bims-auttor 2024-12-08 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2024–12–08
38 papers selected by
Viktor Korolchuk, Newcastle University

Mitophagy in gynecological malignancies: roles, advances, and therapeutic potential.
Autophagy as a way to remove DNA lesions.
USP19 potentiates autophagic cell death via inhibiting mTOR pathway through deubiquitinating NEK9 in pancreatic cancer.
Structural Basis for the Pathogenicity of Parkin Catalytic Domain Mutants.
Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in alzheimer disease.
Hapalindole Q suppresses autophagosome-lysosome fusion by promoting YAP1 degradation via chaperon-mediated autophagy.
Targeting autophagy in HCC treatment: exploiting the CD147 internalization pathway.
Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease.
TMEM16A Activation Inhibits Autophagy in Dorsal Root Ganglion Cells, Which is Associated with the p38 MAPK/mTOR Pathway.
Autophagy-related gene BECN1 single nucleotide polymorphisms in diseases.
Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
Osthole induces accumulation of impaired autophagosome against pancreatic cancer cells.
Selective autophagy receptor hinders antitumor immunity.
Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.
Inhibiting autophagy enhances idarubicin chemosensitivity and induces immune escape in FAT1-low-expressing AML cells.
SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.
JAK2/ULK1 axis promotes cervical cancer progression by autophagy induction and SRPK1 phosphorylation.
Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.
Imperatorin promotes melanin degradation in keratinocytes through facilitating autophagy via the PI3K/Akt signaling pathway.
Inhibition of XIST restrains paclitaxel resistance in breast cancer cells by targeting hsa-let-7d-5p/ATG16L1 through regulation of autophagy.
Effects of autophagy on the selective death of human breast cancer cells exposed to plasma-activated Ringer's lactate solution.
Triolein alleviates ischemic stroke brain injury by regulating autophagy and inflammation through the AKT/mTOR signaling pathway.
Trehalose extricates impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model.
The essential role of TTC28 in maintaining chromosomal stability via HSPA8 chaperone-mediated autophagy.
FBXW8 suppresses PDCoV proliferation via the NPD52-dependent autophagic degradation of a viral nucleocapsid protein.
Dopamine-conjugated extracellular vesicles induce autophagy in Parkinson's disease.
Dynamic regulation of proximal tubular autophagy from injury to repair after ischemic kidney damage.
Caspase-8-dependent autophagy regulates neutrophil infiltration in oral squamous cell carcinoma.
Glutamine is critical for the maintenance of type 1 conventional dendritic cells in normal tissue and the tumor microenvironment.
MAM-mediated mitophagy and endoplasmic reticulum stress: the hidden regulators of ischemic stroke.
The Greatwall-Endosulfine-PP2A/B55 pathway regulates entry into quiescence by enhancing translation of Elongator-tunable transcripts.
Women in Autophagy: an initiative to promote gender parity in science.
Rapamycin administration causes a decrease in muscle contractile function and systemic glucose intolerance concomitant with reduced skeletal muscle Rictor, the mTORC2 component, expression independent of energy intake in young rats.
Discovery of a molecular glue for EGFR degradation.
Ruscus aculeatus extract promotes RNase 7 expression through ERK activation following inhibition of late-phase autophagy in primary human keratinocytes.
TIGAR coordinates senescence-associated secretory phenotype via lysosome repositioning and α-tubulin deacetylation.
Calcium connects lysosomal damage to stress granule formation.
Myeloid cell-specific loss of NPC1 in mice recapitulates microgliosis and neurodegeneration in patients with Niemann-Pick type C disease.

Cell Death Discov. 2024 Dec 05. 10(1): 488

Mitophagy in gynecological malignancies: roles, advances, and therapeutic potential.

Jiao Wang, Dandan Wang.

Mitophagy is a process in which impaired or dysfunctional mitochondria are selectively eliminated through the autophagy mechanism to maintain mitochondrial quality control and cellular homeostasis. Based on specific target signals, several mitophagy processes have been identified. Defects in mitophagy are associated with various pathological conditions, including neurodegenerative disorders, cardiovascular diseases, metabolic diseases, and cancer. Mitophagy has been shown to play a critical role in the pathogenesis of gynecological malignancies and the development of drug resistance. In this review, we have summarized and discussed the role and recent advances in understanding the therapeutic potential of mitophagy in the development of gynecological malignancies. Therefore, the valuable insights provided in this review may serve as a basis for further studies that contribute to the development of novel treatment strategies and improved patient outcomes.

DOI: https://doi.org/10.1038/s41420-024-02259-x
Autophagy. 2024 Dec 05. 0

Autophagy as a way to remove DNA lesions.

Yuchen Lei, Daniel J Klionsky.

DOI: https://doi.org/10.1080/15548627.2024.2434784
Cell Death Differ. 2024 Dec 03.

USP19 potentiates autophagic cell death via inhibiting mTOR pathway through deubiquitinating NEK9 in pancreatic cancer.

Guangfu Wang, Shangnan Dai, Jin Chen, Kai Zhang, Chenyu Huang, Jinfan Zhang, Kunxin Xie, Fuye Lin, Huijuan Wang, Yong Gao, Lingdi Yin, Kuirong Jiang, Yi Miao, Zipeng Lu.

The ubiquitin-specific protease (USP) family is the largest and most diverse deubiquitinase (DUBs) family and plays a significant role in maintaining cell homeostasis. Dysregulation of USPs has been associated with carcinogenesis of various tumors. We identified that USP19 was downregulated in pancreatic tumor tissues and forced expression of USP19 diminished tumorigenicity of pancreatic cancer. Mechanistically, USP19 directly interacts with and stabilized NEK9 via inhibiting K48-specific polyubiquitination process on NEK9 protein at K525 site through its USP domain. Moreover, NEK9 phosphorylates the regulatory associated protein of mTOR (Raptor) at Ser792 and links USP19 to the inhibition of mTORC1 signaling pathway, which further leads to autophagic cell death of pancreatic cancer cells. Inhibition of autophagy by Atg5 knockdown or lysosome inhibitor bafilomycin A1 abolished the decreased malignant phenotype of USP19- and NEK9-overexpressing cancer cells. Importantly, USP19 expression exhibits a positive correlation with NEK9 expression in clinical samples, and low USP19 or NEK9 expression is associated with a worse prognosis. This study revealed that USP19-mediated NEK9 deubiquitylation is a regulatory mechanism for mTORC1 inhibition and provides a therapeutic target for diseases involving mTORC1 dysregulation.

DOI: https://doi.org/10.1038/s41418-024-01426-y
J Biol Chem. 2024 Dec 02. pii: S0021-9258(24)02553-5. [Epub ahead of print] 108051

Structural Basis for the Pathogenicity of Parkin Catalytic Domain Mutants.

Julian P Wagner, Véronique Sauvé, Anshu Saran, Kalle Gehring.

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson's disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several PD mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor-element of parkin (REP) and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin-E2∼Ub complex.

DOI: https://doi.org/10.1016/j.jbc.2024.108051
Autophagy. 2024 Dec 05.

Upregulation of ISG15 induced by MAPT/tau accumulation represses autophagic flux by inhibiting HDAC6 activity: a vicious cycle in alzheimer disease.

Qian Liu, Xin Wang, Zhi-Ting Fang, Jun-Ning Zhao, Xue-Xiang Rui, Bing-Ge Zhang, Ye He, Rui-Juan Liu, Jian Chen, Gao-Shang Chai, Gong-Ping Liu.

