SREBP-1c impairs ULK1 sulfhydration-mediated autophagic flux to promote hepatic steatosis in high-fat-diet-fed mice.

A metabolic imbalance between lipid synthesis and degradation can lead to hepatic lipid accumulation, a characteristic of patients with non-alcoholic fatty liver disease (NAFLD). Here, we report that high-fat-diet-induced sterol regulatory element-binding protein (SREBP)-1c, a key transcription factor that regulates lipid biosynthesis, impairs autophagic lipid catabolism via altered H2S signaling. SREBP-1c reduced cystathionine gamma-lyase (CSE) via miR-216a, which in turn decreased hepatic H2S levels and sulfhydration-dependent activation of Unc-51-like autophagy-activating kinase 1 (ULK1). Furthermore, Cys951Ser mutation of ULK1 decreased autolysosome formation and promoted hepatic lipid accumulation in mice, suggesting that the loss of ULK1 sulfhydration was directly associated with the pathogenesis of NAFLD. Moreover, silencing of CSE in SREBP-1c knockout mice increased liver triglycerides, confirming the connection between CSE, autophagy, and SREBP-1c. Overall, our results uncover a 2-fold mechanism for SREBP-1c-driven hepatic lipid accumulation through reciprocal activation and inhibition of hepatic lipid biosynthesis and degradation, respectively.

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study.

BACKGROUND: Activation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro. METHODS: Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels. RESULTS: The parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin. CONCLUSIONS: Collectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.

Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity.

Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.

Gomisin N Exerts Anti-liver Cancer Effects and Regulates PI3K-Akt and mTOR-ULK1 Pathways in Vitro.

Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.

Scaffold proteins in bulk and selective autophagy.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases.

Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo, enhances translation, and modulates splicing.

Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.

The role of the key autophagy kinase ULK1 in hepatocellular carcinoma and its validation as a treatment target.

Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC. Abbreviations: 3MA: 3-methyladenine; ADV: AutoDock Vina; ATP: adenosine triphosphate; EdU: 5-ethynyl-2'-deoxyuridine; ESI: electrospray ionization; HCC: hepatocellular carcinoma; IC50: half maximal inhibitory concentration; KD: kinase domain; q.o.d., every other day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.

Circ_0009910 promotes imatinib resistance through ULK1-induced autophagy by sponging miR-34a-5p in chronic myeloid leukemia.

BACKGROUND: The occurrence in drug resistance of chronic myeloid leukemia (CML) was accompanied by autophagy activation. Abnormal circular RNAs (circRNAs) participated in this progression. This study attempted to investigate the potential role of circ_0009910 in imatinib resistance of CML cells. METHODS: The expression of circ_0009910 and miR-34a-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The characterization of circ_0009910 was investigated using oligo (dT)18 primers, Actinomycin D and RNase R. Cell viability (IC50 value) and apoptosis were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry assay, respectively. The relative protein expression was quantified by western blot. The relationship among miR-34a-5p, circ_0009910 and ULK1 was predicted by online bioinformatics tool, and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). RESULTS: The expression of circ_0009910 was up-regulated in the serum of imatinib-resistance CML patients and K562/R cells, and associated with unfavorable clinicopathologic features. Circ_0009910 in K562 and K562/R cells was mainly localized in the cytoplasm. Circ_0009910 knockdown inhibited cell proliferation and autophagy, but induced apoptosis in K562/R cells. Circ_0009910 targeted miR-34a-5p to regulate ULK1. MiR-34a-5p depression rescued the effects of circ_0009910 knockdown on apoptosis and autophagy in K562/R cells. CONCLUSION: Circ_0009910 accelerated imatinib-resistance in CML cells by modulating ULK1-induced autophagy via targeting miR-34a-5p, providing a potential target in imatinib resistance of CML.

MAPK15 is part of the ULK complex and controls its activity to regulate early phases of the autophagic process.

Autophagy, a pathway for bulk protein degradation and removal of damaged organelles, represents one of the major responses of cells to stress, thereby exerting a strict control on their correct functioning. Consequently, this process has been involved in the pathogenesis and therapeutic responses of several human diseases. Mitogen-activated protein (MAP) kinase 15 (MAPK15) is an atypical member of the MAP kinase family that recently emerged as a key modulator of autophagy and, through this, of cell transformation. Still, no information is available about signaling pathways mediating the effect of MAPK15 on this process, nor is it known which phase of autophagosome biogenesis is affected by this MAP kinase. Here, we demonstrate that MAPK15 stimulated 5'-AMP-activated protein kinase-dependent activity of UNC-51-like kinase 1 (ULK1), the only protein kinase among the ATG-related proteins, toward downstream substrates and signaling intermediates. Importantly, MAPK15 directly interacted with the ULK1 complex and mediated ULK1 activation induced by starvation, a classical stimulus for the autophagic process. In turn, ULK1 and its highly homologous protein ULK2 are able to transduce MAPK15 signals stimulating early phases of autophagosomal biogenesis in a multikinase cascade that offers numerous potential targets for future therapeutic intervention in cancer and other autophagy-related human diseases.

Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy.

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.

The ER Contact Proteins VAPA/B Interact with Multiple Autophagy Proteins to Modulate Autophagosome Biogenesis.

