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BioRxiv : the Preprint Server For... Apr 2024Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we...
Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.
PubMed: 38903076
DOI: 10.1101/2024.04.02.587808 -
Experimental Neurology Jun 2024Ischemic stroke is a disease associated with high morbidity and disability rates; however, its pathogenesis remains elusive, and treatment options are limited....
Ischemic stroke is a disease associated with high morbidity and disability rates; however, its pathogenesis remains elusive, and treatment options are limited. Ferroptosis, an iron-dependent form of cell death, represents a novel avenue for investigation. The objective of this study was to explore the role of melatonin in MCAO-induced ferroptosis and elucidate its underlying molecular mechanism. To simulate brain damage and neuronal injury caused by ischemic stroke, we established a mouse model of MCAO and an HT-22 cell model of OGD/R. The therapeutic efficacy of melatonin was assessed through measurements of infarct size, brain edema, and neurological scores. Additionally, qRT-PCR, WB analysis, and Co-IP assays were employed to investigate the impact of melatonin on ferroptosis markers such as NCOA4 and FTH1 expression levels. Confocal microscopy was utilized to confirm the colocalization between ferritin and lysosomes. Furthermore, we constructed a SIRT6 siRNA model to validate the regulatory effect exerted by SIRT6 on NCOA4 as well as their binding interaction. The present study provides initial evidence that melatonin possesses the ability to mitigate neuronal damage induced by MCAO and OGD/R. Assessment of markers for oxidative damage and ferroptosis revealed that melatonin effectively inhibits intracellular Fe2+ levels, thereby suppressing ferroptosis. Additionally, our findings demonstrate that melatonin modulates the interaction between FTH1 and NCOA4 via SIRT6, influencing ferritin autophagy without affecting cellular macroautophagy. These findings provide reliable data support for the promotion and application of melatonin in clinical practice.
PubMed: 38901754
DOI: 10.1016/j.expneurol.2024.114868 -
Biochimica Et Biophysica Acta.... Jun 2024The pancreas is a glandular organ with both endocrine and exocrine functions. Researchers have investigated the roles of several Rab proteins, which are major regulators...
The pancreas is a glandular organ with both endocrine and exocrine functions. Researchers have investigated the roles of several Rab proteins, which are major regulators of membrane trafficking, in pancreatic exocytosis of zymogen granules in exocrine cells, also known as acinar cells. However, detailed molecular mechanisms mediated by Rab proteins are not fully understood. RASEF/Rab45 is an atypical Rab GTPase that contains N-terminal EF-hand and coiled-coil domains, as well as a C-terminal Rab-GTPase domain. In this study, we investigated the in vivo role of RASEF in pancreatic acinar cells using RASEF-knockout (KO) mice. Morphological analyses revealed that pancreatic acinar cells in RASEF-KO mice had an increased number of zymogen granules and abnormal formations of organelles, such as the endoplasmic reticulum (ER) and lysosomes. Biochemical analyses showed that ER proteins were decreased, but digestive enzymes were increased in the RASEF-KO pancreas. Moreover, trypsinogen was activated and co-localized with the endo-lysosomal marker LAMP1 in RASEF-KO pancreas. Upon cerulein administration to induce acute pancreatitis, impaired enzyme release from the pancreas was observed in the serum of RASEF-KO mice. These findings suggest that RASEF likely regulates the formation and sorting of zymogen granules and secretion of digestive enzymes by pancreatic acinar cells.
PubMed: 38901651
DOI: 10.1016/j.bbadis.2024.167310 -
Current Medical Science Jun 2024Icariin (ICA) has a good neuroprotective effect and can upregulate neuronal basal autophagy in naturally aging rats. Mitochondrial dysfunction is associated with brain...
OBJECTIVE
Icariin (ICA) has a good neuroprotective effect and can upregulate neuronal basal autophagy in naturally aging rats. Mitochondrial dysfunction is associated with brain aging-related neurodegenerative diseases. Abnormal opening of the mitochondrial permeability transition pore (mPTP) is a crucial factor in mitochondrial dysfunction and is associated with excessive autophagy. This study aimed to explore that ICA protects against neuronal injury by blocking the mPTP opening and down-regulating autophagy levels in a D-galactose (D-gal)-induced cell injury model.
