No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
JNK regulates FoxO-dependent autophagy in neurons
Abstract
The cJun N-terminal kinase (JNK) signal transduction pathway is implicated in the regulation of neuronal function. JNK is encoded by three genes that play partially redundant roles. Here we report the creation of mice with targeted ablation of all three Jnk genes in neurons. Compound JNK-deficient neurons are dependent on autophagy for survival. This autophagic response is caused by FoxO-induced expression of Bnip3 that displaces the autophagic effector Beclin-1 from inactive Bcl-XL complexes. These data identify JNK as a potent negative regulator of FoxO-dependent autophagy in neurons.
... We next aimed to explore possible mechanisms to explain the induction of FOXOs and TFEB regulated by D-BHB. SIRT1 and SIRT2 are NAD + -dependent deacetylases that are involved in the regulation of autophagy; additionally, SIRT2 is highly expressed in the nervous system [38], as confirmed in cultured neurons using a specific antibody against Then, using qRT-PCR, the expression of genes downstream these transcription factors [34][35][36][37] was determined. An increase in mRNA of lysosomal proteases (Ctsb and Ctsd) and lysosomal transmembrane proteins (Lamp1 and Lamp2) was observed in neurons treated with D-BHB for 24 and 48 h ( Figure 7D). ...
... Together, the dat suggest the stimulation of autophagy in the brain of mice treated with the KD, as prev ously reported [45]. Then, we aimed to investigate whether the KD induces changes in the content of the mitophagy proteins Parkin, BNIP3 and NIX/BNIP3L, which are regulated by FOXO1 and FOXO3a [21,36,46,47]. Parkin is an E3 ubiquitin ligase that is recruited from the cytosol to the outer mitochondrial membrane (OMM) by PINK1 when the mitochondrial membrane is depolarized and mitochondria are damaged. ...
Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-β-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic–lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.
... Snake venom did not affect the phosphorylation of the mTOR downstream targets p70 ribosomal protein S6 kinase (p70S6K Thr389, Ser371), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, Thr 37/46), and ULK ( Figure 4A and 4B), indicating that snake venom induces autophagy via an mTOR-independent pathway. JNK is reportedly involved in mTORindependent autophagy [25][26][27][28]. Therefore, JNK activation following snake venom treatment has been investigated. ...
... JNK signaling is critical for the induction of mTOR-independent autophagy. JNK regulates cell proliferation, cell differentiation, apoptosis, and autophagy and is activated by extracellular signals [25][26][27]33]. Hence, the phosphorylation of JNK was increased in CRCs treated with snake venom. ...
Snake venom contains many proteins that help treat or prevent thrombosis, cardiovascular disease, and cancer, and many studies have been reported in this regard. It has recently been reported that autophagy exerts anticancer effects by inducing tumor cell death and inhibiting cell growth. In this study, we investigated the effect of snake venom on autophagy. Unlike normal colon cells, LC3-II protein levels and LC3 puncta accumulation are increased in snake venom-treated colorectal cancer cells. Inhibition of autophagy by treating cells with hydroxychloroquine, an autophagy inhibitor, prevented snake venom-induced cell death, indicating that snake venom indeed induces autophagic cell death in human colorectal cancer cells. In addition, we demonstrated that activated JNK, and not mTOR signaling, is an upstream effector controlling autophagy. Pretreatment with SP600125, a JNK inhibitor, reversed snake venom-induced autophagy and cell death, indicating that JNK plays a critical role in snake venom-induced autophagy. This study demonstrated that snake venom can function as an anticarcinogenby induction autophagy.
... For example, flavonoids in grapefruit juice demonstrated potent inhibition of the cytochrome P450 (CYP) protein family, critical for drug metabolism. The abrupt inhibition of CYP may potentially lead to excessive buildup of drugs increasing toxicity [336][337][338]. Regardless, polyphenols taken exclusively were harmless either in short, medium, or long-term supplementation in humans [339][340][341][342][343][344][345], which certainly encourages their application. ...
... Despite its crucial role in regulating cell survival and cell death via autophagy and apoptosis, MAPK signaling is demonstrated to be involved in pexophagy and mitophagy in yeast cells [224,333]. During increased oxidative stress in a cell, c-Jun N-terminal kinases (JNK) signaling, a type of MAPK signaling, gets highly activated which in turn activates FOXOs, BECN1 mediated autophagy, and c-Jun and c-Fos transcription factors mediate expression of autophagy-related genes [334][335][336]. However, prolonged chronic oxidative damage in a cell may trigger apoptosis through JNK signaling [337]. ...
... First Toy, that blocks ERN1-mediated unconventional splicing of XBP1 and thereby, prevents the occurrence of functional XBP1s, similarly impacted endogenous XBP1s and PINK1 expressions and influenced mitophagy markers and effectors in vivo. Second, overexpressed and endogenous XBP1s indeed modulated autophagy (LC3-II:LC3-I, SQSTM1, BECN1) and mitophagy (PRKN, UB [59], TIMM23, TOMM20) markers and effectors. Third, the depletion of endogenous PINK1 fully mimicked phenotypic modulations triggered by XBP1s depletion. ...
... By contrast, XBP1s was also shown to repress autophagy. Thus, XBP1s downregulates FOXO1 [63,64], a protein that enhances autophagy in human cancer cell lines [59,65]. Further, Hetz et al. show that Xbp1 depletion leads to an aggravation of experimental amyotrophic lateral sclerosis (ALS) due to an enhancement of autophagy in motor neurons [66]. ...
Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.
Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1
... Conversely, inhibition of autophagy by lysosomal inhibitors causes an increase in ROS-induced cytotoxicity [105,106]. ROS can directly induce autophagy by activating protein kinases, including AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK), which are essential for the activation of autophagy [107][108][109]. For example, ROS directly oxidize Cys299/Cys304 of AMPK and activate its kinase activity without reducing the cellular ATP levels [110]. ...
... Conversely, inhibition of autophagy by lysosomal inhibitors causes an increase in ROS-induced cytotoxicity [105,106]. ROS can directly induce autophagy by activating protein kinases, including AMP-activated protein kinase (AMPK) and c-Jun Nterminal kinase (JNK), which are essential for the activation of autophagy [107][108][109]. For example, ROS directly oxidize Cys299/Cys304 of AMPK and activate its kinase activity without reducing the cellular ATP levels [110]. ...
Autophagy, a main degradation pathway for maintaining cellular homeostasis, and redox homeostasis have recently been considered to play protective roles in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Increased levels of reactive oxygen species (ROS) in neurons can induce mitochondrial damage and protein aggregation, thereby resulting in neurodegeneration. Oxidative stress is one of the major activation signals for the induction of autophagy. Upon activation, autophagy can remove ROS, damaged mitochondria, and aggregated proteins from the cells. Thus, autophagy can be an effective strategy to maintain redox homeostasis in the brain. However, the interaction between redox homeostasis and autophagy is not clearly elucidated. In this review, we discuss recent studies on the relationship between redox homeostasis and autophagy associated with neurodegenerative diseases and propose that autophagy induction through pharmacological intervention or genetic activation might be a promising strategy to treat these disorders.
... Many studies have shown that FOXO1 induces autophagy in cardiomyocytes and cancer cells. FOXO1 has been identified as a gene that encodes for a transcription factor involved in modulating autophagy in neurons (Xu et al., 2011). ...
Recently, large-scale scRNA-seq datasets have been generated to understand the complex signaling mechanisms within the microenvironment of Alzheimer’s Disease (AD), which are critical for identifying novel therapeutic targets and precision medicine. However, the background signaling networks are highly complex and interactive. It remains challenging to infer the core intra- and inter-multi-cell signaling communication networks using scRNA-seq data. In this study, we introduced a novel graph transformer model, PathFinder, to infer multi-cell intra- and inter-cellular signaling pathways and communications among multi-cell types. Compared with existing models, the novel and unique design of PathFinder is based on the divide-and-conquer strategy. This model divides complex signaling networks into signaling paths, which are then scored and ranked using a novel graph transformer architecture to infer intra- and inter-cell signaling communications. We evaluated the performance of PathFinder using two scRNA-seq data cohorts. The first cohort is an APOE4 genotype-specific AD, and the second is a human cirrhosis cohort. The evaluation confirms the promising potential of using PathFinder as a general signaling network inference model.
... The JNK pathway is activated in response to a variety of stimuli, such as oxidative stress, inflammation, DNA damage, and cytoskeleton remodeling (11)(12)(13). JNK pathways are reported to promote the expression of various autophagy related genes including Atg5, Atg7 and Beclin-1 directly or through FoxOs, thereby activating autophagy (14). Besides, JNK signaling plays an important role in T cell activation and proliferation (15,16). ...
Background
Acute rejection (AR) after liver transplantation (LT) remains an important factor affecting the prognosis of patients. CD8⁺ T cells are considered to be important regulatory T lymphocytes involved in AR after LT. Our previous study confirmed that autophagy mediated AR by promoting activation and proliferation of CD8⁺ T cells. However, the underlying mechanisms regulating autophagy in CD8⁺ T cells during AR remain unclear.
Methods
Human liver biopsy specimens of AR after orthotopic LT were collected to assess the relationship between JNK and CD8⁺ T cells autophagy. The effect of JNK inhibition on CD8⁺ T cells autophagy and its role in AR were further examined in rats. Besides, the underlying mechanisms how JNK regulated the autophagy of CD8⁺ T cells were further explored.
Results
The expression of JNK is positive correlated with the autophagy level of CD8⁺ T cells in AR patients. And similar findings were obtained in rats after LT. Further, JNK inhibitor remarkably inhibited the autophagy of CD8⁺ T cells in rat LT recipients. In addition, administration of JNK inhibitor significantly attenuated AR injury by promoting the apoptosis and downregulating the function of CD8⁺ T cells. Mechanistically, JNK may activate the autophagy of CD8⁺ T cells through upregulating BECN1 by inhibiting the formation of Bcl-2/BECN1 complex.
Conclusion
JNK signaling promoted CD8⁺ T cells autophagy to mediate AR after LT, providing a theoretical basis for finding new drug targets for the prevention and treatment of AR after LT.
... Reduced insulin receptor and insulin-like growth factor-1 receptor signaling decreased Aβ toxicity in a rodent model, which might be induced by FOXOs, especially by FOXO1 and FOXO3 [157]. FOXO1 is involved in the autophagy of neurons [158], and the rs7981045 SNP variant of FOXO1 is associated with poor responses to acetylcholine esterase inhibitor treatment in patients with AD [159]. MiR-181a is an miRNA associated with cognitive function in pentylenetetrazol-induced epileptic rats [160], and miR-181a expression is reduced in APP -/PS1 -mice. ...
Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) are comorbidities that result from the sharing of common genes. The molecular background of comorbidities can provide clues for the development of treatment and management strategies. Here, the common genes involved in the development of the two diseases and in memory and cognitive function are reviewed. Network clustering based on protein-protein interaction networks identified tightly connected gene clusters that have an impact on memory and cognition among the comorbidity genes of AD and T2DM. Genes with functional implications were intensively reviewed, and relevant evidence was summarized. Gene information will be useful in the discovery of biomarkers and the identification of therapeutic targets for AD.
