway . Generalradation ,65,66. Mradation . These sRaptor in mouse leads to various tissue-specific diseases, such as heart failure [Raptor-knockout promotes liver cancer development [Raptor is the unique subunit of the mTORC1 complex and acts as a scaffolding protein to determine mTORC1 activity and substrate specificity ,5,9,10.  failure , hepatic failure , and mus failure . Unexpecelopment . Similarelopment . JNK, RSelopment ,75,76,77elopment . Recentlelopment . Other Pelopment ,81. Sungelopment .In 2007, three independent groups identified PRAS40 as a Raptor-interacting protein that acts as an inhibitor to block insulin/Rheb-mediated activation of mTORC1 signaling ,84,85. ITSC complex, composed of TSC1, TSC2, and TBC1D7, is a negative regulator of mTORC1 signaling . MutatioRheb is a small GTPase that is enriched on multiple endomembrane compartments including the lysosomal membrane where it interacts with and activates mTORC1. Farnesylation of Rheb in the C-terminal CaaX motif is required for its lysosomal localization ,106. In GTP hydrolysis, leading to inactivation of mTORC1 signaling [RNF152 knockout leads to hyperactivation of mTORC1 [The activation of the Rag heterodimer is the key step for mTORC1 lysosomal recruitment and activation. Notably, RagC is frequently mutated in follicular lymphoma  and the ignaling ,115. Howf mTORC1 . In contf mTORC1 .Ragulator interacts with Rag GTPases and is necessary for localizing the Rag proteins and mTORC1 to the lysosomal surface for activation. Among the five components of the Ragulator complex, myristoylation and palmitoylation in the N-terminus of p18 serve as a membrane localization signal for tethering Ragulator to the lysosomal surface . MoreoveThe GATOR1 complex is composed of three subunits including DEPDC5, NPRL2, and NPRL3, while the GATOR2 complex consists of WDR24, WDR59, MIOS, SEH1L, and SEC13 . KISCTORFLCN leads to cardiac hypertrophy and dysfunction in mice [GTP hydrolysis in the presence of amino acids [FLCN is considered as a tumor suppressor, mutations in which are the drivers of Birt–Hogg–Dubé hereditary cancer syndrome . Moreove in mice . FLCN funo acids . FLCN wano acids , S406/S5no acids , and S30no acids . FLCN hano acids ,132. HowTo date, three types of cytosolic amino acid sensors for the mTORC1 pathway have been identified: the leucine sensor Sestrin2, arginine sensor CASTOR1, and S-adenosylmethionine (SAM) sensor SAMTOR . MechaniAs a S/T protein kinase, mTORC1 exerts its biological functions on cell growth and metabolism by phosphorylating multiple downstream substrates, most of which have been demonstrated to play a role in various types of human diseases, such as cancers and cardiovascular and neurodegenerative diseases . Here, wThe S6K kinases (S6K1 and S6K2) are critical downstream effectors of mTORC1 in controlling fundamental cellular processes, such as transcription, translation, metabolism, and cell growth . PhosphoULK1, a gate keeper for autophagy initiation, is a key downstream effector mediating the role of mTORC1 signaling in autophagy. Upon glucose starvation, AMPK activates ULK1 by directly phosphorylating S317/S377 to induce autophagy. Conversely, under nutrient- and energy-rich conditions, mTORC1 directly phosphorylates ULK1 at S757 to disrupt ULK1 interaction with AMPK, leading to autophagy inhibition . RecentlGrb10 is an adapter protein that interacts with tyrosine kinase receptors, such as insulin receptor . mTORC1-In searching the mTOR-interacting proteins, Rictor was identified as a novel mTOR-associated component, which also binds to mLST8 and confers a rapamycin-insensitive feature to mTORC2 . AcetylaLater studies characterized Sin1 as another essential component of the mTORC2 complex, the depletion of which abolishes mTORC2 complex formation ,158. SinIt has been reported that the Sin1-PH domain binds to PIP3 and subsequently releases its inhibition on the mTOR kinase domain, leading to mTORC2 activation in response to growth factors . Thus, tGDP at S192 by GSK3 mediates its interaction with RasCGTP to promote mTORC2 activation [Like mTORC1, several small GTPases have been identified recently to directly regulate mTORC2 kinase activity. The Rho family of GTPase Rac1 directly binds to mTOR through the C-terminal region and promotes the activity of both mTORC1 and mTORC2 independent of its nucleotide binding states and PI3K pathway . Of the tivation . These sAs a major substrate, Akt is phosphorylated at S473 by mTORC2 , which cBesides Akt, PKCs and SGK1 are also the downstream effectors of mTORC2. Phosphorylation of PKCα by mTORC2 in the turn motif promotes its maturation, stabilization, and activation, which controls