METHODS
A cell model of neuronal injury was established in rat pheochromocytoma cells (PC12 cells) treated with 200 mmol/L D-gal for 48 h. In this cell model, PC12 cells were pre-treated with different concentrations of ICA for 24 h. MTT was used to detect cell viability. Senescence associated β-galactosidase (SA-β-Gal) staining was used to observe cell senescence. Western blot analysis was performed to detect the expression levels of a senescence-related protein (p21), autophagy markers (LC3B, p62, Atg7, Atg5 and Beclin 1), mitochondrial fission and fusion-related proteins (Drp1, Mfn2 and Opa1), and mitophagy markers (Pink1 and Parkin). The changes of autophagic flow were detected by using mRFP-GFP-LC3 adenovirus. The intracellular ultrastructure was observed by transmission electron microscopy. Immunofluorescence was used to detect mPTP, mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (mtROS) and ROS levels. ROS and apoptosis levels were detected by flow cytometry.
RESULTS
D-gal treatment significantly decreased the viability of PC12 cells, and markedly increased the SA-β-Gal positive cells as compared to the control group. With the D-gal stimulation, the expression of p21 was significantly up-regulated. Furthermore, D-gal stimulation resulted in an elevated LC3B II/I ratio and decreased p62 expression. Meanwhile, autophagosomes and autolysosomes were significantly increased, indicating abnormal activation of autophagy levels. In addition, in this D-gal-induced model of cell injury, the mPTP was abnormally open, the ROS generation was continuously increased, the MMP was gradually decreased, and the apoptosis was increased. ICA effectively improved mitochondrial dysfunction to protect against D-gal-induced cell injury and apoptosis. It strongly inhibited excessive autophagy by blocking the opening of the mPTP. Cotreatment with ICA and an mPTP inhibitor (cyclosporin A) did not ameliorate mitochondrial dysfunction. However, the protective effects were attenuated by cotreatment with ICA and an mPTP activator (lonidamine).
CONCLUSION
ICA inhibits the activation of excessive autophagy and thus improves mitochondrial dysfunction by blocking the mPTP opening.
PubMed: 38900385
DOI: 10.1007/s11596-024-2892-0 -
ACS Nano Jun 2024Aluminum salts still remain as the most popular adjuvants in marketed human prophylactic vaccines due to their capability to trigger humoral immune responses with a good...
Aluminum salts still remain as the most popular adjuvants in marketed human prophylactic vaccines due to their capability to trigger humoral immune responses with a good safety record. However, insufficient induction of cellular immune responses limits their further applications. In this study, we prepare a library of silicon (Si)- or calcium (Ca)-doped aluminum oxyhydroxide (AlOOH) nanoadjuvants. They exhibit well-controlled physicochemical properties, and the dopants are homogeneously distributed in nanoadjuvants. By using Hepatitis B surface antigen (HBsAg) as the model antigen, doped AlOOH nanoadjuvants mediate higher antigen uptake and promote lysosome escape of HBsAg through lysosomal rupture induced by the dissolution of the dopant in the lysosomes in bone marrow-derived dendritic cells (BMDCs). Additionally, doped nanoadjuvants trigger higher antigen accumulation and immune cell activation in draining lymph nodes. In HBsAg and varicella-zoster virus glycoprotein E (gE) vaccination models, doped nanoadjuvants induce high IgG titer, activations of CD4 and CD8 T cells, cytotoxic T lymphocytes, and generations of effector memory T cells. Doping of aluminum salt-based adjuvants with biological safety profiles and immunostimulating capability is a potential strategy to mediate robust humoral and cellular immunity. It potentiates the applications of engineered adjuvants in the development of vaccines with coordinated immune responses.
PubMed: 38899978
DOI: 10.1021/acsnano.4c02685 -
Autophagy Jun 2024When exposed to new experiences or changes in the environment, neurons rapidly remodel their synaptic structure and function in a process called activity-induced...