  Alzheimer disease (AD), a prevalent neurodegenerative condition in the elderly, is marked by a deficit in macroautophagy/autophagy, leading to intracellular MAPT/tau accumulation. While ISG15 (ISG15 ubiquitin like modifier) has been identified as a regulator of selective autophagy in ataxia telangiectasia (A-T), its role in AD remains unexplored. Our study reveals elevated ISG15 levels in the brains of patients with sporadic AD and AD models in vivo and in vitro. ISG15 overexpression in cells and the hippocampus inhibited HDAC6 (histone deacetylase 6) activity through C-terminal LRLRGG binding to HDAC6. Consequently, this increased CTTN (cortactin) acetylation, disrupted CTTN and F-actin recruitment to lysosomes, and impaired autophagosome (AP)-lysosome (LY) fusion. These disruptions led to MAPT/tau accumulation, synaptic damage, neuronal loss, and cognitive deficits. Conversely, ISG15 knockdown in our HsMAPT (human MAPT) pathology model restored HDAC6 activity, promoted AP-LY fusion, and improved cognitive function. This study identifies ISG15 as a key regulator of autophagic flux in AD, suggesting that targeting ISG15-mediated autophagy could offer therapeutic potential for AD.

Keywords:  Alzheimer disease; ISG15; MAPT; autophagy; cognition

DOI:  https://doi.org/10.1080/15548627.2024.2431472
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2400809121

Hapalindole Q suppresses autophagosome-lysosome fusion by promoting YAP1 degradation via chaperon-mediated autophagy.

Yali Wu, Shaonan Wang, Zhicong Guo, Min Sun, Zhen Xu, Yu Du, Fahui Zhu, Yajuan Su, Zhou Xu, Yi Xu, Xu Gong, Ruan Fang, Jiaojiao Hu, Yan Peng, Zhaowen Ding, Cong Liu, Ang Li, Weiwei He.

  Autophagy is a conserved catabolic process crucial for maintaining cellular homeostasis and has emerged as a promising therapeutic target for many diseases. Mechanistically novel small-molecule autophagy regulators are highly desirable from a pharmacological point of view. Here, we report the macroautophagy-inhibitory effect of hapalindole Q, a member of the structurally intriguing but biologically understudied hapalindole family of indole terpenoids. This compound promotes the noncanonical degradation of Yes-associated protein 1 (YAP1), the downstream effector of the Hippo signaling pathway, via chaperone-mediated autophagy, disrupting proper distribution of Rab7 and suppressing autophagosome-lysosome fusion in macroautophagy. Its binding to YAP1 is further confirmed by using biophysical techniques. A preliminary structure-activity relationship study reveals that the hapalindole Q scaffold, rather than the isothiocyanate group, is essential for YAP1 binding and degradation. This work not only identifies a macroautophagy inhibitor with a distinct mechanism of action but also provided a molecular scaffold for direct targeting of YAP1, which may benefit the development of therapeutics for both autophagy-related and Hippo-YAP-related diseases.

Keywords:  YAP1 degradation; autophagy inhibitor; chaperone-mediated autophagy; natural product

DOI:  https://doi.org/10.1073/pnas.2400809121
Cell Commun Signal. 2024 Dec 03. 22(1): 583

Targeting autophagy in HCC treatment: exploiting the CD147 internalization pathway.

Meirui Qian, Ziyu Wan, Xue Liang, Lin Jing, Huijie Zhang, Heyao Qin, Wenli Duan, Ruo Chen, Tianjiao Zhang, Qian He, Meng Lu, Jianli Jiang.

   BACKGROUND/AIMS: Chemotherapy resistance in liver cancer is a major clinical issue, with CD147 playing a vital role in this process. However, the specific mechanisms underlying these processes remain largely unknown. This study investigates how CD147 internalization leads to cytoprotective autophagy, contributing to chemotherapy resistance in hepatocellular carcinoma (HCC).
METHODS: Utilizing bioinformatics methods for KEGG pathways enrichment and screening key molecules associated with chemotherapy resistance through analyses of GEO and TCGA databases. An overexpression/knockdown system was used to study how CD147 internalization leads to autophagy in vitro and in vivo. The process was observed using microscopes, and molecular interactions and autophagy flux were analyzed. Analyzing the internalization of CD147 intracellular domains and the interaction with G3BP1 in clinical chemotherapy recurrence HCC tissues by immunohistochemistry, tissue immunofluorescence, and mass spectrometry. A tumor xenograft mice model was used to study cytoprotective autophagy induced by CD147 and test the effectiveness of combining cisplatin with an autophagy inhibitor in nude mice models.
RESULTS: In our study, we identified the tumor-associated membrane protein CD147, which implicated in chemoresistance lysosome pathways, by evaluating its protein degree value and betweenness centrality using Cytoscape. Our findings revealed that CD147 undergoes internalization and interacts with G3BP1 following treatment with cisplatin and methyl-β-cyclodextrin, forming a complex that is transported to lysosomes via Rab7A. Notably, higher doses of cisplatin enhanced CD147-mediated lysosomal transport while concurrently inhibiting SG assembly. The CD147-G3BP1 complex additionally inhibits mTOR activity, promoting autophagy and augmenting chemoresistance in hepatoma cells. In vivo studies investigations and analyses of clinical samples revealed that elevated internalization of CD147 is associated with chemotherapy recurrence in liver cancer and the maintenance of stem cells. Mice experiments found that the combined administration of cisplatin and hydroxychloroquine enhanced the efficacy of treatment.
CONCLUSIONS: This study reveals that CD147 internalization and CD147-G3BP1 complex translocation to lysosomes induce cytoprotective autophagy, reducing chemotherapy sensitivity by suppressing mTOR activity. It is also shown that chemotherapy drugs combined with autophagy inhibitors can improve the therapeutic effect of cancer, providing new insights into potential targeted therapeutic approaches in treating HCC.

Keywords:  CD147; Chemoresistance; Cytoprotective autophagy; G3BP1; SG

DOI:  https://doi.org/10.1186/s12964-024-01956-5
PLoS One. 2024 ;19(12): e0314858

Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease.

Lisa Marie P Evans, Joseph Gawron, Fraser J Sim, M Laura Feltri, Leandro N Marziali.

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

DOI: https://doi.org/10.1371/journal.pone.0314858
Cell Mol Neurobiol. 2024 Dec 04. 45(1): 1

TMEM16A Activation Inhibits Autophagy in Dorsal Root Ganglion Cells, Which is Associated with the p38 MAPK/mTOR Pathway.

Shuyun Yang, Hui Shang, Yuruo Zhang, Jingsong Qiu, Zheyi Guo, Yong Ma, Yuhang Lan, Shaoyang Cui, Hongshuang Tong, Guocai Li.

  Transmembrane member 16A (TMEM16A) exhibits a negative correlation with autophagy, though the underlying mechanism remains elusive. This study investigates the mechanism between TMEM16A and autophagy by inducing autophagy in DRG neuronal cells using Rapamycin. Results indicated that TMEM16A interference augmented cell viability and reduced Rapamycin-induced apoptosis. Autophagosome formation increased with TMEM16A interference but decreased upon overexpression. A similar increase in autophagosomes was observed with SB203580 treatment. Furthermore, TMEM16A interference suppressed Rapamycin-induced gene and protein expression of p38 MAPK and mTOR, whereas overexpression had the opposite effect. These findings suggest that TMEM16A activation inhibits autophagy in DRG cells, which is associated with the p38 MAPK/mTOR pathway, offering a potential target for mitigating neuropathic pain (NP).

Keywords:  Autophagy; Dorsal root ganglia; Neuropathic pain; P38 MAPK; TMEM16A; mTOR

DOI:  https://doi.org/10.1007/s10571-024-01507-z
Mol Cell Biochem. 2024 Dec 05.

Autophagy-related gene BECN1 single nucleotide polymorphisms in diseases.

Sargeet Kaur, Jitendraa Vashistt, Ajay Kumar, Jyoti Parkash, Harish Changotra.