... Many studies have shown that FOXO is associated with autophagy in various cell types from vertebrates to invertebrates (46,47). FOXO is involved in regulating neuronal autophagy in mice (48) and muscle autophagy in D. melanogaster (49). FOXO promotes the transcription of autophagy-related genes, such as Atg1, Atg4, Atg6, Atg8, and Atg12 (18)(19)(20). ...
Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.
... Many studies have shown that FOXO1 induces autophagy in cardiomyocytes and cancer cells. FOXO1 was also reported to be a gene encodes transcription factor that plays a role in autophagy modulation in neurons 33 . ...
Recently, large-scale scRNA-seq datasets have been generated to understand the complex and poorly understood signaling mechanisms within microenvironment of Alzheimer Disease (AD), which are critical for identifying novel therapeutic targets and precision medicine. Though a set of targets have been identified, however, it remains a challenging to infer the core intra- and inter-multi-cell signaling communication networks using the scRNA-seq data, considering the complex and highly interactive background signaling network. Herein, we introduced a novel graph transformer model, PathFinder, to infer multi-cell intra- and inter-cellular signaling pathways and signaling communications among multi-cell types. Compared with existing models, the novel and unique design of PathFinder is based on the divide-and-conquer strategy, which divides the complex signaling networks into signaling paths, and then score and rank them using a novel graph transformer architecture to infer the intra- and inter-cell signaling communications. We evaluated PathFinder using scRNA-seq data of APOE4-genotype specific AD mice models and identified novel APOE4 altered intra- and inter-cell interaction networks among neurons, astrocytes, and microglia. PathFinder is a general signaling network inference model and can be applied to other omics data-driven signaling network inference.
... Previously, FoxO1-mediated transcriptional regulation of several autophagy-related genes is established in neuronal development and neurodegenerative diseases. [80][81][82][83] Because autophagy plays a vital role in JEV infection, we tested whether JEV infection has any significant effect on the AKT-FoxO1 axis. Interestingly, we observed that the infection of Neuro-2a cells with JEV attenuated the phosphorylation of AKT and its downstream target FoxO1 ( Figures 3I and S2A). ...
... c-Jun N-terminal kinase (JNK) is a MAPK signaling molecule that is activated in response to various stimuli, including oxidative stress (Davis, 2000;Weston and Davis, 2007), and plays an essential role in apoptosis (Dhanasekaran and Reddy, 2008). In mammals, there are three isoforms of JNK: JNK1, JNK2, and JNK3, which have overlapping and distinct functions (Sabapathy et al., 2004;Bode and Dong, 2007;Xu et al., 2011). Imidazole ketone erastin (IKE) is a derivative of erastin, a ferroptosis inducer that is more soluble and stable in cells than erastin (Larraufie et al., 2015;Zhang Y et al., 2019). ...
Ferroptosis is an iron-dependent form of non-apoptotic cell death characterized by the generation of excess reactive oxygen species (ROS) and lipid peroxidation. However, it remains largely unknown whether signaling pathways, such as the ROS-activated mitogen-activated protein kinase (MAPK) pathways, affect ferroptosis. The
c-Jun N-terminal kinase (JNK), a MAPK signaling molecule that plays an essential role in apoptosis, is activated in response to various stimuli, including oxidative stress. In this study, we examined the functional role of JNK in cell death induced by imidazole ketone erastin (IKE), a potent ferroptosis inducer, using gene knockout technology. We found that JNK1/2 double-knockout cells, but not JNK1 or JNK2 single-knockout cells, were much more tolerant to IKE-induced cell death compared to control cells. We further demonstrated that IKE-induced increase in CTH and HMOX1, key genes in ferroptosis, in control cells was substantially suppressed by the depletion of JNK1 and JNK2. These results suggested that JNK1 and JNK2 play an important, overlapping role in ferroptosis. Our findings advance our understanding of the JNK pathway, which could contribute to future pharmacological developments in ferroptosis-related diseases such as cancer and neurodegeneration.
... One possible explanation for this finding is that transcriptional regulation may be involved in TTX-induced autophagy, as shown previously in several cell types (Fullgrabe et al., 2016;Sakamaki et al., 2018;Di Malta et al., 2019), including neurons (Xu et al., 2011;Nikoletopoulou et al., 2017;Pan et al., 2021). To test this possibility, we measured the expression of several autophagy-related genes, including Ulk1 (involved in autophagosome Figure 11. ...
Activity-dependent changes in protein expression are critical for neuronal plasticity, a fundamental process for the processing and storage of information in the brain. Among the various forms of plasticity, homeostatic synaptic up-scaling is unique in that it is induced primarily by neuronal inactivity. However, precisely how the turnover of synaptic proteins occurs in this homeostatic process remains unclear. Here, we report that chronically inhibiting neuronal activity in primary cortical neurons prepared from E18 Sprague-Dawley rats (both sexes) induces autophagy, thereby regulating key synaptic proteins for up-scaling. Mechanistically, chronic neuronal inactivity causes dephosphorylation of ERK and mTOR, which induces TFEB-mediated cytonuclear signaling and drives transcription-dependent autophagy to regulate αCaMKII and PSD95 during synaptic up-scaling. Together, these findings suggest that mTOR-dependent autophagy, which is often triggered by metabolic stressors such as starvation, is recruited and sustained during neuronal inactivity in order to maintain synaptic homeostasis, a process that ensures proper brain function and if impaired can cause neuropsychiatric disorders such as autism.
SIGNIFICANCE STATEMENT:
In the mammalian brain, protein turnover is tightly controlled by neuronal activation to ensure key neuronal functions during long-lasting synaptic plasticity. However, a long-standing question is how this process occurs during synaptic up-scaling, a process that requires protein turnover but is induced by neuronal inactivation. Here, we report that mTOR-dependent signaling—which is often triggered by metabolic stressors such as starvation—is “hijacked” by chronic neuronal inactivation, which then serves as a nucleation point for TFEB cytonuclear signaling that drives transcription-dependent autophagy for up-scaling. These results provide the first evidence of a physiological role of mTOR-dependent autophagy in enduing neuronal plasticity, thereby connecting major themes in cell biology and neuroscience via a servo loop that mediates autoregulation in the brain.
... SCFAs can readily enter the circulation from the gut and are transported across the BBB via monocarboxylate transporters (mCTs). Butyric acid, a metabolite of intestinal flora, inhibits histone deacetylase (HDAC) activity and induces forkhead box o (FOXO) to cause cell deaths through the transcriptional regulation of apoptotic genes, and its induction of autophagy genes has been shown as a key mechanism for neurological function protection (Xu et al., 2011). In addition, Parkinson's α-Syn aggregation is reduced in a FOXO-dependent manner with butyrate, which has a direct neuroprotective potential (Koh et al., 2012;Beharry et al., 2014). ...
Background: Parkinson’s disease (PD) is the most common neurodegenerative disease closely related to the immune system, among whose prodromes constipation is a representative symptom. Recent Randomized Controlled Trials (RCTs) have proved that probiotics can be used to effectively treat PD constipation, but the results are inconsistent. We performed a meta-analysis to assess the efficacy and safety of probiotic therapy on Parkinson’s constipation.
Methods: Questions about the research focus were constructed based on the Participants, Intervention, Comparison and Outcomes (PICO) Criteria . We searched electronic databases such as PubMed, Web of Science, EMBASE, Scopus, EBSCO, Cochrane and Google Scholar until March 2022 for eligible literatures. Our primary endpoints were stool frequency, stool consistency, the number of laxatives uses, UPDRS-III scores and adverse events.
Results: 12 eligible studies (n = 818 patients) met the inclusion and endpoint criteria. Meta-analysis results showed that constipation symptoms were improved after probiotic treatment, including an increased stool frequency (WMD = 0.94, 95% CI:0.53 to 1.34; OR = 3.22, 95% CI:1.97–5.29), an improved stool consistency (WMD = 1.46, 95% CI:0.54–2.37), a reduced use of laxatives (WMD = −0.72, 95%CI: −1.04 to−0.41), and also a reduced Parkinson’s UPDRS-III score (WMD = −6.58, 95%CI: −12.02 to −1.14); there was no significant difference in total adverse events (OR = 0.82, 95%CI:0.39–1.72).
Conclusion: Our analysis suggests that probiotics can be used to improve the constipation and motor symptoms for patients with Parkinson’s constipation, possibly by reducing the inflammatory response and improving gut-brain axis neuron function, whose safety also proved to be good.
... The transcriptional activity of FOXO3 is regulated by its translocation from the cytosol to the nucleus and vice versa. Indeed, in the presence of growth factors, FOXO3 is phosphorylated by Akt, which causes its cytoplasmic retention and consequent inhibition of its transcriptional activity [66][67][68]. ...
Autophagy is an evolutionarily conserved and tightly regulated process that plays an important role in maintaining cellular homeostasis. It involves regulation of various genes that function to degrade unnecessary or dysfunctional cellular components, and to recycle metabolic substrates. Autophagy is modulated by many factors, such as nutritional status, energy level, hypoxic conditions, endoplasmic reticulum stress, hormonal stimulation and drugs, and these factors can regulate autophagy both upstream and downstream of the pathway. In cancer, autophagy acts as a double-edged sword depending on the tissue type and stage of tumorigenesis. On the one hand, autophagy promotes tumor progression in advanced stages by stimulating tumor growth. On the other hand, autophagy inhibits tumor development in the early stages by enhancing its tumor suppressor activity. Moreover, autophagy drives resistance to anticancer therapy, even though in some tumor types, its activation induces lethal effects on cancer cells. In this review, we summarize the biological mechanisms of autophagy and its dual role in cancer. In addition, we report the current understanding of autophagy in some cancer types with markedly high incidence and/or lethality, and the existing therapeutic strategies targeting autophagy for the treatment of cancer.
... 43 A comparable study has suggested that Akt-induced FoxO1 phosphorylation excludes FoxO1 from the nucleus to cytoplasm, resulting in the enhancement of FoxO1-Atg7 interaction and promoting autophagy activation in the cytoplasm. 43,65 The AMPK-FoxO-autophagy pathway contributes to cell viability with the essential adaptation mechanism, maintaining cellu- 69 and activates AMPK via SESN to promote autophagy activation. 50,51 Apart from binding to the promotors to upregulate the expression of autophagy-related genes, FoxOs independently activate autophagy without transactivation functions. ...