actin polymerization, cell shape, and mobility ,183,184.Dysregulation of mTOR signaling is tightly associated with many human diseases, such as obesity, diabetes, cancers, and neuronal disorders , making Despite TORKi being able to block mTORC2-dependent phosphorylation of Akt-S473, its inhibition on mTORC1 may release the negative feedback regulation of PI3K/PDK1/Akt signaling ,198, conAs the major downstream effector of PI3K/mTOR signaling, Akt plays a central role in promoting cell survival and apoptosis. Given hyperactivation of Akt is a hallmark of cancers, targeting Akt for cancer therapy has been extensively investigated . Many AkAs the key events in signaling transduction, PTMs including phosphorylation, ubiquitination, methylation, and acetylation, among others, provide a tight and reversible regulation of mTOR signaling. Aberrancies in these PTM-related enzymes may lead to the aberrant activation of the mTOR pathway. Therefore, targeting these mTOR-related regulators would offer an option for treating mTOR-associated human diseases. Below, we primarily focus on kinases, E3 ubiquitin ligases, and deubiquitinases, which are key regulators of the mTOR pathway.As S/T kinases, RSKs regulate diverse cellular processes by phosphorylating many downstream substrates . NotablyJNKs including JNK1, JNK2, and JNK3 in mammals are predominantly activated by environmental stress and regulate many physiological processes, such as cell proliferation, apoptosis, immune response, inflammation, and neuronal plasticity . Their hThe PIM kinase family containing three members, PIM1–3, and plays a critical role in cell proliferation, apoptosis, and migration by directly phosphorylating a number of downstream targets, such as MYC, CDC25, p21/p27, and TSC2. Overexpression of PIM kinases has been observed in a variety of human cancers and is associated with poor prognosis and drug resistance in prostate cancer and breast cancer . As therOther mTOR pathway-associated kinases including cdc2 , IKKβ 2, AMPK 2, LATS 2, ULK1 2, and GSKAs described above, a dozen E3 ligases and DUBs have been identified to regulate mTOR signaling by controlling protein stability, enzymatic activity, and complex formation. Aberrancies in these E3s/DUBs are frequently observed in human diseases, which may be in part responsible for the dysregulation of mTOR signaling. FBXW7 mutations [FBXW7, a cullin-based SCF type of E3 ubiquitin ligase, plays an important role in various biological processes such as cell cycle transition, cell proliferation, apoptosis, metastasis, and immune response . FBXW7 iutations . Beside Parkin belongs to the RING-between-RING (RBR) family of E3 ubiquitin ligases that regulate a variety of cellular processes including mitochondrial quality control, anti-oxidative stress, and mitophagy. Mutations in Parkin lost its protective role on the survival of dopaminergic neurons, which is the major cause of autosomal recessive parkinsonism. Notably, mTOR signaling exerts either a neuroprotective or neurotoxic role depending on PD models and has been linked to PD. It will be interesting to investigate whether Parkin-mediated ubiquitination and activation of mTOR signaling play a role in PD. Moreover, Parkin is an emerging tumor suppressor as it is frequently lost in cancers including breast, lung, colorectal, and ovarian cancers . ConsistOther E3 ubiquitin ligases including Skp2  and KLHLOTUD7B belongs to the ovarian tumor (OUT) family of deubiquitinases and prefers to hydrolyze the K63-linked ubiquitin chain . OTUD7B Despite the dramatic advance in understanding the roles and regulations of E3 ubiquitin ligases/DUBs in human diseases, only a few of their inhibitors are clinically used or in clinical trials . In the Since identification of the mTOR kinase in the 1990s, significant progress has been made in the mTOR research field, notably mTORC1 signaling. Many new components and regulators were discovered to make up the delicate mTOR network. Given the essential role of the mTOR pathway in many fundamental biological processes, complete loss of most mTOR components has been proved to be embryonically lethal in mouse, such as mTOR, Raptor, mLST8, Rictor, Sin1, Rheb, and RagA . TherefoTo date, more than 200 types of PTMs have been identified and play key roles in regulating protein functions in all signaling pathways. However, only a few kinds of PTMs have been validated in a dozen mTOR components and linked to the dysregulation of mTOR signaling. Therefore, identifying functional PTMs on mTOR components would be of great interest in future studies, which may provide more strategies for therapeutically targeting mTOR signaling in clinic.