  Autophagy is a cytoprotective process that operates within a cell to maintain cellular homeostasis. An array of multiple proteins is involved to mediate this conserved cellular process. Among these, Beclin 1 protein encoded by BECN1 gene plays a crucial role during the initiation of autophagy. It acts as a molecular platform onto which multiple proteins interact to mediate autophagy initiation. The functioning of such proteins has reportedly been influenced by the molecular markers such as Single Nucleotide Polymorphisms (SNPs) present within the encoding gene. The SNPs within the autophagy gene have been known to influence the functioning of autophagy proteins which further is involved in various diseases. Studies have reported that the SNPs within the BECN1 are involved in various diseases. This report outlines the findings of all the existing research on the role of SNPs within BECN1.

Keywords:  Autophagy; Beclin 1; Intronic variants; Regulatory variants; Single nucleotide polymorphisms

DOI:  https://doi.org/10.1007/s11010-024-05177-9
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2417390121

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

Kelsey L Hickey, Alexandra Panov, Enya Miguel Whelan, Tillman Schäfer, Arda Mizrak, Ron R Kopito, Wolfgang Baumeister, Rubén Fernández-Busnadiego, J Wade Harper.

  A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Keywords:  protein aggregates; protein quality control; protein turnover; ubiquitin-proteasome system

DOI:  https://doi.org/10.1073/pnas.2417390121
Sci Rep. 2024 12 04. 14(1): 30163

Osthole induces accumulation of impaired autophagosome against pancreatic cancer cells.

Ningna Weng, Sujuan Lv, Hong Chen, Hanchen Zheng, Tong Lin, Qing Zhu, Kai Zhu, Sha Huang.

  Pancreatic cancer (PC) is one of the most aggressive malignancies worldwide, and few effective therapeutics are available. Osthole (OST), a natural coumarin, has been proven to be a potential anticancer compound. In this study, we found that OST significantly inhibited PC growth both in vitro and in vivo. Notably, the anti-PC effect of OST is mediated by excessive autophagosome accumulation and consequent apoptosis in PC cells. Mechanistically, OST increased the number of autophagosomes via two pathways. First, OST induced autophagy initiation by suppressing the Akt/mTOR signaling pathway. Second, OST induced autophagic arrest by blocking autophagosome-lysosome fusion. Consistently, inhibition of autophagy initiation restored PC cell growth, whereas autophagic flux inhibitors exacerbated the antitumor effect of OST in PC cells, suggesting a cytotoxic role of OST-induced autophagosome accumulation. In addition, we found that OST upregulates the expression of activating transcription factor 3 (ATF3), resulting in inactivation of the Akt/mTOR signaling pathway and autophagy initiation in PC cells. ATF3 overexpression increased the activation of autophagy, and inhibition of ATF3 expression decreased autophagy. Taken together, our study provides new insights into the OST-induced growth inhibitory effect on PC cells, suggesting a promising potential therapeutic role of OST for PC treatment.

Keywords:  ATF3; Autophagy; Osthole; Pancreatic cancer; mTOR

DOI:  https://doi.org/10.1038/s41598-024-81911-z
Trends Cancer. 2024 Dec 02. pii: S2405-8033(24)00259-0. [Epub ahead of print]

Selective autophagy receptor hinders antitumor immunity.

Jiao Liu, Rui Kang, Daolin Tang.

  Autophagy has a dual role in tumor progression and therapy, influenced by specific receptors and cargo selection. Recent research published in Cell by Herhaus et al. identifies immunity-related GTPase Q (IRGQ) as a novel autophagy receptor that facilitates immune evasion in hepatocellular carcinoma (HCC) by degrading histocompatibility complex class I (MHC-I) molecules, highlighting a potential target to enhance immunotherapy.

Keywords:  antigen presentation; autophagy receptor; hepatocellular carcinoma; peptide degradation; tumor immune evasion

DOI:  https://doi.org/10.1016/j.trecan.2024.11.004
Proc Natl Acad Sci U S A. 2024 Dec 24. 121(52): e2419079121

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons.

Frances V Hundley, Miguel A Gonzalez-Lozano, Lena M Gottschalk, Aslan N K Cook, Jiuchun Zhang, Joao A Paulo, J Wade Harper.

  Plasma membrane protein degradation and recycling are regulated by the endolysosomal system, wherein endocytic vesicles bud from the plasma membrane into the cytoplasm and mature into endosomes and then degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface and influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we develop a toolkit for capturing EEA1-positive endosomes (termed Endo-IP) and TMEM192-positive lysosomes (termed Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrate its utility by revealing the endolysosomal protein landscapes for stem cells and cortical-like iNeurons, and profiling endosomes in response to potassium-mediated neuronal depolarization. Through global profiling of endocytic cargo, we identify hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Keywords:  endosome; iNeuron; lysosome; proteomics; stem cells

DOI:  https://doi.org/10.1073/pnas.2419079121
Int Immunopharmacol. 2024 Nov 29. pii: S1567-5769(24)02006-X. [Epub ahead of print]144 113484

Inhibiting autophagy enhances idarubicin chemosensitivity and induces immune escape in FAT1-low-expressing AML cells.

Nan Zhang, Yu-Ting Wang, Su-Si Dai, Fang-Yi Fan, Ling Qiu, Hai Yi, Yong-Jian Yang.

   OBJECTIVE: Both Autophagy and FAT atypical cadherin 1 (FAT1) regulates the chemosensitivity and immune escape of tumour cells. Our previous paper showed that FAT1 decreased acute myeloid leukemia (AML) autophagy by inhibiting the TGFβ-Smad2/3 pathway. This study builds upon our previous paper and aims to explore whether FAT1-inhibited autophagy is involved in regulating chemosensitivity and immune escape in AML.
METHODS: We validated the inhibitory effect of FAT1 on AML autophagy through western blot, qPCR, and luciferase reporter assays. In addition, we explored the effect of FAT1-inhibited autophagy on idarubicin (IDA) sensitivity and AML immune escape through caspase-3 activity analysis, trypan blue exclusion assays, and flow cytometry.
RESULTS: We demonstrated for the first time that the autophagy inhibitor chloroquine (CQ) enhances the cytotoxic effect of IDA on FAT1-low-expressing (FAT1-L) AML cells. We also found that CQ weakened CD8+ T cell infiltration in FAT1-L AML cells. Further research revealed that CQ upregulated PD-L1 protein levels by decreasing its autophagic degradation and that the PD-L1 inhibitor atezolizumab reversed the decrease in CD8+ T cell infiltration caused by CQ in FAT1-L AML cells. In addition, we found that FAT1 decreased autophagy related 10 (ATG10) transcription, leading to decreased AML autophagy.
CONCLUSIONS: These results revealed that in FAT1-L AML cells, inhibiting autophagy by CQ enhances the cytotoxic effect of IDA, but leads to immune escape, resulting in AML recurrence. Our study supports the use of a combination of autophagy and PD-L1 inhibitors with IDA to increase the cytotoxic effect of IDA while inhibiting AML recurrence.

Keywords:  AML; ATG10; Autophagy; FAT1; Immune escape; PD-L1

DOI:  https://doi.org/10.1016/j.intimp.2024.113484
Cell Death Dis. 2024 Dec 05. 15(12): 881

SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

Alice Zhao, Laura Maple, Juwei Jiang, Katie N Myers, Callum G Jones, Hannah Gagg, Connor McGarrity-Cottrell, Ola Rominiyi, Spencer J Collis, Greg Wells, Marufur Rahman, Sarah J Danson, Darren Robinson, Carl Smythe, Chun Guo.