Autophagy is designated as a biological recycling process to maintain cellular homeostasis by the sequestration of damaged proteins and organelles in plasma and cargo delivery to lysosomes for degradation and reclamation. This organelle recycling process promotes chondrocyte homeostasis and has been previously implicated in osteoarthritis (OA). Autophagy is widely involved in regulating chondrocyte degeneration markers such as MMPs, ADAMSTs and Col10 in chondrocytes. The critical autophagy‐related (ATG) proteins have now been considered the protective factor against late‐onset OA. The current research field proposes that the autophagic pathway is closely related to chondrocyte activity. However, the mechanism is complex yet needs precise elaboration. This review concluded that FoxO1, a forkhead O family protein, which is a decisive mediator of autophagy, facilitates the pathological process of osteoarthritis. Diverse mechanisms regulate the activity of FoxO1 and promote the initiation of autophagy, including the prominent AMPK and Sirt‐2 cellular pathways. FoxO1 transactive is regulated by phosphorylation and acetylation processes, which modulates the downstream ATGs expression. Furthermore, FoxO1 induces autophagy by directly interacting with ATGs proteins, which control the formation of autophagosomes and lysosomes fusion. This review will discuss cutting‐edge evidence that the FoxO–autophagy pathway plays an essential regulator in the pathogenesis of osteoarthritis.
... In adult neural stem and progenitor cells, ablation of FoxO3 reduces Bnip3 and Bnip3L expression and mitochondrial turnover but increases aggregate levels [33]. FoxO1 induction of Bnip3 was also observed in neurons lacking JNK [93] and in skeletal muscle [94]. Overexpression of Sirt1 deactivates FoxO1 and FoxO3 through deacetylation, thereby suppressing Bnip3 [94]. ...
Mitochondria play essential roles in cellular energetics, biosynthesis, and signaling transduction. Dysfunctional mitochondria have been implicated in different diseases such as obesity, diabetes, cardiovascular disease, nonalcoholic fatty liver disease, neurodegenerative disease, and cancer. Mitochondrial homeostasis is controlled by a triad of mitochondrial biogenesis, dynamics (fusion and fission), and autophagy (mitophagy). Studies have underscored FoxO transcription factors as key mitochondrial regulators. Specifically, FoxOs regulate mitochondrial biogenesis by dampening NRF1-Tfam and c-Myc-Tfam cascades directly, and inhibiting NAD-Sirt1-Pgc1α cascade indirectly by inducing Hmox1 or repressing Fxn and Urod. In addition, FoxOs mediate mitochondrial fusion (via Mfn1 and Mfn2) and fission (via Drp1, Fis1, and MIEF2), during which FoxOs elicit regulatory mechanisms at transcriptional, posttranscriptional (e.g. via miR-484/Fis1), and posttranslational (e.g. via Bnip3-calcineurin mediated Drp1 dephosphorylation) levels. Furthermore, FoxOs control mitochondrial autophagy in the stages of autophagosome formation and maturation (e.g. initiation, nucleation, and elongation), mitochondria connected to and engulfed by autophagosome (e.g. via PINK1 and Bnip3 pathways), and autophagosome-lysosome fusion to form autolysosome for cargo degradation (e.g. via Tfeb and cathepsin proteins). This article provides an up-to-date view of FoxOs regulating mitochondrial homeostasis and discusses the potential of targeting FoxOs for therapeutics.
... In our study, both total JNK (1/2) and phosphorylated JNK (1/2) were increased significantly by AA, and this elevation could be suppressed by a JNK inhibitor, which also altered the expression of cell cycle-related proteins. FoxO transcription factors (FoxO1, FoxO3a, FoxO4 and FoxO6), which are downstream of the JNK signaling pathway, are important transcriptional regulators of target genes like Cyclin D, cyclin E, p21 and p27 that function in cell proliferation, differentiation and the cell cycle [15,[44][45][46][47][48][49]. We observed that total FoxO1, FoxO3a and phosphorylated FoxO1/3a were elevated upon AA-induced RAW264.7 cell cycle arrest. ...
Background: Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. Results: AA exposure reduced the viability and number of macrophages in a dose-and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose-and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Conclusion: Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.
... Recent experiments have confirmed that JNK activation responds to oxidative stress by inhibiting FOXO-induced autophagy related gene expression, leading to reduced clearance of cell debris and aggregative proteins [67]. Thus, the JNK pathway plays a deleterious role in neurological impairment in neurodegenerative models by promoting apoptosis and inhibiting autophagy [68]. Under oxidative stress caused by aberrant protein synthesis, aggregation, or mitochondrial dysfunction, neurons or glial cells regulate nuclear translocation and activity of FOXO1 through PI3K/Akt and JNK pathways, hence mediating the pathological process of neurodegeneration. ...
Alzheimer’s disease (AD) and Huntington’s disease (HD) are destructive worldwide diseases. Efforts have been made to elucidate the process of these two diseases, yet the pathogenesis remains elusive as it involves a combination of multiple factors, including genetic and environmental ones. To explore the potential role of forkhead box O1 (FOXO1) in the development of AD and HD, we identified 1,853 differentially expressed genes (DEGs) from 19,414 background genes in both the AD&HD/control and FOXO1-low/high groups. Four coexpression modules were predicted by the weighted gene coexpression network analysis (WGCNA), among which blue and turquoise modules had the strongest correlation with AD&HD and high expression of FOXO1. Functional enrichment analysis showed that DEGs in these modules were enriched in phagosome, cytokine-cytokine receptor interaction, cellular senescence, FOXO signaling pathway, pathways of neurodegeneration, GABAergic synapse, and AGE-RAGE signaling pathway in diabetic complications. Furthermore, the cross-talking pathways of FOXO1 in AD and HD were jointly determined in a global regulatory network, such as the FOXO signaling pathway, cellular senescence, and AGE-RAGE signaling pathway in diabetic complications. Based on the performance evaluation of the area under the curve of 85.6%, FOXO1 could accurately predict the onset of AD and HD. We then identified the cross-talking pathways of FOXO1 in AD and HD, respectively. More specifically, FOXO1 was involved in the FOXO signaling pathway and cellular senescence in AD; correspondingly, FOXO1 participated in insulin resistance, insulin, and the FOXO signaling pathways in HD. Next, we use GSEA to validate the biological processes in AD&HD and FOXO1 expression. In GSEA analysis, regulation of protein maturation and regulation of protein processing were both enriched in the AD&HD and FOXO1-high groups, suggesting that FOXO1 may have implications in onset and progression of these two diseases through protein synthesis. Consequently, a high expression of FOXO1 is a potential pathogenic factor in both AD and HD involving mechanisms of the FOXO signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and cellular senescence. Our findings provide a comprehensive perspective on the molecular function of FOXO1 in the pathogenesis of AD and HD.
... Therefore, the expression of core autophagy-related genes that are transcriptionally regulated by FoXo1 were analyzed. The results revealed that ATG5, BECN1 and MAP1LC3B were significantly upregulated in response to PTX treatment, consistent with previous reports that nuclear FoXo1 transcriptionally activates ATG5 and BECN1 to promote autophagy (41). after inhibition of FoXo1 in combination with PTX treatment, ATG5, VPS34, ATG4B, BECN1 and MAP1LC3B were markedly downregulated. ...
Triple‑negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX‑induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA‑MB‑231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX‑induced autophagy through a transcriptional activation pattern in MDA‑MB‑231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3‑kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3β. Knocking down FOXO1 attenuated the survival of MDA‑MB‑231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC.
... Therefore, we detected the expression of core autophagyrelated genes that are transcriptionally regulated by FOXO1. The results showed that ATG5, BECN1 and MAP1LC3B were signi cantly up-regulated when treatment with PTX, which was consistent with previous study where nuclear FOXO1 transcriptionally activated ATG5 and BECN1 to promote autophagy [37]. After inhibition of FOXO1 combined with PTX treatment, ATG5, BECN1, VPS34 and MAP1LC3B were markedly down-regulated. ...
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and often produces resistance to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX-induced tumor cell death and targeting autophagy is promising to improve the efficacy of tumor chemotherapy in recent years. Here, we reported that PTX induced both apoptosis and autophagy of MDA-MB-231 cells, and inhibition of autophagy enable to promote apoptotic cell death. Furthermore, we found that FOXO1 enhanced PTX-induced autophagy by a transcriptional activation pattern in MDA-MB-231 cells, which was associated with its downstream target genes ATG5 , VPS34 , BECN1 and MAP1LC3B . The knockdown of FOXO1 attenuated the survival of MDA-MB-231 cells under the PTX treatment. These findings will be beneficial to improve the treatment efficacy of PTX and to develop the autophagic target therapy of TNBC.
... However, recent investigations have demonstrated that the nucleus is a major regulator of autophagy [6]. The nuclear translocation of transcription factors such as Gln3 [7], TFEB [8], and FOXO [9] regulates the expression of autophagy-related genes and enhances autophagy. In addition, identification of the microRNAs associated with the autophagic process indicates that nuclear events are key mechanisms for autophagy regulation [10][11][12][13]. ...
Autophagy is an evolutionally conserved process that recycles aged or damaged intracellular components through a lysosome-dependent pathway. Although this multistep process is propagated in the cytoplasm by the orchestrated activity of the mTOR complex, phosphatidylinositol 3-kinase, and a set of autophagy-related proteins (ATGs), recent investigations have suggested that autophagy is tightly regulated by nuclear events. Thus, it is conceivable that the nucleolus, as a stress-sensing and -responding intranuclear organelle, plays a role in autophagy regulation, but much is unknown concerning the nucleolar controls in autophagy. In this report, we show a novel nucleolar–cytoplasmic axis that regulates the cytoplasmic autophagy process: nucleolar protein NOP53 regulates the autophagic flux through two divergent pathways, the ZKSCAN3-dependent and -independent pathways. In the ZKSCAN3-dependent pathway, NOP53 transcriptionally activates a master autophagy suppressor ZKSCAN3, thereby inhibiting MAP1LC3B/LC3B induction and autophagy propagation. In the ZKSCAN3-independent pathway, NOP53 physically interacts with histone H3 to dephosphorylate S10 of H3, which, in turn, transcriptionally downregulates the ATG7 and ATG12 expressions. Our results identify nucleolar protein NOP53 as an upstream regulator of the autophagy process.
... In mouse muscle cells, some of the Atg genes including Lc3b, and Gabarapl1 are directly targeted and activated by FOXO3 as indicated by chromatin immunoprecipitation (ChIP) assays [62,63]. In neurons, transcription of Atg genes such as Atg3, Atg5, and Atg12 is activated by FOXO1 [64]. ...