Infectious bursal disease virus (IBDV) causes acute, highly contagious, immunosuppressive, and lethal infectious disease in young chickens and mainly infects the bursa of Fabricius (BF). To investigate interactions between IBDV and its host, RNA sequencing was applied to analyze the responses of the differentially expressed transcriptional profiles of BF infected by very virulent IBDV (vvIBDV).In total, 317 upregulated and 94 downregulated mRNAs were found to be significantly differentially expressed in infected chickens, compared to controls. Long non-coding RNA (lncRNA) and circular RNA (circRNA) alterations were identified in IBDV-infected chickens, and significantly different expression was observed in 272 lncRNAs and 143 circRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to assess the functions of significantly dysregulated genes, which showed that the JAK-STAT signaling pathway, the NOD-like receptor signaling pathway, and apoptosis may be activated by IBDV infection. We predicted interactions between differentially expressed genes and produced lncRNA-mRNA and circRNA-miRNA-mRNA regulator network.The present study identified the expression profiles of mRNAs, lncRNAs, and circRNAs during vvIBDV infection and provides new insights into the pathogenesis of IBDV and antiviral immunity of the host. Birnaviridae; it can cause acute, highly contagious, and immunosuppressive disease in chickens aged 3–6 weeks, leading to high mortality and considerable economic losses [Infectious bursal disease virus (IBDV), a non-enveloped double-stranded RNA virus, is a member of the family c losses , 2. Seroc losses , 5. The High-throughput sequencing technology and proteomic approaches have been used for this purpose, and various types of non-coding RNAs (ncRNAs) are also gaining increasing attention in this regard , includiAlthough many studies have been conducted to assess the effects of host lncRNAs on IBDV infection and its underlying molecular mechanisms , 19–21, In total, 589,776,342 raw reads were obtained from the control and IBDV-infected bursa tissues. After data filtering and quality control, 584,284,990 clean reads of high quality were retained, and the proportion of clean reads ranged from 98.99 to 99.13%  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to analyze the 411 differentially expressed mRNAs and the target genes of the differentially expressed lncRNAs and circRNAs to examine the functions of gene dysregulation during IBDV infection. GO enrichment of the biological processes (BPs), cellular components (CCs), and molecular functions (MFs) of the differentially expressed mRNAs, lncRNAs, and circRNAs is shown in Figs. KEGG is the main public pathway-related database, and it has been used previously to determine the significantly enriched pathways of dysregulated gene products during IBDV infection , 24. TheSTAT1, STAT3, STAT4, TRIM25, and IFIH1 , TRIM25, IFIT1, Mx1; of STAT1, STAT4, and of Toll-like receptors, including TLR3 and TLR7, is increased in IBDV-infected chickens or DF-1 or DT40 cells [STAT1, STAT3, STAT4, TRIM25, IFIT1, and Mx1 in the bursal tissue, and our results were in line with those of previous studies. In STAT1, a member of the STAT family, phosphorylation induces the expression of interferon-stimulating genes through a series of signal transduction steps to elicit antiviral mechanisms [STAT1 can be regulated by many differentially expressed lncRNAs, suggesting that STAT1 may be an important regulator during IBDV infection in chickens. Additionally, STAT3, STAT4, TRIM25, IFIT1, and Mx1 may be involved in interactions between vvIBDV and the host.Host cells counteract the invasion of viral particles through a cascade of mechanisms, and IBDV infection elicits the expression of various genes. Previous study reported that the expression of interferon regulatory factor 8  by binding viral RNA-activated RIG-I [Mx1, OAS, and IFIH1, through the JAK-STAT signaling pathway [STAT1, STAT3, STAT4, IFIH1 and TRIM25, might play a vital role in the IBD anti-viral response. Intriguingly, gene targeting studies have shown that STAT1 target genes can promote antagonizing proliferation and inflammation; however, STAT3 was the opposite [The potential functions of lncRNAs are commonly predicted according to their target genes. Increasing evidence suggests that lncRNAs have important roles in adaptive or innate immune responses –43. In aed RIG-I , 45. It  pathway , 46. Theopposite , 48. TheSTAT1 and IRF7 by binding to miR-1587-x and miR-4507-y respectively, suggesting that these circRNAs also play an important role in IBDV pathogenicity.CircRNAs, a newly discovered class of ncRNAs, can affect the prognosis of diseases, especially tumors , 50 by rSTAT1, STAT3, STAT4, IFIH1 and TRIM25 expression. Our results