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, and its reversal, deSUMOylation by SUMO proteases like Sentrin-specific proteases (SENPs), are crucial for initiating cellular responses to hypoxia. However, their roles in subsequent adaptation processes to hypoxia such as mitochondrial autophagy (mitophagy) remain unexplored. Here, we show that general SUMOylation, particularly SUMO2/3 modification, suppresses mitophagy under both normoxia and hypoxia. Furthermore, we identify deSUMO2/3-ylation enzyme SENP3 and mitochondrial Fission protein 1 (FIS1) as key players in hypoxia-induced mitophagy (HIM), with SUMOylatable FIS1 acting as a crucial regulator for SENP3-mediated HIM regulation. Interestingly, we find that hypoxia promotes FIS1 SUMO2/3-ylation and triggers an interaction between SUMOylatable FIS1 and Rab GTPase-activating protein Tre-2/Bub2/Cdc16 domain 1 family member 17 (TBC1D17), which in turn suppresses HIM. Therefore, we propose a novel SUMOylation-dependent pathway where the SENP3-FIS1 axis promotes HIM, with TBC1D17 acting as a fine-tuning regulator. Importantly, the SENP3-FIS1 axis plays a protective role against hypoxia-induced cell death, highlighting its physiological significance, and hypoxia-inducible FIS1-TBC1D17 interaction is detectable in primary glioma stem cell-like (GSC) cultures derived from glioblastoma patients, suggesting its disease relevance. Our findings not only provide new insights into SUMOylation/deSUMOylation regulation of HIM but also suggest the potential of targeting this pathway to enhance cellular resilience under hypoxic stress.

DOI: https://doi.org/10.1038/s41419-024-07271-8
Oncogene. 2024 Dec 04.

JAK2/ULK1 axis promotes cervical cancer progression by autophagy induction and SRPK1 phosphorylation.

Qiuhong Duan, Wei Wang, Hua Xiong, Juanjuan Xiao, Han Xiao, Feng Zhu, Hui Lu.

Cervical cancer is the most common gynecologic cancer. Autophagy is involved in the progression of CCa. ULK1 is a crucial kinase in autophagy initiation. However, few studies have investigated the role of ULK1 phosphorylation at tyrosine residues in the progression of CCa, and the underlying mechanism remains elusive. In this study, we demonstrated that JAK2 is a novel upstream kinase that phosphorylates ULK1 at the tyrosine site. JAK2 interacts with and phosphorylates ULK1 at Tyr1007. The phosphorylation of ULK1 at Y1007 increases its activity and stability, activates autophagy, and promotes the progression of CCa. We further showed that the phosphorylation of ULK1 at Y1007 is a predictive marker of CCa patient outcome. Furthermore, we identified SRPK1 as a potential downstream substrate of ULK1 to promote the progression of CCa. Our research sheds light on the molecular mechanism of CCa progression, through JAK2/ULK1 axis, and emphasizes the phosphorylation of ULK1 at Y1007 as a predictor of CCa.

DOI: https://doi.org/10.1038/s41388-024-03246-3
J Biol Chem. 2024 Nov 29. pii: S0021-9258(24)02547-X. [Epub ahead of print] 108045

Quiescent cells maintain active degradation-mediated protein quality control requiring proteasome, autophagy and nucleus-vacuole junctions.

Dina Franić, Mihaela Pravica, Klara Zubčić, Shawna Miles, Antonio Bedalov, Mirta Boban.

  Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

Keywords:  autophagy; inclusions; nucleus-vacuole junction; proteasome; protein aggregation; protein degradation; protein quality control; quiescence; spatial sequestration; ubiquitin

DOI:  https://doi.org/10.1016/j.jbc.2024.108045
Arch Dermatol Res. 2024 Dec 05. 317(1): 70

Imperatorin promotes melanin degradation in keratinocytes through facilitating autophagy via the PI3K/Akt signaling pathway.

Pan Huang, Zhibo Yang, Haizhen Wang, Chang Wang, Meijunzi Luo, Rong Zhou, Yi Pan.

   PURPOSE: Melanin's pivotal role in skin protection and its overproduction leading to hyperpigmentation disorders highlight the necessity of regulating melanogenesis, with autophagy identified as a key degradation pathway. Imperatorin, a compound from Angelica dahurica, has been revealed to reduce melanin in epidermal keratinocytes, with the specific effects and mechanisms unknown. The purpose of this study was to investigate the mechanism by which imperatorin, reduces melanin production in HaCaT cells, with a focus on its potential role in promoting autophagy and regulating the PI3K/Akt signaling pathway.
METHODS: The study used HaCaT cells to investigate the effects of imperatorin on melanin production, autophagy, and PI3K/Akt signaling. Melanin content was measured using a spectrophotometric method. Protein levels of PMEL, ATG1, ATG5, and LC3B II were assessed by Western blotting. Autophagy was further visualized by GFP-LC3B puncta formation. The autophagy inhibitor 3-MA, the PI3K/Akt inhibitor LY294002 and PI3K/Akt activator 740 Y-P were used to assess the role of autophagy and PI3K/Akt signaling in imperatorin's effects. Cell viability was monitored to ensure that imperatorin's effects were not due to cytotoxicity.
RESULTS: Imperatorin reduced melanin content in HaCaT cells in a dose-dependent manner without compromising cell viability. This reduction in melanin was accompanied by decreased levels of PMEL protein, a key player in melanosome formation. Additionally, imperatorin promoted autophagy in HaCaT cells, as evidenced by increased levels of autophagy-associated markers ATG1, ATG5, and LC3B II, as well as an increase in GFP-LC3B puncta. The autophagy inhibitor 3-MA partially reversed the effects of imperatorin on both autophagy markers and PMEL levels, indicating that autophagy plays a crucial role in imperatorin's depigmentation action. Furthermore, imperatorin inhibited Akt and mTOR phosphorylation, which are downstream targets of PI3K/Akt signaling, enhancing autophagy and further reducing melanin levels. The PI3K/Akt inhibitor LY294002 amplified imperatorin's effects on PI3K and Akt phosphorylation, autophagy, and melanin levels. While, PI3K/Akt activator 740 Y-P reversed imperatorin's effects on these factors.
CONCLUSIONS: Imperatorin reduces melanin in HaCaT cells via promoting autophagy and melanin degradation, possibly via the PI3K/Akt signaling. Taken together, imperatorin has the therapeutic potential for the treatment of hyperpigmentation disorders.

Keywords:  Autophagy; Hyperpigmentation; Imperatorin; Keratinocytes; Melanin; The PI3K/Akt signaling pathway

DOI:  https://doi.org/10.1007/s00403-024-03559-z
Cell Signal. 2024 Dec 03. pii: S0898-6568(24)00509-6. [Epub ahead of print] 111534

Inhibition of XIST restrains paclitaxel resistance in breast cancer cells by targeting hsa-let-7d-5p/ATG16L1 through regulation of autophagy.

Yueyue Wang, Wenhao Pei, Yuping Yang, Chaoqun Xia, Qiang Zhang, Zhijun Geng, Xiuru Shi, Fengchao Wang.

  Breast cancer is a fatal malignant tumor in women worldwide. The development of paclitaxel resistance remains a challenge. Autophagy is considered to have a significant part in the chemotherapeutic stress mechanism. This study aimed to investigate the function of long non-coding RNA (lncRNA) in breast cancer cell chemoresistance and autophagy. The paclitaxel (PTX)-resistant breast cancer cells were established. The function of X-inactive specific transcript (XIST) was demonstrated using in vitro and in vivo experiments. Transmission electron microscope (TEM) was used to observe autophagy vesicles. Protein and mRNA levels were determined using western blotting and quantitative real time polymerase chain reaction (qRT-PCR). We discovered that autophagic activity was correlated with chemoresistance in PTX-resistant breast cancer cells. In vitro and in vivo studies showed that XIST inhibition reduced cell resistance to paclitaxel, caused autophagy to be suppressed by regulating hsa-let-7d-5p and ATG16L1 expression. Mechanically, threonine protein kinase B (PKB; also known as AKT) - mammalian target of rapamycin (mTOR) pathway was activated when knockdown of XIST, while was reversed by inhibition of hsa-let-7d-5p. Our results verified that XIST played a significant role in developing chemoresistance via mediating autophagy in PTX-resistant breast cancer cells. It may be a potential target for breast cancer treatment strategies.