Macroautophagy (hereafter autophagy) is a cellular recycling process through which cytoplasmic contents are delivered into the lysosome/vacuole for degradation by double-membrane organelles, autophagosomes. Autophagy is essential for cell survival under stress; however, too much autophagy can also be detrimental. Autophagy activity can be regulated by modulating the size or the number of autophagosomes. Although there are more than 40 autophagy-related (ATG) genes that have been identified, it is not fully understood how most of these genes contribute to these aspects of regulation. Autophagy is highly conserved among eukaryotic cells, and its molecular machinery has been best characterized in the budding yeast Saccharomyces cerevisiae. In my thesis studies, I use budding yeast as the model organism, taking advantage of its utility in genetic/genomic screening; in addition, high-throughput sequencing and powerful autophagy assays have been developed uniquely in the yeast system, to explore how autophagy is modulated through transcriptional regulation. When I joined the Klionsky lab, I became involved in the study of a negative transcriptional regulator of autophagy, Ume6. Deletion of the UME6 gene results in an increase in the size, but not the number, of autophagosomes by increasing the expression of Atg8. From a subsequent genetic screen for autophagy modulators, I identified another transcription repressor of autophagy, Pho23. Intriguingly, Pho23 ended up being characterized as a specific regulator of the number, but not the size, of autophagosomes, or it can be viewed as controlling the rate of autophagosome formation by regulating Atg9 expression. These studies support a model whereby the size and numbers of autophagosomes are independently regulated through precise transcriptional regulation of different ATG genes. Collaborating with Amélie Bernard, a postdoc in the lab, to further explore the transcriptional regulation network of autophagy, we analyzed 139 yeast strains each deleted for a single gene encoding a transcription factor; we profiled the transcription of several ATG genes in each strain. Through this screen we identified Gcn4, Slf1, Gat1 and Gln3 as transcriptional activators, and Spt10, Fyv5, and Rph1 as transcriptional repressors of autophagy. We also further investigated the detailed molecular mechanisms of the regulation of autophagy by Rph1.
... Thus, any PTM that disrupts the interaction between BECN1 and its inhibitory partners has the potential for stimulating autophagy and vice versa. For example, DAPK (death-associated protein kinase) phosphorylates BECN1 and MAPK8/ JNK1 (mitogen-activated protein kinase 8) phosphorylates BCL2 [125,[128][129]. In both cases the interaction between BECN1 and BCL2 is disrupted, allowing BECN1 to associate with PIK3C3 and induce autophagy. ...
Macroautophagy (hereafter autophagy), literally defined as a type of self-eating, is a dynamic cellular process in which cytoplasm is sequestered within a unique compartment termed the phagophore. Upon completion, the phagophore matures into a double-membrane autophagosome that fuses with the lysosome or vacuole, allowing degradation of the cargo. Autophagy is involved in various aspects of cell physiology, and its dysregulation is associated with a range of diseases. Thus, Understanding of molecular mechanism and the regulation of autophagy is important for exploring potential therapy of diseases. The most prominent feature of autophagy is the formation of a double-membrane sequestering compartment, the phagophore; this transient organelle surrounds part of the cytoplasm and matures into an autophagosome, which subsequently fuses with the vacuole or lysosome to allow degradation of the cargo. Much attention has focused on the process involved in phagophore nucleation and expansion, but many questions remain. Here, we identified the yeast protein Icy2, which we now name Atg41, as playing a role in autophagosome formation. Atg41 interacts with the transmembrane protein Atg9, a key component involved in autophagosome biogenesis, and both proteins display a similar localization profile. Under autophagy-inducing conditions the expression level of Atg41 increases dramatically and is regulated by the transcription factor Gcn4. This work provides further insight into the mechanism of Atg9 function and the dynamics of sequestering membrane formation during autophagy. Autophagy is a tightly controlled cellular process by which cytosolic proteins, Studies have revealed the molecular mechanism of transcriptional regulation of autophagy-related (ATG) genes upon nutrient deprivation. However, little is known about their translational regulation. Here we found that Dhh1, a DExD/H-box RNA helicase, is required for efficient translation of Atg1 and Atg13, two proteins essential for autophagy induction. Dhh1 directly associates with ATG1 and ATG13 mRNAs under nitrogen starvation conditions. The structured regions shortly after the start codons of the two mRNAs are necessary for their regulation by Dhh1. Moreover, Eap1, an EIF4E binding protein, physically interacts with Dhh1 to facilitate the delivery of the Dhh1-ATG mRNA complex to the translation initiation machinery. These results suggest a model for how some ATG genes bypass the general translational suppression that occurs during nitrogen starvation to maintain a proper level of autophagy. The molecular mechansim of autophagosome formation has been an interesting topic over years due to its importance to autophagy process. This thesis enlarges the knowledge of Atg9 cycling system in yeast, which provides insight for understanding membrane donation process in autophagy. Diffterent levels of autophagy control are essential for cellular homestasis. This thesis, by investigating both transcriptional and translational regulation of autophagy, provides insight in understanding autophagy control. This will help further investigation on diseases resulting from autophagy dysfunction and their connections to autophagy regulation.
... Alternatively, nutrient availability activates anabolic pathways, such as insulin signaling, that directly attenuate autophagy (Codogno and Meijer, 2005;Mammucari et al., 2007;Meijer and Codogno, 2008;Russell et al., 2014). Regulation of autophagy by nutrient plenty occurs through two primary mechanisms: (1) phosphorylation events facilitated by the kinase mTORC1, and (2) induction of gene expression via the Forkhead box O (FOXO) family of transcription factors (Mammucari et al., 2007;Zhao et al., 2007;Liu et al., 2009;Xu et al., 2011;Sanchez et al., 2012;Xiong et al., 2012;Di Malta et al., 2019; Figure 1). Downstream of mTORC1 lies the ULK1 complex, required for initiation of autophagy. ...
Insulin is a paramount anabolic hormone that promotes energy-storage in adipose tissue, skeletal muscle and liver, and these responses are significantly attenuated in insulin resistance leading to type 2 diabetes. Contrasting with insulin’s function, macroautophagy/autophagy is a physiological mechanism geared to the degradation of intracellular components for the purpose of energy production, building-block recycling or tissue remodeling. Given that both insulin action and autophagy are dynamic phenomena susceptible to the influence of nutrient availability, it is perhaps not surprising that there is significant interaction between these two major regulatory mechanisms. This review examines the crosstalk between autophagy and insulin action, with specific focus on dysregulated autophagy as a cause or consequence of insulin resistance.
... responses in various human cells [54,55,56]. In the brain, the JNK pathway is fundamental in mediating physiological responses such as neuronal autophagy [57] and brain development, shown to be activated by TGF-beta via TAK1 and JNK [58], and pathological responses such as neuronal apoptosis [59], traumatic brain injuries [60,61] and microglia response to autoimmune inflammation [62]. The exact context-specific mechanisms that regulate JNK pathways in physiological and pathological responses in the various cell types are still not fully understood. ...
To differentiate between conditions of health and disease, current pathway enrichment analysis methods detect the differential expression of distinct biological pathways. System-level model-driven approaches, however, are lacking. Here we present a new methodology that uses a dynamic model to suggest a unified subsystem to better differentiate between diseased and healthy conditions. Our methodology includes the following steps: 1) detecting connections between relevant differentially expressed pathways; 2) construction of a unified in silico model, a stochastic Petri net model that links these distinct pathways; 3) model execution to predict subsystem activation; and 4) enrichment analysis of the predicted subsystem. We apply our approach to the TGF-beta regulation of the autophagy system implicated in autism. Our model was constructed manually, based on the literature, to predict, using model simulation, the TGF-beta-to-autophagy active subsystem and downstream gene expression changes associated with TGF-beta, which go beyond the individual findings derived from literature. We evaluated the in silico predicted subsystem and found it to be co-expressed in the normative whole blood human gene expression data. Finally, we show our subsystem’s gene set to be significantly differentially expressed in two independent datasets of blood samples of ASD (autistic spectrum disorders) individuals as opposed to controls. Our study demonstrates that dynamic pathway unification can define a new refined subsystem that can significantly differentiate between disease conditions.
... FOXO1 is frequently reported to be closely associated with autophagy. Activated FOXO1 enhances autophagy by increasing the expression of numerous autophagyrelated genes in various cells (Zhao et al., 2007(Zhao et al., , 2008Sengupta et al., 2009;Xu et al., 2011;Warr et al., 2013;Chi et al., 2016;Wang et al., 2018;. Activation of the FOXO family is regulated by complex mechanisms in various cells and tissues, while acetylation is one of them. ...
Vascular aging plays a pivotal role in the morbidity and mortality of elderly people. Decrease in autophagy leads to acceleration of vascular aging, while increase in autophagy leads to deceleration of vascular aging. And emerging evidence indicates that acetylation plays an important role in autophagy regulation; therefore, recent research has focused on an in-depth analysis of the mechanisms underlying this regulation. In this review, current knowledge on the role of acetylation of autophagy-related proteins and the mechanisms by which acetylation including non-autophagy-related acetylation and autophagy related acetylation regulate vascular aging have been discussed. We conclude that the occurrence of acetylation modification during autophagy is a fundamental mechanism underlying autophagy regulation and provides promising targets to retard vascular aging.
... The results suggested that inhibition of JNK is the key mechanism of action. JNK activation by ROS production was found to mediate autophagy initiation, 41,42 which leads to caspase-dependent apoptosis. 25,[43][44][45] In addition, a previous study suggested that JNK mediates autophagic cell death in Bax/Bak double-knockout cells, a pathway independent of apoptosis, 46 which is in line with the other studies suggesting caspase-independent autophagic cell death mediated by JNK. ...
Nobiletin was found to protect against acute myocardial infarction (AMI)‐induced cardiac function decline and myocardial remodeling, although the dose–effect relationship and underlying pathways remained unclear. In the current research, different doses of Nobiletin (7.5, 15 and 30 mg/kg/day) were administered to AMI rat model for 21 days. Survival rate, echocardiography, and histological analysis were assessed in vivo. In addition, MTT assay, flow cytometry, and Western blotting were conducted to explore Nobiletin's cytotoxicity and antiapoptotic effect on H9C2 cells. Mechanistically, the activation of MAPK effectors and p38 in vivo was studied. The results showed medium‐ and high‐dose Nobiletin could significantly improve survival rate and cardiac function and reduce the area of infarction and cardiac fibrosis. Medium dose showed the best protection on cardiac functions, whereas high dose showed the best protective effect on cellular apoptosis and histological changes. JNK activation was significantly inhibited by Nobiletin in vivo, which could help to explain the partial contribution of autophagy to AMI‐induced apoptosis and the discrepancy on dose–effect relationships. Together, our study suggested that JNK inhibition plays an important role in Nobiletin‐induced antiapoptotic effect in myocardial infarction, and medium‐dose Nobiletin demonstrated the strongest effect in vivo.
... Beclin1 can initiate autophagosome formation by binding Vps34 and Vps15, thereby forming the class III PI3K complex to induce autophagy. Previous studies have shown that a number of transcription factors are involved in regulation of Beclin1 expression, including p65, FoxO1, FoxO3, c-Jun, and E2F1 (Copetti et al., 2009;Li et al., 2009;Wang et al., 2010;Xu et al., 2011;Sanchez et al., 2012). On the contrary, as a Bcl-2-homology (BH)-3 domain only protein, Beclin1 interacts with other apoptotic Bcl-2 family members, which in turn regulates autophagy (Oberstein et al., 2007). ...
Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured in vitro, FSH treatment increased the level of the autophagic marker, LC3-II, as well as increased the number of autophagic vacuoles in GCs. The underlying molecular mechanism and biological functions were then investigated in porcine GCs. Our results demonstrated that FSH could upregulate Beclin1 levels in porcine GCs; however, this effect was blocked by LY294002 (a PI3K/AKT inhibitor) and SP600125 (SAPK/JNK inhibitor). Further research confirmed that the transcriptional factor, c-Jun, was phosphorylated by FSH, then translocated into the nucleus from the cytoplasm and bound to the BECLIN1 promoter region, and that LY294002, SP600125, or c-Jun knockdown prevented the increase in Beclin1 levels induced by FSH. Interestingly, inhibition of autophagy using chloroquine or SP600125 decreased progesterone production in porcine GCs treated with FSH, although the expression of StAR and P450scc was not disturbed. Moreover, FSH treatment reduced the average number and size of LDs in porcine GCs, but these effects were eliminated by knocking down the key autophagy genes, ATG5 and BECLIN1; in addition, the effect of FSH on promoting progesterone secretion by the cells was also reduced significantly. Based on the above results, we concluded that FSH promoted progesterone production by enhancing autophagy through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs, independent of the classical steroidogenic pathway.
... In mammals, certain foxo isoforms show transcriptional regulation of cytoskeletal genes during development (de la Torre-Ubieta et al., 2010). FOXOs are also implicated in regulating autophagy in neurons under Parkinson's disease models (Xu et al., 2011;Pino et al., 2014;Schaffner et al., 2018). It has also been shown that aging results in the activation of pro-inflammatory signals and proteotoxic stress in mice brains, and the depletion of neuronal FOXOs causes premature occurrences of these events and induces motor function impairment (Hwang et al., 2018). ...
The transcription factor foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. We profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. We also observed changes to the axonal cytoskeleton and an accumulation of late endosomes in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, we identify several downstream factors mediated through foxo-regulated NMJ homeostasis, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. Our work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling.
... Similarly, phosphorylation of the pro-survival protein Mcl-1 by JNK on residues Thr163 and Ser121 promotes apoptosis [369,370] while phosphorylation of Ser64 enhances its anti-apoptotic activity [371]; phosphorylation by ERK1/2 exhibited a similar dual effect [372]. The putative phosphorylation of the N-terminal domain of Smac/DIABLO by JNK mediates its release from the mitochondria to ubiquitinylate the inhibitors of apoptosis (IAPs) meant for degradation, thereby maintaining the apoptotic process [373][374][375]. ...
The major role of mitochondria is to provide cells with energy, but no less important are their roles in responding to various stress factors and the metabolic changes and pathological processes that might occur inside and outside the cells. The post-translational modification of proteins is a fast and efficient way for cells to adapt to ever changing conditions. Phosphorylation is a post-translational modification that signals these changes and propagates these signals throughout the whole cell, but it also changes the structure, function and interaction of individual proteins. In this review, we summarize the influence of kinases, the proteins responsible for phosphorylation, on mitochondrial biogenesis under various cellular conditions. We focus on their role in keeping mitochondria fully functional in healthy cells and also on the changes in mitochondrial structure and function that occur in pathological processes arising from the phosphorylation of mitochondrial proteins.
Forkhead box O (FOXO) family proteins are expressed in various cells, and play crucial roles in cellular metabolism, apoptosis, and aging. FOXO1-null mice exhibit embryonic lethality due to impaired endothelial cell (EC) maturation and vascular remodeling. However, FOXO1-mediated genome-wide regulation in ECs remains unclear. Here, we demonstrate that VEGF dynamically regulates FOXO1 cytosol-nucleus translocation. FOXO1 re-localizes to the nucleus via PP2A phosphatase. RNA-seq combined with FOXO1 overexpression/knockdown in ECs demonstrated that FOXO1 governs the VEGF-responsive tip cell-enriched genes, and further inhibits DLL4-NOTCH signaling. Endogenous FOXO1 ChIP-seq revealed that FOXO1 binds to the EC-unique tip-enriched genes with co-enrichment of EC master regulators, and the condensed chromatin region as a pioneer factor. We identified new promoter/enhancer regions of the VEGF-responsive tip cell genes regulated by FOXO1: ESM1 and ANGPT2. This is the first study to identify cell type-specific FOXO1 functions, including VEGF-mediated tip cell definition in primary cultured ECs.
Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) are comorbidities that result from the sharing of common genes. The molecular background of comorbidities can provide clues for the development of treatment and management strategies. Here, the common genes involved in the development of the two diseases and in memory and cognitive function are reviewed. Network clustering based on protein–protein interaction network identified tightly connected gene clusters that have an impact on memory and cognition among the comorbidity genes of AD and T2DM. Genes with functional implications were intensively reviewed and relevant evidence summarized. Gene information will be useful in the discovery of biomarkers and the identification of tentative therapeutic targets for AD and T2DM.
It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.
Autophagy involves the sequestration and delivery of cytoplasmic materials to lysosomes, where proteins, lipids, and organelles are degraded and recycled. According to the way the cytoplasmic components are engulfed, autophagy can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy. Recently, many studies have found that autophagy plays an important role in neurological diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, neuronal excitotoxicity, and cerebral ischemia. Autophagy maintains cell homeostasis in the nervous system via degradation of misfolded proteins, elimination of damaged organelles, and regulation of apoptosis and inflammation. AMPK-mTOR, Beclin 1, TP53 , endoplasmic reticulum stress, and other signal pathways are involved in the regulation of autophagy and can be used as potential therapeutic targets for neurological diseases. Here, we discuss the role, functions, and signal pathways of autophagy in neurological diseases, which will shed light on the pathogenic mechanisms of neurological diseases and suggest novel targets for therapies.
Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.
Upregulation and aggregation of the pre-synaptic protein, α-synuclein plays a key role in Parkinson's disease (PD) and mitochondrial dysfunction was surmised to be an upstream event in the disease pathogenesis. Emerging reports identified the role of nitazoxanide (NTZ), an anti-helminth drug, in enhancing mitochondrial oxygen consumption rate (OCR) and autophagy. In the present study, we have examined the mitochondrial effects of NTZ in mediating cellular autophagy and subsequent clearance of both endogenous and pre-formed aggregates of α-synuclein in cellular model of PD. Our results demonstrate that the mitochondrial uncoupling effects of NTZ results in the activation of AMPK and JNK, which in-turn leads to the enhancement of cellular autophagy. Also,1-methyl-4-phenylpyridinium (MPP+) mediated decrease in autophagic flux with a concomitant increase in the α-synuclein levels were ameliorated in cells treated with NTZ. However, in cells lacking functional mitochondria (ρ0 cells), NTZ did not mitigate MPP+ mediated alterations in the autophagic clearance of α-synuclein, indicating that the mitochondrial effects of NTZ play a crucial role in the clearance of α-synuclein by autophagy. Also, the ability of AMPK inhibitor, compound C, in abrogating NTZ mediated enhancement in the autophagic flux and α-synuclein clearance highlight the pivotal role of AMPK in NTZ mediated autophagy. Further, NTZ per se enhanced the clearance of preformed α-synuclein aggregates that were exogenously added to the cells. Overall, the results of our present study suggest that NTZ activates macroautophagy in cells due to its uncoupling effects on mitochondrial respiration via activation of AMPK-JNK pathway resulting in the clearance of both endogenous and pre-formed α-synuclein aggregates. As NTZ happens to possess good bioavailability and safety profile, considering this drug for PD based on its mitochondrial uncoupling and autophagy enhancing properties for mitigating mitochondrial reactive oxygen species (ROS) and α-synuclein toxicity appears to be a promising therapeutic option.
The endoplasmic reticulum (ER) plays an important role in the regulation of protein synthesis. Alterations in the folding capacity of the ER induce stress, which activates three ER sensors that mediate the unfolded protein response (UPR). Components of the pathways regulated by these sensors have been shown to regulate autophagy. The last corresponds to a mechanism of self‐eating and recycling important for proper cell maintenance. Ultraviolet radiation (UV) is an external damaging stimulus that is known for inducing oxidative stress, and DNA, lipid, and protein damage. Many controversies exist regarding the role of UV‐inducing ER stress or autophagy. However, a connection between the three of them hasn’t been addressed. In this review, we will discuss the contradictory theories regarding the relationships between UV radiation with the induction of ER stress and autophagy, as well as hypothetic connections between UV, ER stress, and autophagy.
Accumulating evidence has shown that Tau aggregates not only seed further tau aggregation within neurons but may also spread to neighboring cells and functionally connected brain regions. This process is referred to as “Tau propagation” and may explain the stereotypic progression of Tau pathology in the brains of Alzheimer’s disease (AD) patients. Tau filaments have distinct cellular and neuroanatomical distributions with morphological and biochemical differences, suggesting the ability to adopt disease-specific molecular conformations. These conformers may give rise to different neuropathological phenotypes, reminiscent of prion strains (strain hypothesis). Autophagy is one of the surveillance systems that contribute to protein homeostasis (proteostasis) through their degradation in lysosomes. The loss of proteostasis occurs with age and in neurodegenerative diseases, such as AD. Defective autophagy has been proposed to contribute to the accumulation of protein aggregates in the elderly brain and as well as the brains of patients with neurodegenerative conditions. In this chapter, we discuss the different Tau propagation mechanisms that may be involved in the cell-to-cell transmission of AD and related tauopathies. The mechanisms by which deficits in autophagic degradative pathways may contribute to the abnormal accumulation of tau in AD are also considered. Furthermore, the issue of pharmacological agents targeting specific tau species to promote the autophagic clearance of Tau from cells will be addressed. We propose our hypothesis that strain-specific autophagic degradation may contribute to the pathogenesis of tauopathies.
Macroautophagy/autophagy is an evolutionarily conserved intracellular lysosomal degradation system for recycling cytoplasmic components, including unnecessary macromolecules, damaged organelles, and aggregated proteins. It is an essential process that maintains cellular and organismal homeostasis in eukaryotes. Autophagy proceeds by formation of a double-membrane autophagosome structure, isolation of cargo into the autophagosome, and degradation of the cargo by fusion with a lysosome. Autophagic dysfunction has been implicated in various diseases, such as cancer, infections, and neurodegenerative diseases. In this chapter, we first summarize the core autophagy machinery that is essential for the formation of autophagosomes and autolysosomes. We then describe intracellular signaling pathways and key relevant molecules involved in the regulation of autophagy. Finally, target-specific autophagy (selective autophagy) will be discussed. Understanding the complicated cellular and molecular mechanisms of autophagy will aid the development of treatments for neurodegenerative diseases such as Alzheimer’s disease.
Aging is the single largest risk factor for many debilitating conditions, including heart diseases, stroke, cancer, diabetes, and neurodegenerative disorders. While far from understood in its full complexity, it is scientifically well-established that aging is influenced by genetic and environmental factors, and can be modulated by various interventions. One of aging's early hallmarks are aberrations in transcriptional networks, controlling for example metabolic homeostasis or the response to stress. Evidence in different model organisms abounds that a number of evolutionarily conserved transcription factors, which control such networks, can affect lifespan and healthspan across species. These transcription factors thus potentially represent conserved regulators of longevity and are emerging as important targets in the challenging quest to develop treatments to mitigate age-related diseases, and possibly even to slow aging itself. This review provides an overview of evolutionarily conserved transcription factors that impact longevity or age-related diseases in at least one multicellular model organism (nematodes, flies, or mice), and/or are tentatively linked to human aging. Discussed is the general evidence for transcriptional regulation of aging and disease, followed by a more detailed look at selected transcription factor families, the common metabolic pathways involved, and the targeting of transcription factors as a strategy for geroprotective interventions.