When exposed to new experiences or changes in the environment, neurons rapidly remodel their synaptic structure and function in a process called activity-induced synaptic remodeling. This process is necessary for transforming transient experiences into stable, lasting memories. The molecular mechanisms underlying acute, activity-dependent synaptic changes are not well understood, partly because processes regulating synaptic plasticity and neurodevelopment are intricately linked. By using an RNAi screen in targeting genes associated with human nervous system function, we found that while macroautophagy (referred to as autophagy) is fundamental for both synapse development and synaptic plasticity, activity-induced synaptic remodeling does not rely on genes associated with lysosomal degradation. These findings suggest a requirement for the unconventional secretory autophagy pathway in regulating synaptic plasticity, wherein autophagosomes, instead of fusing with lysosomes for degradation, fuse with the plasma membrane to release their contents extracellularly. To test this hypothesis, we knocked down Sec22, Snap29, and Rab8, molecular components required for secretory autophagy, all of which disrupted structural and functional plasticity. Additionally, by monitoring autophagy, we demonstrated that neuronal activity suppresses degradative autophagy to shift the pathway toward secretory autophagy release. Our work unveils secretory autophagy as a novel trans-synaptic signaling mechanism crucial for activity-induced synaptic remodeling.
PubMed: 38899624
DOI: 10.1080/15548627.2024.2370179 -
ELife Jun 2024The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn)...
The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.
PubMed: 38899618
DOI: 10.7554/eLife.98649 -
Autophagy Jun 2024In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form....
In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in and , core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases.: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.
PubMed: 38899385
DOI: 10.1080/15548627.2024.2366122 -
Dalton Transactions (Cambridge, England... Jun 2024In this work we disclose a new family of biscyclometallated Ir(III) complexes of the general formula [Ir(C^N)(N^N)]Cl (IrL1-IrL5), where HC^N is 1-phenyl-β-carboline...
In this work we disclose a new family of biscyclometallated Ir(III) complexes of the general formula [Ir(C^N)(N^N)]Cl (IrL1-IrL5), where HC^N is 1-phenyl-β-carboline and N^N ligands (L1-L5) are different diimine ligands that differ from each other in the number of aromatic rings fused to the bipyridine scaffold. The photophysical properties of IrL1-IrL5 were thoroughly studied, and theoretical calculations were performed for a deeper comprehension of the respective variations along the series. All complexes exhibited high photostability under blue light irradiation. An increase in the number of aromatic rings led to a reduction in the HOMO-LUMO band gap causing a red-shift in the absorbance bands. Although all the complexes generated singlet oxygen (O) in aerated aqueous solutions through a photocatalytic process, IrL5 was by far the most efficient photosensitizer. Consequently, IrL5 was highly active in the photocatalytic oxidation of NADH. The formation of aggregates in DMSO at a high concentration (25 mM) was confirmed using different techniques, but was proved to be negligible in the concentration range of biological experiments. Moreover, ICP-MS studies proved that the cellular uptake of IrL2 and IrL3 is much better relative to that of IrL1, IrL4 and IrL5. The antiproliferative activity of IrL1-IrL5 was investigated in the dark and under blue light irradiation against different cancer cell lines. Complexes IrL1-IrL4 were found to be cytotoxic under dark conditions, while IrL5 turned out to be weakly cytotoxic. Despite the low cellular uptake of IrL5, this derivative exhibited a high increase of cytotoxicity upon blue light irradiation resulting in photocytotoxicity indexes (PI) up to 38. IrL1-IrL4 showed lower photocytotoxicity indexes ranging from 1.3 to 17.0. Haemolytic experiments corroborated the compatibility of our complexes with red blood cells. Confocal microscopy studies proved their accumulation in mitochondria, leading to mitochondrial membrane depolarization, and ruled out their localization in lysosomes. Overall, the mitochondria-targeted activity of IrL5, which inhibits considerably the viability of cancer cells upon blue light irradiation, allows us to outline this PS as a new alternative to traditional chemotherapeutic agents.
PubMed: 38899369
DOI: 10.1039/d4dt00390j -
Journal of Experimental & Clinical... Jun 2024Though tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge...
BACKGROUND
Though tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated.
METHODS
High-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts.
RESULTS
PRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer.
CONCLUSIONS
PRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer.
Topics: Tamoxifen; Humans; Female; Breast Neoplasms; Animals; Receptors, Prolactin; Mice; Immunotoxins; Xenograft Model Antitumor Assays; Cell Line, Tumor; Drug Resistance, Neoplasm; Cell Proliferation; Apoptosis
PubMed: 38898487
DOI: 10.1186/s13046-024-03099-4