Keywords:  Autophagy; Breast cancer; Chemoresistance; XIST

DOI:  https://doi.org/10.1016/j.cellsig.2024.111534
Free Radic Res. 2024 Dec 03. 1-12

Effects of autophagy on the selective death of human breast cancer cells exposed to plasma-activated Ringer's lactate solution.

Taishi Yamakawa, Ayako Tanaka, Camelia Miron, Kae Nakamura, Hiroaki Kajiyama, Shinya Toyokuni, Masaaki Mizuno, Masaru Hori, Hiromasa Tanaka.

  Plasma-activated Ringer's lactate (PAL) solution prepared by irradiating an intravenous solution with a non-equilibrium atmospheric pressure plasma is a potential new cancer therapy having no side effects. However, the induction of autophagy to avoid cell death has been confirmed to occur following exposure to PAL solution. It is thought that the antitumor effect of PAL solution could be weakened by this process, which is meant to maintain homeostasis in cells and assists tumorigenesis. Thus, it would be helpful to devise PAL-based cancer therapies that inhibit autophagy. Unfortunately, it is not yet clear which substances in PAL solution promote autophagy. The present work examined the mechanism by which PAL solution induces autophagy when treating MCF-7 human breast cancer cells. Autophagy was found to be temporarily induced upon exposure to PAL solution, suggesting that this effect contributes to cell proliferation. Although autophagy is associated with reactive oxygen and nitrogen species and/or acidic environments, in this study, significant autophagy was observed using a PAL solution diluted 1/256x without these stressors. Acetate, glyoxylate and 2,3-dimethyltartrate in the PAL solution were determined to promote autophagy. Interestingly, 2,3-dimethyltartrate was found to either induce cell death or autophagy depending on the concentration.

Keywords:  2,3-dimethyltartrate; Autophagy; breast cancer cells; non-equilibrium atmospheric pressure plasma; plasma-activated ringer’s lactate solution

DOI:  https://doi.org/10.1080/10715762.2024.2433965
Mol Med. 2024 Dec 06. 30(1): 242

Triolein alleviates ischemic stroke brain injury by regulating autophagy and inflammation through the AKT/mTOR signaling pathway.

Chaoqun Wang, Yuntao Li, Yonggang Zhang, Daniel Smerin, Lijuan Gu, Shuting Jiang, Xiaoxing Xiong.

   BACKGROUND: Triolein, a symmetric triglyceride exhibiting anti-inflammatory and antioxidant properties, has demonstrated potential in mitigating cellular damage. However, its therapeutic efficacy in ischemic stroke (IS) and underlying molecular mechanisms remain elusive. Given the critical roles of inflammation and autophagy in IS pathogenesis, this study aimed to elucidate the effects of triolein in IS and investigate its mechanism of action.
METHODS: We evaluated the impact of triolein using both in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) and in vivo middle cerebral artery occlusion (MCAO/R) models. Neurological function and cerebral infarct volume were assessed 72 h post-reperfusion. Autophagy was quantified through monodansyl cadaverine (MDC) labeling of autophagic vesicles and Western blot analysis of autophagy-related proteins. Microglial activation was visualized via immunofluorescence, while inflammatory cytokine expression was quantified using RT-qPCR. The cytoprotective effect of triolein on OGD/R-induced HT22 cells was evaluated using Cell Counting Kit-8 and lactate dehydrogenase release assays. The involvement of the Protein kinase B/Mechanistic target of rapamycin kinase (AKT/mTOR) pathway was assessed through Western blot analysis.
RESULTS: Triolein administration significantly reduced infarct volume, enhanced neurological recovery, and attenuated M1 microglial activation and inflammation in MCAO/R-induced mice. Western blot analysis and MDC labeling revealed that triolein exerted an inhibitory effect on post-IS autophagy. Notably, in the BV2-induced OGD/R model, triolein demonstrated an autophagy-dependent suppression of the inflammatory response. Furthermore, triolein inhibited the activation of the AKT/mTOR signaling pathway, consequently attenuating autophagy and mitigating the post-IS inflammatory response.
CONCLUSIONS: This study provides novel evidence that triolein exerts neuroprotective effects by inhibiting post-stroke inflammation through an autophagy-dependent mechanism. Moreover, the modulation of the AKT/mTOR signaling pathway appears to be integral to the neuroprotective efficacy of triolein. These findings elucidate potential therapeutic strategies for IS management and warrant further investigation.

Keywords:  AKT/ mTOR signaling pathway; Autophagy; Inflammation; Ischemic stroke; Neuroprotection

DOI:  https://doi.org/10.1186/s10020-024-00995-5
Stem Cell Res Ther. 2024 Dec 05. 15(1): 464

Trehalose extricates impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model.

Divyani Nayak, Shivapriya Shivakumar, Rohit Shetty, K N Prashanthi, Arkasubhra Ghosh, Nallathambi Jeyabalan, Koushik Chakrabarty.

   BACKGROUND: Patient-derived induced pluripotent stem cell (iPSCs) represents a powerful tool for elucidating the underlying disease mechanisms. Macular corneal dystrophy (MCD) is an intractable and progressive bilateral corneal disease affecting the corneal stroma due to mutation/s in carbohydrate sulfotransferase 6 (CHST6) gene. The underlying molecular mechanisms leading to MCD are unclear due to a lack of human contextual model and limited access to affected corneal stromal keratocytes (CSKs) from MCD patients. This has restricted the current treatment option for MCD to restorative corneal transplantation thereby lending itself to the use of iPSCs.
METHODS: induced pluripotent stem cells (iPSCs) were generated from two MCD patients and a healthy participant by senai virus based reprogramming of the peripheral mononuclear blood cells (PBMCs). The iPSCs were characterized based on the expression of pluripotent markers and formation of embryoid bodies possessing tri-lineage potential. Directed differentiation of the iPSCs to corneal stromal keratocytes (CSKs) was done via intermediate induction of neural crest cells. The iCSKs were characterized by immunocytochemistry and qPCR. Proteostat staining of the iCSKs was done to validate the disease phenotype invitro. Expression of autophagy markers in the iCSKs and JC staining were visualized by immunochemistry and live-cell imaging in trehalose treated iCSKs.
RESULTS: We show that the MCD iPSC-derived CSKs (MCDiCSKs) exhibits impaired autophagy assessed by the profiles of autophagy-associated proteins (LAMP1, LC3II/I, p62 and Beclin-1) and mitochondrial membrane potential. Significantly higher protein aggregates in MCDiCSKs was seen compared with the control, which could be rescued upon autophagy modulation. Hence, we treated MCD-iCSKs with trehalose (autophagy inducer) and showed that it protects MCD-iCSKs from mitochondrial dysfunction and maintains autophagic degradation.
CONCLUSION: Our study highlights the possible pathological mechanisms involved in MCD. We found trehalose ameliorate the impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model, which could be a potential alternative for MCD management.

Keywords:  Autophagy; Corneal stromal cells; Differentiation; Macular corneal dystrophy; Mitochondrial damage; Pluripotent stem cells

DOI:  https://doi.org/10.1186/s13287-024-04016-4
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2409447121

The essential role of TTC28 in maintaining chromosomal stability via HSPA8 chaperone-mediated autophagy.

Ge Zhang, Meiyi Xiang, Liankun Gu, Jing Zhou, Baozhen Zhang, Wei Tian, Dajun Deng.