All cells and intracellular components are remodeled and recycled in order to replace the old and damaged cells. Autophagy is a process by which damaged and unwanted cells are degraded in the lysosomes. There are three different types of autophagy, and these include macroautophagy, microautophagy and chaperonemediated autophagy. Autophagy has an effect on adaptive and innate immunity, suppression of any tumour and the elimination of various microbial pathogens. The process of autophagy has both positive and negative effects and this pertains to any specific disease or its stage of progression. Autophagy involves various processes which are controlled by various signaling pathways, such as Jun N-terminal kinase, GSK3, ERK1, Leucine-rich repeat kinase 2, and PTEN-induced putative kinase 1 and parkin RBR E3. Protein kinases are also important for the regulation of autophagy as they regulate the process of autophagy either by activation or inhibition. In the present review, we discuss the kinase catalyzed phosphorylated reactions, the kinase inhibitors, types of protein kinase inhibitors and their binding properties to protein kinase domains, the structures of active and inactive kinases, and the hydrophobic spine structures in active and inactive protein kinase domains. The intervention of autophagy by targeting specific kinases may form the mainstay of treatment of many diseases and lead the road to future drug discovery.
Autophagy is a cellular catabolic pathway. During autophagy, cargo targeted for degradation is engulfed in double-membrane vesicles and delivered to the lysosome, where hydrolytic enzymes produce recycled metabolites. Autophagy is a necessary component in cellular stress response and is therefore regulated at several levels. This chapter reviews the modulation of autophagy at the posttranslational, transcriptional, and epigenetic regulatory levels. We discuss the role of phosphorylation by the nutrient-sensitive kinases mTOR and AMPK in the induction of autophagy and the role of ubiquitin-mediated degradation in restraining autophagy. We outline the role of the MiT/TFE family of proteins in regulating autophagy–lysosomal genes and how the regulation of MiT/TFE protein localization and stability controls lysosomal activity. We also describe the role of transcription factors, such as the FOXO, ZKSCAN3, and FOXO, in activating or repressing autophagy. Lastly, we describe how epigenetic remodeling through histone modification and regulation of protein translation contributes to regulation of autophagy activity. Altogether, we aim to show how multiple systems interact to ensure controlled activation of autophagy in response to stress and how autophagy regulation is a critical component in the orchestration of metabolism.
L’insuffisance cardiaque (IC) est une cause majeure de mortalité dans les pays industrialisés. Bien que les mécanismes intrinsèques de l’IC soient complexes, il a été démontré que le stress oxydant était un déterminant essentiel dans la progression de la dysfonction ventriculaire au cours de la pathologie. En ce sens, l’équipe s’est intéressée à la monoamine oxydase A (MAO-A), une enzyme située au niveau de la membrane externe mitochondriale, qui constitue la principale voie de dégradation des catécholamines et de la sérotonine. Au cours des dernières années, la MAO-A a été identifiée comme une source importante de stress oxydant dans le cœur. Le but de cette thèse a été de mieux caractériser les mécanismes d’action de la MAO-A, du stress oxydant et de ses effecteurs dans l’altération cardiaque, ce qui pourrait constituer une avancée importante dans la compréhension des mécanismes physiopathologiques de l’IC. Dans une première partie, nous nous sommes intéressés à l’infarctus du myocarde (IDM) chronique qui constitue la cause principale d’IC. Nous avons observé que la MAO-A était activée au cours de l’ischémie chronique chez des patients ainsi que chez des souris, et que cette activation participait au remodelage délétère post-IDM. D’un point de vue mécanistique, nous avons montré que l’activation de la MAO-A favorisait l’accumulation mitochondriale de 4-hydroxynonénal (4-HNE), un aldéhyde très réactif, via une peroxydation lipidique intra-mitochondriale. Cette accumulation a conduit à la fixation du 4-HNE sur certaines cibles mitochondriales, favorisant une surcharge calcique dans l’organite, une altération de la chaîne respiratoire et un déficit énergétique, qui participent au remodelage délétère post-IDM. Cette étude a donc permis de mettre en évidence le rôle clé de la dysfonction mitochondriale dans les dommages induits par la MAO-A. Dans une deuxième partie, nous avons cherché à caractériser les conséquences de la suractivation de la MAO-A sur le contrôle qualité mitochondrial à long terme. Les mitochondries dysfonctionnelles sont normalement éliminées par la voie autophagie-lysosome, mais des études ont montré que cette voie était progressivement altérée dans l’IC. Nos travaux ont montré que l’activation chronique de la MAO-A entrainait une altération de l’acidification lysosomale et un défaut de translocation nucléaire du facteur de transcription EB (TFEB), un « master régulateur » de l'autophagie et de la biogenèse des lysosomes.
Background:
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Autophagy plays a vital role in NAFLD development and progression. We aimed to establish a novel autophagy-related gene (ARG) signature as a therapeutic target in NAFLD patients based on high-throughput sequencing data.
Methods:
ARGs obtained from the HAMdb and high-sequencing data obtained from the Gene Expression Omnibus (GEO) database were analyzed to identify differentially expressed ARGs (DEARGs) between normal and NASH tissues. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to explore potential biological and pathological functions of DEARGs. The protein-protein interaction (PPI) network of the DEARGs was established through the STRING website, and visualized by Cytoscape. In addition, hub genes were validated by an independent dataset GSE89632. Finally, we performed Gene Set Variation Analysis (GSVA) pathway-related analysis to identify the pivotal signaling pathways and genes for the progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH).
Results:
A total of 76 DEARGs were identified in the GSE126848 dataset, of which 45 genes were upregulated and 31 genes were downregulated. GO analysis showed that the biological functions of DEARGs focused primarily on autophagy, cellular response to external stimulus, fibroblast proliferation, late endosome, and ubiquitin protein ligase binding. KEGG pathway analysis showed that these DEARGs were mainly involved in the apoptosis, PI3K-Akt signaling pathway, and estrogen signaling pathway. Among DEGs, 9 most closely related genes were identified from the PPI network. Furthermore, NOS3, IGF1, VAMP8, FOS, and HMOX1 were verified in the GSE89632 dataset. At last, the MAPK signal pathway was identified as important pathway, and JUN was identified as a key gene involved in the progression from NAFL to NASH.
Conclusion:
This study may provide credible molecular biomarkers in terms of screening and diagnosis for NAFLD. Meanwhile, it also serves as a basis for exploring the molecular mechanisms underlying the progression of NAFL to NASH.
Microtubules are non-covalent polymers of αβ-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential in vivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.
Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological, biochemical and cell biological experiments, we found a new pathogenic pathway that is affected in SBMA and results in compromised FAT. PolyQ-AR inhibits FAT in a human cell line and in squid axoplasm through a pathway that involves activation of cJun N-terminal kinase (JNK) activity. Active JNK phosphorylated kinesin-1 heavy chains and inhibited kinesin-1 microtubule-binding activity. JNK inhibitors prevented polyQ-AR-mediated inhibition of FAT and reversed suppression of neurite formation by polyQ-AR. We propose that JNK represents a promising target for therapeutic interventions in SBMA.
We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.
Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis.
We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association
of SAPK with the anti-apoptotic Bcl-xL protein. SAPK phosphorylates Bcl-xL on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-xL, a mutant Bcl-xL with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced
apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with
Bcl-xL in the apoptotic response to genotoxic stress.
Research in autophagy continues to accelerate, and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose. There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
c-Jun N-terminal kinases (JNKs) (comprising JNK1-3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1(-/-) DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.
Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule
stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate
microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis,
and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in
both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun
N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus
of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization
and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to
localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs,
to control neuronal dendritogenesis.
Programmed cell death is a crucial process in the normal development and physiology of metazoans, and it can be divided into several categories that include type I death (apoptosis) and type II death (autophagic cell death). The Bcl-2 family proteins are well-characterized regulators of apoptosis, among which multidomain pro-apoptotic members (such as Bax and Bak) function as a mitochondrial gateway at which various apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we have previously reported that these cells still die by autophagy in response to various death stimuli. In this study, we found that jun N-terminal kinase (JNK) was activated in etoposide- and staurosporine-treated, but not serum-starved, Bax/Bak DKO cells, and that autophagic cell death was suppressed by the addition of a JNK inhibitor and by a dominant-negative mutant of JNK. Studies with sek1(-/-)mkk7(-/-) cells revealed that disruption of JNK prevented the induction of autophagic cell death. Co-activation of JNK and autophagy induced autophagic cell death. Activation of JNK occurred downstream of the induction of autophagy, and was dependent on the autophagic process. These results indicate that JNK activation is crucial for the autophagic death of Bax/Bak DKO cells.
Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die
after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately
elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of
two key antiapoptotic proteins, Bcl-xL and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase
has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced
Bcl-xL/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal
mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-xL and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which
was blocked by a phosphodefective Bcl-xL mutant but not by a phosphomimetic Bcl-xL mutant, confirming Bcl-xL as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation,
from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-xL/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic
Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.
Bcl2-modifying factor (Bmf) is a member of the BH3-only group of proapoptotic proteins. To test the role of Bmf in vivo, we
constructed mice with a series of mutated Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation by the c-Jun NH2-terminal kinase (JNK) on Ser74, or mimic Bmf phosphorylation on Ser74. We report that the loss of Bmf causes defects in uterovaginal development, including an imperforate vagina and hydrometrocolpos.
We also show that the phosphorylation of Bmf on Ser74 can contribute to a moderate increase in levels of Bmf activity. Studies of compound mutants with the related gene Bim demonstrated that Bim and Bmf exhibit partially redundant functions in vivo. Thus, developmental ablation of interdigital
webbing on mouse paws and normal lymphocyte homeostasis require the cooperative activity of Bim and Bmf.
Selected vulnerability of neurons in Huntington's disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntington's disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntington's disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntington's disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt-induced inhibition of FAT.
An emerging theme in molecular neurobiology is the discovery of post-mitotic functions for proteins classically associated with mitotic transition in cycling cells. Although neurons have departed the cell cycle, they surprisingly express molecules in the cell cycle apparatus throughout development. The major mitotic cyclin-dependent kinase Cdk1 plays a critical role during the period of naturally occurring neuronal death in the nervous system and has been suggested to contribute to the pathogenesis of neurodegenerative diseases. However, the mechanisms by which Cdk1 promotes neuronal apoptosis are incompletely understood. A recent report by Yuan et al., (2008) has identified a direct relationship between this mitotic kinase and forkhead transcription factor FOXO1, a protein previously implicated in cell death, DNA damage repair and tumor suppression. Here we will discuss the key findings of this report and consider the implications of this mechanism to the regulation of other signal transduction pathways in brain development and diseases.
Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.
Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12-Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.
Interphase cultured monkey kidney (TC-7) cells contain distinct subsets of cellular microtubules (MTs) enriched in posttranslationally detyrosinated (Glu) or tyrosinated (Tyr) alpha tubulin (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779-789). To determine the relative stability of these subsets of MTs, we subjected TC-7 cells to treatments that slowly depolymerized MTs. We found Glu MTs to be more resistant than Tyr MTs to depolymerization by nocodazole in living cells, and to depolymerization by dilution in detergent-permeabilized cell models. However, in cold-treated cells, Glu and Tyr MTs did not differ significantly in their stability. Digestion of permeabilized cell models with pancreatic carboxypeptidase A, to generate Glu MTs from endogenous Tyr MTs, did not significantly alter the resistance of the endogenous Tyr MTs toward dilution-induced depolymerization. Furthermore, in human fibroblasts that contained no distinct Glu MTs, we observed a population of nocodazole-resistant MTs. These data suggest that Glu MTs possess enhanced stability against end-mediated depolymerization, yet detyrosination alone appears to be insufficient to confer this enhanced stability.
Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.
The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.
The glucocorticoid receptor (Gr, encoded by the gene Grl1) controls transcription of target genes both directly by interaction with DNA regulatory elements and indirectly by cross-talk with other transcription factors. In response to various stimuli, including stress, glucocorticoids coordinate metabolic, endocrine, immune and nervous system responses and ensure an adequate profile of transcription. In the brain, Gr has been proposed to modulate emotional behaviour, cognitive functions and addictive states. Previously, these aspects were not studied in the absence of functional Gr because inactivation of Grl1 in mice causes lethality at birth (F.T., C.K. and G.S., unpublished data). Therefore, we generated tissue-specific mutations of this gene using the Cre/loxP -recombination system. This allowed us to generate viable adult mice with loss of Gr function in selected tissues. Loss of Gr function in the nervous system impairs hypothalamus-pituitary-adrenal (HPA)-axis regulation, resulting in increased glucocorticoid (GC) levels that lead to symptoms reminiscent of those observed in Cushing syndrome. Conditional mutagenesis of Gr in the nervous system provides genetic evidence for the importance of Gr signalling in emotional behaviour because mutant animals show an impaired behavioural response to stress and display reduced anxiety.
The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence
of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis
in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnkgenes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling
pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic
signal transduction through the JNK pathway.
c-Jun N-terminal kinases (JNKs) typically respond strongly to stress, are implicated in brain development, and are believed to mediate neuronal apoptosis. Surprisingly, however, JNK does not respond characteristically to stress in cultured cerebellar granule (CBG) neurons, a widely exploited CNS model for studies of death and development, despite the regulation of its substrate c-Jun. To understand this anomaly, we characterized JNK regulation in CBG neurons. We find that the specific activity of CBG JNK is elevated considerably above that from neuron-like cell lines (SH-SY5Y, PC12); however, similar elevated activities are found in brain extracts. This activity does not result from cellular stress because the stress-activated protein kinase p38 is not activated. We identify a minor stress-sensitive pool of JNK that translocates with mitogen-activated protein kinase kinase-4 (MKK4) into the nucleus. However, the major pool of total activity is cytoplasmic, residing largely in the neurites, suggesting a non-nuclear role for JNK in neurons. A third JNK pool is colocalized with MKK7 in the nucleus, and specific activities of both increase during neuritogenesis, nuclear JNK activity increasing 10-fold, whereas c-Jun expression and activity decrease. A role for JNK during differentiation is supported by modulation of neuritic architecture after expression of dominant inhibitory regulators of the JNK pathway. Channeling of JNK signaling away from c-Jun during differentiation is consistent with the presence in the nucleus of the JNK/MKK7 scaffold protein JNK-interacting protein, which inhibits JNK-c-Jun interaction. We propose a model in which distinct pools of JNK serve different functions, providing a basis for understanding multifunctional JNK signaling in differentiating neurons.
Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIM(EL), a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIM(EL) in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim(-/-) knockout mice die more slowly after NGF withdrawal.
Mitogen-activated protein kinases (MAPK) are activated by phosphorylation on Thr and Tyr by MAPK kinases. Two MAPK kinases (MKK4 and MKK7) can activate the c-Jun NH(2)-terminal kinase (JNK) group of MAPK in vitro. JNK is phosphorylated preferentially on Tyr by MKK4 and on Thr by MKK7. Targeted gene-disruption studies in mice were performed to examine the role of MKK4 and MKK7 in vivo. Simultaneous disruption of the Mkk4 and Mkk7 genes was required to block JNK activation caused by exposure of cells to environmental stress. In contrast, disruption of the Mkk7 gene alone was sufficient to prevent JNK activation caused by proinflammatory cytokines. These data demonstrate that MKK4 and MKK7 serve different functions in the JNK signal transduction pathway.
Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal-induced death. Here, we have investigated the involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors in the regulation of BIM expression by NGF. We find that overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, we find that FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, we show that FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons.
Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.
c-Jun N-terminal kinase (JNK) signaling is an important contributor to stress-induced apoptosis, but it is unclear whether JNK and its isoforms (JNK1, JNK2, and JNK3) have distinct roles in cerebral ischemia. Here we show that JNK1 is the major isoform responsible for the high level of basal JNK activity in the brain. In contrast, targeted deletion of Jnk3 not only reduces the stress-induced JNK activity, but also protects mice from brain injury after cerebral ischemia-hypoxia. The downstream mechanism of JNK3-mediated apoptosis may include the induction of Bim and Fas and the mitochondrial release of cytochrome c. These results suggest that JNK3 is a potential target for neuroprotection therapies in stroke.
The c-Jun NH2-terminal kinase (JNK) is activated when cells are exposed to ultraviolet (UV) radiation. However, the functional consequence of JNK activation in UV-irradiated cells has not been established. It is shown here that JNK is required for UV-induced apoptosis in primary murine embryonic fibroblasts. Fibroblasts with simultaneous targeted disruptions of all the functional Jnk genes were protected against UV-stimulated apoptosis. The absence of JNK caused a defect in the mitochondrial death signaling pathway, including the failure to release cytochrome c. These data indicate that mitochondria are influenced by proapoptotic signal transduction through the JNK pathway.
Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.
We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.
c-Jun N-terminal kinase (JNK) signaling is an important contributor to stress-induced apoptosis, but it is unclear whether JNK and its isoforms (JNK1, JNK2, and JNK3) have distinct roles in cerebral ischemia. Here we show that JNK1 is the major isoform responsible for the high level of basal JNK activity in the brain. In contrast, targeted deletion of Jnk3 not only reduces the stress-induced JNK activity, but also protects mice from brain injury after cerebral ischemia-hypoxia. The downstream mechanism of JNK3-mediated apoptosis may include the induction of Bim and Fas and the mitochondrial release of cytochrome c. These results suggest that JNK3 is a potential target for neuroprotection therapies in stroke
Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis. We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association of SAPK with the anti-apoptotic Bcl-x(L) protein. SAPK phosphorylates Bcl-x(L) on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-x(L), a mutant Bcl-x(L) with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with Bcl-x(L) in the apoptotic response to genotoxic stress.
In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-β in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.
Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress
causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by
glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the
c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore
requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal
integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.
XG-102 (formerly D-JNKI1), a TAT-coupled dextrogyre peptide which selectively inhibits the c-Jun N-terminal kinase, is a powerful neuroprotectant in mouse models of middle cerebral artery occlusion (MCAo) with delayed intracerebroventricular injection. We aimed to determine whether this neuroprotection could also be achieved by intravenous injection of XG-102, which is a more feasible approach for future use in stroke patients. We also tested the compatibility of the compound with recombinant tissue plasminogen activator (rtPA), commonly used for intravenous thrombolysis and known to enhance excitotoxicity.
Male ICR-CD1 mice were subjected to a 30-min-suture MCAo. XG-102 was injected intravenously in a single dose, 6 h after ischemia. Hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mM) deprivation for 30 min. rtPA was added after ischemia and before XG-102 administration, both in vitro and in vivo.
The lowest intravenous dose achieving neuroprotection was 0.0003 mg/kg, which reduced the infarct volume after 48 h from 62 +/- 19 mm(3) (n = 18) for the vehicle-treated group to 18 +/- 9 mm(3) (n = 5, p < 0.01). The behavioral outcome was also significantly improved at two doses. Addition of rtPA after ischemia enhanced the ischemic damage both in vitro and in vivo, but XG-102 was still able to induce a significant neuroprotection.
A single intravenous administration of XG-102 several hours after ischemia induces a powerful neuroprotection. XG-102 protects from ischemic damage in the presence of rtPA. The feasibility of systemic administration of this promising compound and its compatibility with rtPA are important steps for its development as a drug candidate in ischemic stroke.
The axonal transport systems have a wide variety of primary roles and secondary responses in neurological disease processes. Recent advances in understanding these roles have built on the increasingly detailed insights into the cell biology of the axon and its supporting cells. Fast transport is a microtubule-based system of bidirectional movement of membranous organelles; the mechanism of translocation of these organelles involves novel proteins, including the recently described protein of fast anterograde transport, kinesin. Slow transport conveys the major cytoskeletal elements, microtubules, and neurofilaments. Several types of structural changes in diseased nerve fibers are understood in terms of underlying transport abnormalities. Altered slow transport of neurofilaments produces changes in axonal caliber (swelling or atrophy) and is involved in some types of perikaryal neurofibrillary abnormality. Secondary changes in slow axonal transport--for example, the reordered synthesis and delivery of cytoskeletal proteins after axotomy--also can produce changes in axonal caliber. Secondary demyelination can be a prominent late consequence of a sustained alteration of neurofilament transport. Impaired fast transport is found in experimental models of distal axonal degeneration (dying back). Retrograde axonal transport provides access to the central nervous system for agents such as polio virus and tetanus toxin, as well as access for known and hypothetical trophic factors. Correlative studies of axonal transport, axonal morphometry, cytoskeletal ultrastructure, and molecular biology of cytoskeletal proteins are providing extremely detailed reconstructions of the pathogenesis of experimental models of neurological disorders. A major challenge lies in the extension of these approaches to clinical studies.
Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is an important component in the pathogenesis of glutamate neurotoxicity.
Morphometric analyses of the immunohistochemical expression of the Clara cell secretory 10-kDa protein (CC10) and surfactant apoproteins A and B (SP-A and -B) were carried out on the developing bronchi and bronchioles of human fetuses and neonates. We analysed the ratio of the number of CC10-positive cells per subepithelial length of the bronchial or bronchiolar basement membrane and found that both the bronchial and the bronchiolar population of CC10-positive cells was significantly higher than that of either SP-A or SP-B. In addition, CC10 was found to be distributed mainly in the bronchiole. CC10-positive cells began to be recognized in the late pseudoglandular phase (15 weeks of gestation) and thereafter gradually increased in the canalicular and terminal sac phases, which correspond to the active development period of the acini or peripheral airways. The earliest expression of SP-A was also noted at 15 weeks of gestation, but its positive epithelial cells were present mainly in the larger bronchi. Double immunohistochemical staining for CC10 and SP-A revealed that the CC10-positive cells lining both the bronchi and bronchioles were different from the SP-A-positive cells. This finding suggests that CC10-positive cells are functionally and developmentally heterogeneous in both fetal and neonatal lungs in humans.