The endoplasmic reticulum (ER) is the site of biogenesis of the isolation membrane (IM, autophagosome precursor) and forms extensive contacts with IMs during their expansion into double-membrane autophagosomes. Little is known about the molecular mechanism underlying the formation and/or maintenance of the ER/IM contact. The integral ER proteins VAPA and VAPB (VAPs) participate in establishing ER contacts with multiple membranes by interacting with different tethers. Here, we demonstrate that VAPs also modulate ER/IM contact formation. Depletion of VAPs impairs progression of IMs into autophagosomes. Upon autophagy induction, VAPs are recruited to autophagosome formation sites on the ER, a process mediated by their interactions with FIP200 and PI(3)P. VAPs directly interact with FIP200 and ULK1 through their conserved FFAT motifs and stabilize the ULK1/FIP200 complex at the autophagosome formation sites on the ER. The formation of ULK1 puncta is significantly reduced by VAPA/B depletion. VAPs also interact with WIPI2 and enhance the formation of the WIPI2/FIP200 ER/IM tethering complex. Depletion of VMP1, which increases the ER/IM contact, greatly elevates the interaction of VAPs with these autophagy proteins. The VAPB P56S mutation, which is associated with amyotrophic lateral sclerosis, reduces the ULK1/FIP200 interaction and impairs autophagy at an early step, similar to the effect seen in VAPA/B-depleted cells. Our study reveals that VAPs directly interact with multiple ATG proteins, thereby contributing to ER/IM contact formation for autophagosome biogenesis.

Influences of different dietary energy level on sheep testicular development associated with AMPK/ULK1/autophagy pathway.

Energy balance is an important feature for spermatozoa production in the testis. The 5'-AMP-activated protein kinase (AMPK) is a sensor of cell energy, has been implicated as a mediator between gonadal function and energy balance. Herein, we intended to determine the physiological effects of AMPK on testicular development in feed energy restricted and compensated pre-pubertal rams. Lambs had restricted feeding for 2 months and then provided compensatory feeding for another 3 months. Feed levels were 100%(control), 15% and 30% of energy restriction (ER) diets, respectively. The results showed that lambs fed the 30% ER diet had significantly lower testicular weight (P < .05) and spermatids number in the seminiferous tubules, but there were no differences between control and 15% ER groups. Meanwhile, 15% ER and 30% ER diets induced testis autophagy and apoptosis through activating AMPK-ULK1(ULK1, Unc-51 like autophagy activating kinase) signal pathway with characterization of increased Beclin-1 and Light chain 3-Ⅱ/Light chain 3-Ⅰ (LC3-II/LC3-I) ratio, up-regulated the ratio of pro-apoptotic Bcl-2-associated X protein (BAX) and anti-apoptotic B-cell lymphoma 2 (Bcl-2), as well as activated AMPK, phosphorylated AMPK(p-AMPK) and ULK1. Furthermore, a compensation of these parameters occurred when the lambs were re-fed with normal energy requirement after restriction. Taken together, dietary energy levels influence testicular development through autophagy and apoptosis interplay mediated by AMPK-ULK1 signal pathway, which also indicates the important role of the actions of AMPK in the testis homeostasis.

Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy.

Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.

The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the ß1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a ß1-Ser108 kinase in cells. Cellular ß1-Ser108 phosphorylation by ULK1 was dependent on AMPK ß-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates ß1-Ser108 on the regulatory ß1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop.

The ULK complex initiates autophagosome formation at phosphatidylinositol synthase-enriched ER subdomains.

In our recent paper, we biochemically analyzed autophagosome-related membranes at the initiation stage of macroautophagy/autophagy using atg knockout (KO) cells and demonstrated that the ULK complex is recruited to 2 distinct membranes: the ER membrane and ATG9A-positive autophagosome precursors. We have also identified phosphatidylinositol synthase (PIS)-enriched ER subdomains as the initiation site of autophagosome formation. Based on these findings, we propose that the ULK complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.

miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1.

Porcine hemagglutinating encephalomyelitis virus (PHEV) invades the central nervous system (CNS) and causes neurodegenerative disease in suckling piglets, but the understanding of its neuropathogenicity for neurological dysfunction remains limited. Here, we report that miR-142-5p is localized to neurons and negatively regulates neuronal morphogenesis in porcine hemagglutinating encephalomyelitis (PHE). This phenotype was mediated by miR-142-5p inhibition of an mRNA encoding unc-51-like-kinase1 (Ulk1), which controls axon outgrowth and dendrite formation. Modulating miR-142-5p activity by microRNA mimics or inhibitors induced neurodegeneration, including stunted axon elongation, unstable dendritic spine formation, and irregular swelling and disconnection in neurites. Relieving Ulk1 mRNA repression in primary cortical neurons by miR-142-5p antagomirs or replication-deficient adenoviruses encoding Ulk1 (Ad5-Ulk1), which improved rescue of nerve injury, restricted viral replication, and increased survival rate in mice underlying PHEV infection. In contrast, disrupting Ulk1 in RNAi-expressing neurons mostly led to significantly shortened axon elongation and/or an abnormally large number of branched dendrites. Taken together, we demonstrated that the abnormal neuronal morphogenesis underlying PHEV infection was mainly caused by functional mRNA repression of the miR-142-5p target Ulk1. Our data revealed that PHEV adapted to use spatiotemporal control of host microRNAs to invade CNS, and provided new insights into the virus-associated neurological dysfunction microenvironment.

FoxO1-AMPK-ULK1 Regulates Ethanol-Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1-PIK3C3 Complex.

BACKGROUND: Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK. METHODS: C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors. RESULTS: Incubation of C2C12 myoblasts with 100 mM EtOH increased the autophagy markers LC3B-II and ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOH altered ULK1 S555 and S757 phosphorylation in a time- and AMPK-dependent manner. The activation of AMPK and ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14-AMBRA1-BECN1-PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH-induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14-3-3θ. Reduction of AMPK using siRNA suppressed the stimulatory effect of EtOH on BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time-dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOH on LC3B-II, SQSTM1, and ATG7 protein expression. CONCLUSIONS: EtOH-induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1-AMPK or indirectly via the FoxO1-AMPK-ULK1 signaling cascade in a mTORC1-independent or mTORC1-dependent manner.