  There are three distinct forms of autophagy, namely, macroautophagy, microautophagy, and HSPA8 chaperone-mediated autophagy (CMA). While macroautophagy is widely recognized as a regulator of chromosomal instability (CIN) through various pathways, the contributions of CMA and microautophagy to CIN remain uncertain. TTC28, a conserved gene in vertebrates, is frequently mutated and down-regulated in numerous human cancers. This study presents findings demonstrating the interaction between human tetratricopeptide repeat domain 28 (TTC28) and heat shock protein member 8 (HSPA8) and lysosomal-associated membrane protein 2A proteins. The tetratricopeptide repeat domains of TTC28 bind to the C-terminal motif (PTIEEVD) in HSPA8, resulting in the subsequent degradation of TTC28 via CMA/microautophagy. Notably, the baseline frequency of micronuclei (FMN) in human cancer cells with TTC28 knockout cells was three times greater than that in cells with wild-type TTC28 (7.7% vs. 2.3%, P = 4.86E-09). Furthermore, the overexpression of Ttc28 mitigated the impact of TTC28 knockout on FMN (11.9% vs. 4.8%, P = 2.83E-11). Our findings also demonstrate that CMA has a protective effect on genome stability and that TTC28 plays an essential role in the effect of CMA. These results were further supported by the quantification of γH2AX and comet analyses and the analysis of The Cancer Genome Atlas data via bioinformatics. Mechanistically, TTC28 regulates mitosis and cytokinesis, which are involved in the maintenance of genome integrity by CMA. In conclusion, our study demonstrated that TTC28 is not only an HSPA8-mediated CMA/microautophagy substrate but also essential for maintaining chromosomal stability via CMA. Comprehensive TTC28 downregulation may lead to CIN in cancer cells.

Keywords:  HSPA8; TTC28; cancer; chaperone-mediated autophagy; chromosomal instability

DOI:  https://doi.org/10.1073/pnas.2409447121
Front Immunol. 2024 ;15 1457255

FBXW8 suppresses PDCoV proliferation via the NPD52-dependent autophagic degradation of a viral nucleocapsid protein.

Likai Ji, Liying Zhou, Ying Wang, Shixing Yang, Yuwei Liu, Xiaochun Wang, Quan Shen, Chenglin Zhou, Juan Xu, Wen Zhang.

  Porcine deltacoronavirus (PDCoV), a newly discovered intestinal coronavirus, has rapidly spread among pigs worldwide and has shown the potential for cross-species infection. However, the interaction mechanism between PDCoV and the host's antiviral response is still poorly understood. In this study, an E3 ubiquitin ligase FBXW8 was explored on PDCoV proliferation. Our findings demonstrate that PDCoV infection increases the expression of FBXW8 through p65-mediated activation of its promoter. We also discovered that FBXW8 suppresses PDCoV replication by directly targeting and inducing the degradation of the PDCoV-encoded nucleocapsid (N) protein. Interestingly, FBXW8 catalyzes the K48-linked polyubiquitination of the PDCoV N protein at a unique lysine-rich region (KR). Furthermore, we observed that the FBXW8-ubiquitinated PDCoV N protein interacts with NDP52, a cargo receptor, leading to autophagic degradation instead of proteasomal degradation. In summary, these findings reveal FBXW8 as a novel host antiviral factor involved in PDCoV infection. It mediates the NDP52-dependent autophagic degradation of the PDCoV N protein. These results provide new insights and a potential target for host defenses against PDCoV.

Keywords:  FBXW8; N protein; NDP52; PDCoV; selective autophagy

DOI:  https://doi.org/10.3389/fimmu.2024.1457255
J Extracell Vesicles. 2024 Dec;13(12): e70018

Dopamine-conjugated extracellular vesicles induce autophagy in Parkinson's disease.

Jae Hoon Sul, Sol Shin, Hark Kyun Kim, Jihoon Han, Junsik Kim, Soyong Son, Jungmi Lee, Seung Hyun Baek, Yoonsuk Cho, Jeongmi Lee, Jinsu Park, Donghoon Ahn, Sunyoung Park, Leon F Palomera, Jeein Lim, Jongho Kim, Chanhee Kim, Seungsu Han, Ka Young Chung, Sangho Lee, Tae-In Kam, Yunjong Lee, Jeongyun Kim, Jae Hyung Park, Dong-Gyu Jo.

  The application of extracellular vesicles (EVs) as vehicles for anti-Parkinson's agents represents a significant advance, yet their clinical translation is hampered by challenges in efficient brain delivery and complex blood-brain barrier (BBB) targeting strategies. In this study, we engineered dopamine onto the surface of adipose-derived stem cell EVs (Dopa-EVs) utilizing a facile, two-step cross-linking approach. This engineering enhanced neuronal uptake of the EVs in primary neurons and neuroblastoma cells, a process shown to be competitively inhibited by dopamine pretreatment and dopamine receptor antibodies. Notably, Dopa-EVs demonstrated increased brain accumulation in mouse Parkinson's disease (PD) models. Therapeutically, Dopa-EVs administration led to the rescue of dopaminergic neuronal loss and amelioration of behavioural deficits in both 6-hydroxydopamine (6-OHDA) and α-Syn PFF-induced PD models. Furthermore, we observed that Dopa-EVs stimulated autophagy evidenced by the upregulation of Beclin-1 and LC3-II. These findings collectively indicate that surface modification of EVs with dopamine presents a potent strategy for targeting dopaminergic neurons in the brain. The remarkable therapeutic potential of Dopa-EVs, demonstrated in PD models, positions them as a highly promising candidate for PD treatment, offering a significant advance over current therapeutic modalities.

Keywords:  Parkinson's disease; autophagy; dopamine; exosome; extracellular vesicles

DOI:  https://doi.org/10.1002/jev2.70018
Cell Mol Biol Lett. 2024 Dec 05. 29(1): 151

Dynamic regulation of proximal tubular autophagy from injury to repair after ischemic kidney damage.

Yuhong Gong, Wei Zhu, Yongqiang Li, Tao Lu, Jiexing Tan, Changsheng He, Luodan Yang, Yufeng Zhu, Li Gong.

   BACKGROUND: The role of proximal tubular autophagy in repairing kidney injury following ischemia remains unclear.
METHODS: In this study, we utilized mice with conditional deletion of the Atg5 gene in proximal tubules and monitored the long-term dynamic regulation of autophagy following ischemic acute kidney injury (AKI).
RESULTS: The results showed that Atg5-deficient proximal tubule epithelial cells exhibited damaged mitochondria, concentric membranes, and lysosomal accumulation 24 h after ischemia/reperfusion. However, 28 days after ischemia/reperfusion, concentric membrane bodies remained, but lysosomal accumulation was no longer observed. Notably, the absence of Atg5 in renal tubular epithelial cells impaired renal function and led to increased tubular cell proliferation and oxidative stress in the early stage of injury. However, during the repair period following AKI, Atg5 deficiency exhibited no significant difference in the expression of proliferating cell nuclear antigen (PCNA) and 4-hydoxynonenal (4HNE), suggesting that the improvement in renal fibrosis associated with Atg5 deficiency is unlikely to result from its effect on cell proliferation or reactive oxygen species levels. Additionally, Atg5 deficiency inhibits the secretion of profibrotic factor fibroblast growth factor 2 (FGF2) from the early stage of renal injury to the recovery stage of AKI, indicating that autophagy-specific regulation of FGF2 secretion is a dynamic process overlapping with other stages of injury. Furthermore, increased co-localization of ATG5 with 4HNE and FGF2 was observed in patient samples.
CONCLUSION: In summary, our results suggest that the dynamic regulation of autophagy on key molecules involved in kidney injury and repair varies with the stage of kidney injury.