Mouse kif5B gene was disrupted by homologous recombination. kif5B-/- mice were embryonic lethal with a severe growth retardation at 9.5-11.5 days postcoitum. To analyze the significance of this conventional kinesin heavy chain in organelle transport, we studied the distribution of major organelles in the extraembryonic cells. The null mutant cells impaired lysosomal dispersion, while brefeldin A could normally induce the breakdown of their Golgi apparatus. More prominently, their mitochondria abnormally clustered in the perinuclear region. This mitochondrial phenotype was reversed by an exogenous expression of KIF5B, and a subcellular fractionation revealed that KIF5B is associated with mitochondria. These data collectively indicate that kinesin is essential for mitochondrial and lysosomal dispersion rather than for the Golgi-to-ER traffic in these cells.
Precursor CD4+ T cells develop into effector Th1 and Th2 cells that play a central role in the immune response. We show that the JNK MAP kinase pathway is induced in Th1 but not in Th2 effector cells upon antigen stimulation. Further, the differentiation of precursor CD4+ T cells into effector Th1 but not Th2 cells is impaired in JNK2-deficient mice. The inability of IL-12 to differentiate JNK2-deficient CD4+ T cells fully into effector Th1 cells is caused by a defect in IFNgamma production during the early stages of differentiation. The addition of exogenous IFNgamma during differentiation restores IL-12-mediated Th1 polarization in the JNK2-deficient mice. The JNK MAP kinase signaling pathway, therefore, plays an important role in the balance of Th1 and Th2 immune responses.
The c-Jun NH2-terminal kinase (JNK) signaling pathway has been implicated in the immune response that is mediated by the activation and
differentiation of CD4 helper T (TH) cells into TH1 and TH2 effector cells. JNK activity observed in wild-type activated TH cells was severely reduced in TH cells fromJnk1
–/– mice. TheJnk1
–/– T cells hyperproliferated, exhibited decreased activation-induced cell death, and preferentially differentiated to TH2 cells. The enhanced production of TH2 cytokines by Jnk1
–/– cells was associated with increased nuclear accumulation of the transcription factor NFATc. Thus, the JNK1 signaling pathway
plays a key role in T cell receptor–initiated TH cell proliferation, apoptosis, and differentiation.
The c-Jun NH2-terminal kinase (Jnk) family is implicated in apoptosis, but its function in brain development is unclear. Here, we address this issue using mutant mice lacking different members of the family (Jnk1, Jnk2, and Jnk3). Mice deficient in Jnk1, Jnk2, Jnk3, and Jnk1/Jnk3 or Jnk2/Jnk3 double mutants all survived normally. Compound mutants lacking Jnk1 and Jnk2 genes were embryonic lethal and had severe dysregulation of apoptosis in brain. Specifically, there was a reduction of cell death in the lateral edges of hindbrain prior to neural tube closure. In contrast, increased apoptosis and caspase activation were found in the mutant forebrain, leading to precocious degeneration. These results suggest that Jnk1 and Jnk2 regulate region-specific apoptosis during early brain development.
Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.
Multiple signal transduction pathways are capable of modifying BCL-2 family members to reset susceptibility to apoptosis. We used two-dimensional peptide mapping and sequencing to identify three residues (Ser70, Ser87, and Thr69) within the unstructured loop of BCL-2 that were phosphorylated in response to microtubule-damaging agents, which also arrest cells at G
2
/M. Changing these sites to alanine conferred more antiapoptotic activity on BCL-2 following physiologic death signals as well as paclitaxel, indicating that phosphorylation is inactivating. An examination of cycling cells enriched by elutriation for distinct phases of the cell cycle revealed that BCL-2 was phosphorylated at the G
2
/M phase of the cell cycle. G
2
/M-phase cells proved more susceptible to death signals, and phosphorylation of BCL-2 appeared to be responsible, as a Ser70Ala substitution restored resistance to apoptosis. We noted that ASK1 and JNK1 were normally activated at G
2
/M phase, and JNK was capable of phosphorylating BCL-2. Expression of a series of wild-type and dominant-negative kinases indicated an ASK1/Jun N-terminal protein kinase 1 (JNK1) pathway phosphorylated BCL-2 in vivo. Moreover, the combination of dominant negative ASK1, (dnASK1), dnMKK7, and dnJNK1 inhibited paclitaxel-induced BCL-2 phosphorylation. Thus, stress response kinases phosphorylate BCL-2 during cell cycle progression as a normal physiologic process to inactivate BCL-2 at G
2
/M.
Neurofilaments comprise three subunit proteins; neurofilament light, middle and heavy chains (NF-L, NF-M and NF-H). The carboxy-terminal domains of NF-M and NF-H form side-arms that project from the filament and that of NF-H contains multiple repeats of the motif lys-ser-pro, the serines of which are targets for phosphorylation. The level of phosphorylation on the lys-ser-pro repeats varies topographically within the cell; in cell bodies and proximal axons, the side-arms are largely non-phosphorylated whereas in more distal regions of axons, the side-arms are heavily phosphorylated. Here we show that stress activated protein kinase 1b (SAPK1b), a major SAPK in neurones will phosphorylate NF-H side-arms both in vitro and in transfected cells. These studies suggest that SAPK1b targets multiple phosphorylation sites within NF-H side-arms. Additionally, we show that glutamate treatment induces activation of SAPK1b in primary cortical neurones and increased phosphorylation of NF-H in cell bodies. This suggests that glutamate causes increased NF-H phosphorylation at least in part by activation of stress activated protein kinases.
MAP kinases are evolutionarily conserved proteins that are activated by a protein kinase cascade, including a MAP kinase kinase kinase, which phosphorylates a MAP kinase kinase, which in turn activates the MAP kinase by phosphorylation on Thr and Tyr residues. The primary sequence surrounding these phosphorylation sites serves to distinguish three major groups of mammalian MAP kinases. These include the Ras-activated ERK MAP kinases, which are characterized by the sequence TEY and the two stress-activated MAP kinases: p38 with the sequence TGY, and the c-Jun NH2-terminal kinases (JNK) with the sequence TPY. This review will focus on the JNK group of MAP kinases.
The cerebellar cortex and its sole output, the Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the ability to generate Purkinje cell-specific mutations in physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP strategy that allows temporally and spatially controlled gene inactivation. Here, we present the characterization of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/pcp-2 protein is expressed exclusively in Purkinje cells and retinal bipolar neurones. Recombination was detected by beta-galactosidase histochemistry in tissues from crosses of the L7/pcp-2:Cre transgenic lines with two different indicator strains, GtROSA26 and ACZL. Purkinje cells in all folia of the cerebellum displayed intense beta-galactosidase staining, whereas only few blue cells were observed in the retina and other parts of the CNS. Thus, these transgenic lines are potentially of great importance for genetic manipulations in cerebellar Purkinje cells.
Not later than two synapses after their arrival in the cerebellar cortex all excitatory afferent signals are subsequently transformed into inhibitory ones. Guaranteed by the exceedingly ordered and stereotyped synaptic arrangement of its cellular elements, the cerebellar cortex transmits this inhibitory result of cerebellar integration exclusively via Purkinje cells (PCs) in a precise temporal succession directly onto the target neurons of the deep cerebellar and vestibular nuclei. Thus the cerebellar cortex seems to produce a temporal pattern of inhibitory influence on these target neurons that modifies their excitatory action in such a way that an activation of muscle fibers occurs which progressively integrates the intended motion into the actual condition of the motoric inventory. In consequence, disturbances that affect this cerebellar inhibition will cause uncoordinated, decomposed and ataxic movements, commonly referred to as cerebellar ataxia. Electrophysiological investigations using different cerebellar mouse mutants have shown that alterations in the cerebellar inhibitory input in the target nuclei lead to diverse neuronal responses and to different consequences for the behavioural phenotype. A dependence between the reconstitution of inhibition and the behavioural outcome seems to exist. Obviously two different basic mechanisms are responsible for these observations: (1) ineffective inhibition on target neurons by surviving PCs; and (2) enhancement of intranuclear inhibition in the deep cerebellar and vestibular nuclei. Which of the two strategies evolves is dependent upon the composition of the residual cell types in the cerebellum and on the degree of PC input loss in a given area of the target nuclei. Motor behaviour seems to deteriorate under the first of these mechanisms whereas it may benefit from the second. This is substantiated by stereotaxic removal of the remaining PC input, which eliminates the influence of the first mechanism and is able to induce the second strategy. As a consequence, motor performance improves considerably. In this review, results leading to the above conclusions are presented and links forged to human cerebellar diseases.
c-Jun is considered a major regulator of both neuronal death and regeneration. Stress in primary cultured CNS neurons induces phosphorylation of c-Jun serines 63 and 73 and increased c-Jun protein. However, total c-Jun N-terminal protein kinase (JNK) activity does not increase, and no satisfactory explanation for this paradox has been available. Here we demonstrate that neuronal stress induces strong activation of JNK2/3 in the presence of constitutively and highly active JNK1. Correspondingly, neurons from JNK1(-/-) mice show lower constitutive activity and considerably higher responsiveness to stress. p38 activity can be completely inhibited without effect on c-Jun phosphorylation, whereas 10 micrometer SB203580 strongly inhibits neuronal JNK2/3, stress-induced c-Jun phosphorylation, induced c-Jun activity, and neuronal death in response to trophic withdrawal stress. Neither constitutive JNK1 activity nor total neuronal JNK activity were significantly affected by this concentration of drug. Thus, neuronal stress selectively activates JNK2/3 in the presence of mechanisms maintaining constitutive JNK1 activity, and this JNK2/3 activity selectively targets c-Jun, which is isolated from constitutive JNK1 activity.
Microtubules (MTs) play an important role in elaboration and maintenance of axonal and dendritic processes. MT dynamics are modulated by MT-associated proteins (MAPs), whose activities are regulated by protein phosphorylation. We found that a member of the c-Jun NH(2)-terminal protein kinase (JNK) subgroup of MAP kinases, JNK1, is involved in regulation of MT dynamics in neuronal cells. Jnk1(-/-) mice exhibit disrupted anterior commissure tract formation and a progressive loss of MTs within axons and dendrites. MAP2 and MAP1B polypeptides are hypophosphorylated in Jnk1(-/-) brains, resulting in compromised ability to bind MTs and promote their assembly. These results suggest that JNK1 is required for maintaining the cytoskeletal integrity of neuronal cells and is a critical regulator of MAP activity and MT assembly.
The c-Jun NH(2)-terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. Here we demonstrate that JNK-stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF-kappaB to increase antiapoptotic gene expression. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Furthermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biological response to a specific challenge.