provide new insights into the pathways and mechanisms that mediate host immune responses to vvIBDV. Further studies are needed to explore the biological functions of LOC107053928, LOC107054815, LOC107053352, and LOC107053557 during vvIBDV infection.Many signaling pathways were found to be involved in IBDV infection, particularly the JAK-STAT signaling pathways; however, further research is needed to assess their effects on the pathogenesis of IBDV infection. Additionally, we constructed lncRNA-mRNA and circRNA-miRNA-mRNA co-expression networks and predicted that LOC107053928, LOC107054815, LOC107053352, and LOC107053557 may affect viral replication by regulating Three-week-old specific-pathogen-free (SPF) White Leghorn chickens were obtained from the Harbin Institute of Veterinary Medicine . A vvIBDV strain isolated from chicken BF and maintained in our laboratory was used in this study (strain LJ-5).3 × 50% embryo lethal dose (ELD) per 0.2 mL via eye-nose drops [Eighteen chickens were randomly assigned to two groups (control group and vvIBDV infection group) with nine individuals per group, and the two groups were housed independently. Chickens in the control group were injected with phosphate-buffered saline , and the vvIBDV infection group chickens were inoculated with the LJ-5 strain virus at a dose of 10se drops , 57. These drops . SamplesTotal RNA was isolated using Trizol reagent , and the purity and integrity of the RNA were measured using a Nanodrop micro-spectrophotometer  and an Agilent 2100 Bioanalyzer , respectively. RNA samples qualified for analyses under the following conditions: concentration ≥ 100 ng/μL, RNA integrity number ≥ 7.0, and RNA 260/280 ratio of 1.8–2.0. For RNA sequencing, 3 μg RNA per sample was used, and ribosomal RNA was removed to generate sequencing libraries using a NEBNext® Ultra™ RNA Library Prep kit . First-strand complementary DNA was synthesized using random hexamer primers and ProtoScript II Reverse Transcriptase, and the second strand was generated using a Second Strand Synthesis Enzyme Mix. Uracil N Glycosylase was used to digest complementary DNA, and Agencourt AMPure XP Beads  were used to purify library fragments to retain DNA fragments of 150–200 bp. The quality of the libraries was evaluated using a High Sensitivity DNA assay Kit (Agilent Technologies), and the libraries were sequenced on an Illumina HiSeq 4000 platform by a commercial service provider . For circRNA sequencing, RNase R  was used on rRNA-depleted RNAs to remove linear RNAs before sequencing library preparation.Raw reads containing adapters or bases of low quality affect assembly and analysis; therefore, to obtain clean high-quality sequences, raw reads were filtered to remove adapters, low-quality reads, and poly-N reads. Clean high-quality data was used for further analyses.Gallus gallus GRCg6a reference genome using TopHat2 (v. 2.1.1) [The short reads alignment tool Bowtie 2 (v. 2.2.8)  was used. 2.1.1) ; Cufflin. 2.1.1)  was usedCNCI (version: 2.0)  and CPC p < 0.05) and predicted target genes (fold change > 2 and p < 0.05) were chosen to construct a co-expression regulatory network, which was visualized using Cytoscape software (v3.6.0) [lncRNAs are involved in many post-transcriptional regulation processes, as are some other small RNAs such as microRNA (miRNA), the regulation of which depends on complementary base pairing. Some antisense lncRNAs may regulate gene silencing, transcription, and mRNA stability. To assess the interactions between lncRNAs and mRNAs, RNAplex software  was used(v3.6.0) . The bin(v3.6.0) , and thehttp://www.geneontology.org/) and KEGG (http://www.kegg.jp/kegg/) databases were used to perform GO and KEGG enrichment analyses, respectively. GO terms and KEGG pathways with p values < 0.05 were considered significantly enriched.To understand the functions of the differentially expressed transcripts, including the mRNA and the target genes of the lncRNAs and circRNAs, the GO , cellular components (CCs), and molecular functions (MFs), respectively.Additional file 11. KEGG pathway analysis of mRNAs.Additional file 12: Figure S2. Kyoto Encyclopedia of Genes and Genomes pathway enrichment for the antisense, cis, and trans roles of the differentially expressed lncRNAs in chicken BF between the two groups; a antisense; b cis; and c trans. The vertical axis shows the pathways, and the horizontal axis indicates the Rich factor. The dot size indicates the number of differentially expressed genes in the pathway, and the coloration corresponds to the Q-value range.Additional file 13. LncRNA-mRNA co-expression relationships.Additional file 14: Table S3. The target genes of lncRNAs.Additional file 15. CircRNA/mRNA-miRNA co-expression relationships.Additional file 16. ARRIVE guidelines checklist.