Keywords:  Acute kidney injury; Autophagy; Interstitial fibrosis; Proximal tubule

DOI:  https://doi.org/10.1186/s11658-024-00663-w
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2406944121

Caspase-8-dependent autophagy regulates neutrophil infiltration in oral squamous cell carcinoma.

Miguel Bernabé-Rubio, Fiona M Watt.

  Oral squamous cell carcinoma (OSCC) is a subtype of head and neck cancer that arises in the multilayered epithelia of the mouth and lips. Although inactivating mutations in CASP8 are frequently found in human OSCC their role in the disease is unknown. To investigate this, we deleted Casp8 in the oral epithelium of adult mice. Loss of Caspase-8 resulted in defects in the tongue epithelial barrier and triggered a neutrophil-rich immune infiltrate distinct from that observed on epidermal Casp8 deletion. Oral Casp8 deletion led to activation of autophagy. Inhibition of autophagy partially rescued epithelial integrity in Casp8-/- mice, while induction of autophagy in wild type mice resulted in oral barrier defects and excessive neutrophil infiltration. On treatment with the carcinogen 4-nitroquinoline-1-oxide Casp8-/- mice showed increased susceptibility to developing oral tumors. Depletion of neutrophils reduced tumor incidence, which correlated with a reduction in reactive oxygen species and decreased epithelial DNA damage. Our findings establish a functional link between epithelial integrity, autophagy, and the tumor immune microenvironment, placing Caspase-8 at the center of these processes.

Keywords:  autophagy; cancer; epithelia; neutrophils

DOI:  https://doi.org/10.1073/pnas.2406944121
Proc Natl Acad Sci U S A. 2024 Dec 10. 121(50): e2412157121

Glutamine is critical for the maintenance of type 1 conventional dendritic cells in normal tissue and the tumor microenvironment.

Graham P Lobel, Nanumi Han, William A Molina Arocho, Michal Silber, Jason Shoush, Michael C Noji, Tsun Ki Jerrick To, Li Zhai, Nicholas P Lesner, M Celeste Simon, Malay Haldar.

  Proliferating tumor cells take up glutamine for anabolic processes, engendering glutamine deficiency in the tumor microenvironment. How this might impact immune cells is not well understood. Using multiple mouse models of soft tissue sarcomas, glutamine antagonists, as well as genetic and pharmacological inhibition of glutamine utilization, we found that the number and frequency of conventional dendritic cells (cDCs) is dependent on microenvironmental glutamine levels. cDCs comprise two distinct subsets-cDC1s and cDC2s, with the former subset playing a critical role in antigen cross-presentation and tumor immunity. While both subsets show dependence on glutamine, cDC1s are particularly sensitive. Notably, glutamine antagonism did not reduce the frequency of DC precursors but decreased the proliferation and survival of cDC1s. Further studies suggest a role of the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway in this process. Taken together, these findings uncover glutamine dependence of cDC1s that is coopted by tumors to escape immune responses.

Keywords:  dendritic cells; glutamine; tumor microenvironment

DOI:  https://doi.org/10.1073/pnas.2412157121
Front Cell Neurosci. 2024 ;18 1470144

MAM-mediated mitophagy and endoplasmic reticulum stress: the hidden regulators of ischemic stroke.

Ziyi Jia, Hongtao Li, Ke Xu, Ruobing Li, Siyu Yang, Long Chen, Qianwen Zhang, Shulin Li, Xiaowei Sun.

  Ischemic stroke (IS) is the predominant subtype of stroke and a leading contributor to global mortality. The mitochondrial-associated endoplasmic reticulum membrane (MAM) is a specialized region that facilitates communication between the endoplasmic reticulum and mitochondria, and has been extensively investigated in the context of neurodegenerative diseases. Nevertheless, its precise involvement in IS remains elusive. This literature review elucidates the intricate involvement of MAM in mitophagy and endoplasmic reticulum stress during IS. PINK1, FUNDC1, Beclin1, and Mfn2 are highly concentrated in the MAM and play a crucial role in regulating mitochondrial autophagy. GRP78, IRE1, PERK, and Sig-1R participate in the unfolded protein response (UPR) within the MAM, regulating endoplasmic reticulum stress during IS. Hence, the diverse molecules on MAM operate independently and interact with each other, collectively contributing to the pathogenesis of IS as the covert orchestrator.

Keywords:  endoplasmic reticulum stress; ischemic stroke (IS); mitochondrial-associated endoplasmic reticulum membrane (MAM); mitophagy; unfolded protein response (UPR)

DOI:  https://doi.org/10.3389/fncel.2024.1470144
Nat Commun. 2024 Dec 05. 15(1): 10603

The Greatwall-Endosulfine-PP2A/B55 pathway regulates entry into quiescence by enhancing translation of Elongator-tunable transcripts.

Javier Encinar Del Dedo, M Belén Suárez, Rafael López-San Segundo, Alicia Vázquez-Bolado, Jingjing Sun, Natalia García-Blanco, Patricia García, Pauline Tricquet, Jun-Song Chen, Peter C Dedon, Kathleen L Gould, Elena Hidalgo, Damien Hermand, Sergio Moreno.

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. In this study, we demonstrate that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications crucial for sustaining the translation programme during entry into quiescence. This mechanism promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, several of these mRNAs encode TORC1 inhibitors, TORC2 activators, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between tRNA epitranscriptome and biased codon usage.

DOI: https://doi.org/10.1038/s41467-024-55004-4
Nat Cell Biol. 2024 Dec 02.

Women in Autophagy: an initiative to promote gender parity in science.

Mericka McCabe, Patricia Boya, Ruey-Hwa Chen, Charleen T Chu, Maria Isabel Colombo, Laura Delgui, Eeva-Liisa Eskelinen, Maho Hamasaki, Malene Hansen, Congcong He, Marja Jäättelä, Adi Kimchi, Claudine Kraft, Mondira Kundu, Alicia Melendez, Sophie Pattingre, Tassula Proikas-Cezanne, Salwa Sebti, Anna Katharina Simon, Anne Simonsen, Sharon A Tooze, Maria Ines Vaccaro, Xiaochen Wang, Eileen White, Yan Zhao, Ana Maria Cuervo.

DOI: https://doi.org/10.1038/s41556-024-01566-w
PLoS One. 2024 ;19(12): e0312859

Rapamycin administration causes a decrease in muscle contractile function and systemic glucose intolerance concomitant with reduced skeletal muscle Rictor, the mTORC2 component, expression independent of energy intake in young rats.

Satoru Ato, Chieri Oya, Riki Ogasawara.

Emerging evidence suggests the potential of rapamycin, an antibiotic from Streptomyces hygroscopicus that functions as a mechanistic target of rapamycin (mTOR) inhibitor, as a mimetic of caloric restriction (CR) for maintaining skeletal muscle health. Several studies showed that rapamycin administration (RAP) reduced appetite and energy intake. However, the physiological and molecular differences between RAP and CR in skeletal muscle are not fully understood. Here we observed the effects of 4 weeks of RAP administration and CR corresponding to the reduction in energy intake produced by RAP administration (PF, paired feeding) on fast glycolytic and slow oxidative muscle in young adult rats. We found that 4 weeks of RAP demonstrated low fast-glycolytic muscle mass with smaller type I and IIb/x myofiber size independent of the energy intake. In addition, PF improved the contractile function of the plantar flexor muscle, whereas RAP did not improve its function. The suppressing response of mTORC1 signaling to RAP is greater in slow-oxidative muscles than in fast-glycolytic muscles. In addition, systemic glucose tolerance was exacerbated by RAP, with reduced expression of Rictor and hexokinase in skeletal muscle. These observations imply that RAP may have a slight but significant negative impact and it obviously different to CR in young adult skeletal muscle.

DOI: https://doi.org/10.1371/journal.pone.0312859
Oncogene. 2024 Dec 03.

Discovery of a molecular glue for EGFR degradation.

Hairui Wang, Hui Wang, Rui Wang, Yuanzhen Li, Zhipeng Wang, Wenshen Zhou, Li Deng, Xiyin Li, Li Zou, Qin Yang, Ren Lai, Xiaowei Qi, Jianyun Nie, Baowei Jiao.

Aberrant expression of epidermal growth factor receptor (EGFR) plays a critical role in the pathogenesis of various tumors, potentially representing a target for therapeutic intervention. Nonetheless, EGFR remains a challenging protein to target pharmacologically in triple-negative breast cancer (TNBC). An emerging approach to address the removal of such proteins is the application of molecular glue (MG) degraders. These compounds facilitate protein-protein interactions between a target protein and an E3-ubiquitin ligase, subsequently leading to protein degradation. Herein, we identified a new MG (CDDO-Me, C-28 methyl ester of 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid), which orchestrated binding between EGFR and KEAP1 (an E3-ubiquitin ligase adapter), thereby initiating the ubiquitination and degradation of EGFR. CDDO-Me directly interacted with the tyrosine kinase (TK) domain of EGFR, resulting in its degradation via an autophagy-dependent lysosomal pathway. Knockdown of KEAP1 decreased the degradation of EGFR by reducing its K63-linked ubiquitination, leading to diminished EGFR colocalization in autophagosomes and lysosomes. Notably, CDDO-Me attenuates TNBC progression by accelerating EGFR degradation in cell-derived xenografts and patient-derived organoid models, highlighting its clinical application potential. Consequently, induction of EGFR degradation through MG degraders represents a viable therapeutic strategy for TNBC.

DOI: https://doi.org/10.1038/s41388-024-03241-8
PLoS One. 2024 ;19(12): e0314873

Ruscus aculeatus extract promotes RNase 7 expression through ERK activation following inhibition of late-phase autophagy in primary human keratinocytes.

Shigeyuki Ono, Akiko Kawasaki, Kotaro Tamura, Yoshihiko Minegishi, Takuya Mori, Noriyasu Ota.

Antimicrobial peptides (AMPs) are crucial for protecting human skin from infection. Therefore, the expression levels of beneficial AMPs such as ribonuclease 7 (RNase 7) must be appropriately regulated in healthy human skin. However, there is limited understanding regarding the regulating AMP expression, especially when using applications directly to healthy human skin. Here, we investigated the effects of the extract of Ruscus aculeatus (RAE), a medicinal plant native to Mediterranean Europe and Africa that is known to have a high safety level, on AMP expression in primary human keratinocytes. Treatment with RAE induced RNase 7 expression, which was suppressed by an extracellular signal-regulated kinase (ERK) inhibitor. The autophagic flux assay and the immunofluorescence analysis of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ and p62 showed that RAE inhibited late-phase autophagy. Moreover, both the inhibition of early-phase autophagy by EX-527, an inhibitor of silent information regulator of transcription 1 (SIRT1) and its enhancement by resveratrol, an activator of SIRT1 inhibited RNase 7 and ERK expression, indicating that autophagosome accumulation is necessary for RAE-induced RNase 7 expression. Additionally, spilacleoside was identified as the active component in RAE. These findings suggest that RAE promotes RNase 7 expression via ERK activation following inhibition of late-phase autophagy in primary human keratinocytes and that this mechanism is a novel method of regulation of AMP expression.

DOI: https://doi.org/10.1371/journal.pone.0314873
Exp Mol Med. 2024 Dec 04.

TIGAR coordinates senescence-associated secretory phenotype via lysosome repositioning and α-tubulin deacetylation.

Hae Yun Nam, Seung-Ho Park, Geun-Hee Lee, Eun-Young Kim, SangEun Lee, Hyo Won Chang, Eun-Ju Chang, Kyung-Chul Choi, Seong Who Kim.

TP53-induced glycolysis and apoptosis regulator (TIGAR) regulates redox homeostasis and provides the intermediates necessary for cell growth by reducing the glycolytic rate. During cellular senescence, cells undergo metabolic rewiring towards the glycolytic pathway, along with the development of the senescence-associated secretory phenotype (SASP), also known as the secretome. We observed that TIGAR expression increased during replicative senescence following the in vitro expansion of human mesenchymal stromal cells (MSCs) and that TIGAR knockout (KO) decreased SASP factors and triggered premature senescence with decelerated progression. Additionally, TIGAR KO impaired flexible lysosomal movement to the perinuclear region and decreased the autophagic flux of MSCs. Research on the mechanism of lysosomal movement revealed that, while native senescent MSCs presented low levels of Ac-α-tubulin (lysine 40) and increased sirtuin 2 (SIRT2) activity compared with those in growing cells, TIGAR KO-MSCs maintained Ac-α-tubulin levels and exhibited decreased SIRT2 activity despite being in a senescent state. The overexpression of SIRT2 reduced Ac-α-tubulin as a protein target of SIRT2 and induced the positioning of lysosomes at the perinuclear region, restoring the cytokine secretion of TIGAR KO-MSCs. Furthermore, TIGAR expression was positively correlated with SIRT2 activity, indicating that TIGAR affects SIRT2 activity partly by modulating the NAD+ level. Thus, our study demonstrated that TIGAR provides a foundation that translates the regulation of energy metabolism into lysosome positioning, affecting the secretome for senescence development. Considering the functional value of the cell-secretome in aging-related diseases, these findings suggest the feasibility of TIGAR for the regulation of secretory phenotypes.

DOI: https://doi.org/10.1038/s12276-024-01362-4
Nat Rev Mol Cell Biol. 2024 Dec 03.

Calcium connects lysosomal damage to stress granule formation.

Kim Baumann.

DOI: https://doi.org/10.1038/s41580-024-00817-w
Sci Transl Med. 2024 Dec 04. 16(776): eadl4616

Myeloid cell-specific loss of NPC1 in mice recapitulates microgliosis and neurodegeneration in patients with Niemann-Pick type C disease.

Lina Dinkel, Selina Hummel, Valerio Zenatti, Mariagiovanna Malara, Yannik Tillmann, Alessio Colombo, Laura Sebastian Monasor, Jung H Suh, Todd Logan, Stefan Roth, Lars Paeger, Patricia Hoffelner, Oliver Bludau, Andree Schmidt, Stephan A Müller, Martina Schifferer, Brigitte Nuscher, Jasenka Rudan Njavro, Matthias Prestel, Laura M Bartos, Karin Wind-Mark, Luna Slemann, Leonie Hoermann, Sebastian T Kunte, Johannes Gnörich, Simon Lindner, Mikael Simons, Jochen Herms, Dominik Paquet, Stefan F Lichtenthaler, Peter Bartenstein, Nicolai Franzmeier, Arthur Liesz, Antje Grosche, Tatiana Bremova-Ertl, Claudia Catarino, Skadi Beblo, Caroline Bergner, Susanne A Schneider, Michael Strupp, Gilbert Di Paolo, Matthias Brendel, Sabina Tahirovic.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1 gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration. Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1 in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we report severe alterations in microglial lipidomic profiles, including the enrichment of bis(monoacylglycero)phosphate, increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hyperactivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a pathological cascade resembling NPC-like phenotypes, including a shortened life span, motor impairments, astrogliosis, neuroaxonal pathology, and increased neurofilament light chain (NF-L), a neuronal injury biomarker. As observed in the mouse model, patients with NPC showed increased NF-L in the blood and microglial hyperactivity, as visualized by TSPO-PET imaging. Reduced TSPO expression in blood-derived macrophages of patients with NPC was measured after N-acetyl-l-leucine treatment, which has been recently shown to have beneficial effects in patients with NPC, suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further investigated for therapeutic targeting and disease monitoring.

DOI: https://doi.org/10.1126/scitranslmed.adl4616