Cayratia albifolia C.L.Li exerts anti-rheumatoid arthritis effect by inhibiting macrophage activation and neutrophil extracellular traps (NETs).

BACKGROUND: Cayratia albifolia C.L.Li (CAC), commonly known as "Jiao-Mei-Gu" in China, has been extensively utilized by the Dong minority for several millennia to effectively alleviate symptoms associated with autoimmune diseases. CAC extract is believed to possess significant anti-inflammatory properties within the context of Dong medicine. However, an in-depth understanding of the specific pharmaceutical effects and underlying mechanisms through which CAC extract acts against rheumatoid arthritis (RA) has yet to be established. METHODS: Twenty-four Sprague-Dawley rats were divided into four groups, with six rats in each group. To induce the collagen-induced arthritis (CIA) model, the rats underwent a process of double immunization with collagen and adjuvant. CAC extract (100 mg/kg) was orally administered to rats. The anti-RA effects were evaluated in CIA rats by arthritis score, hind paw volume and histopathology analysis. Pull-down assay was conducted to identify the potential targets of CAC extract from RAW264.7 macrophage lysates. Moreover, mechanism studies of CAC extract were performed by immunofluorescence assays, real-time PCR and Western blot. RESULTS: CAC extract was found to obviously down-regulate hind paw volume of CIA rats, with diminished inflammation response and damage. 177 targets were identified from CAC extract by MS-based pull-down assay. Bioinformatics analysis found that these targets were mainly enriched in macrophage activation and neutrophils extracellular traps (NETs). Additionally, we reported that CAC extract owned significant anti-inflammatory activity by regulating PI3K-Akt-mTOR signal pathway, and inhibited NETosis in response to PMA. CONCLUSIONS: We clarified that CAC extract significantly attenuated RA by inactivating macrophage and reducing NETosis via a multi-targets regulation.

Enhancement of LC3-associated efferocytosis for the alleviation of intestinal inflammation.

LC3-associated phagocytosis (LAP) is an instrumental machinery for the clearance of extracellular particles including apoptotic cells for the alleviation of inflammation. While pharmacological approaches to modulate LAP for inflammation regulation have been poorly explored, in our study we identified a novel compound, columbamine (COL), which can trigger LAP and enhance efferocytosis in an animal model of colitis to attenuate inflammation. We found that COL directly binds to and biasedly activates FPR2 (formyl peptide receptor 2) to promote efferocytosis and alleviate colitis. Biochemically, COL induces an interaction between RAC1 and the PIK3C3/VPS34-RUBCN/RUBICON complex, stimulating LC3-associated efferocytosis. These findings provide a novel interpretation of the potential roles of LAP in regulating inflammatory bowel disease (IBD), reveal the relationship between G protein-coupled receptors (GPCRs) and LAP, and highlight the role of RAC1 in regulating the PIK3C3/VPS34-RUBCN complex in LAP.

Molecular Modulators and Receptors of Selective Autophagy: Disease Implication and Identification Strategies.

Autophagy is a highly conserved physiological process that maintains cellular homeostasis by recycling cellular contents. Selective autophagy is based on the specificity of cargo recognition and has been implicated in various human diseases, including neurodegenerative diseases and cancer. Selective autophagy receptors and modulators play key roles in this process. Identifying these receptors and modulators and their roles is critical for understanding the machinery and physiological function of selective autophagy and providing therapeutic value for diseases. Using modern researching tools and novel screening technologies, an increasing number of selective autophagy receptors and modulators have been identified. A variety of Strategies and approaches, including protein-protein interactions (PPIs)-based identification and genome-wide screening, have been used to identify selective autophagy receptors and modulators. Understanding the strengths and challenges of these approaches not only promotes the discovery of even more such receptors and modulators but also provides a useful reference for the identification of regulatory proteins or genes involved in other cellular mechanisms. In this review, we summarize the functions, disease association, and identification strategies of selective autophagy receptors and modulators.

Mic19 depletion impairs endoplasmic reticulum-mitochondrial contacts and mitochondrial lipid metabolism and triggers liver disease.

Endoplasmic reticulum (ER)-mitochondria contacts are critical for the regulation of lipid transport, synthesis, and metabolism. However, the molecular mechanism and physiological function of endoplasmic reticulum-mitochondrial contacts remain unclear. Here, we show that Mic19, a key subunit of MICOS (mitochondrial contact site and cristae organizing system) complex, regulates ER-mitochondria contacts by the EMC2-SLC25A46-Mic19 axis. Mic19 liver specific knockout (LKO) leads to the reduction of ER-mitochondrial contacts, mitochondrial lipid metabolism disorder, disorganization of mitochondrial cristae and mitochondrial unfolded protein stress response in mouse hepatocytes, impairing liver mitochondrial fatty acid ß-oxidation and lipid metabolism, which may spontaneously trigger nonalcoholic steatohepatitis (NASH) and liver fibrosis in mice. Whereas, the re-expression of Mic19 in Mic19 LKO hepatocytes blocks the development of liver disease in mice. In addition, Mic19 overexpression suppresses MCD-induced fatty liver disease. Thus, our findings uncover the EMC2-SLC25A46-Mic19 axis as a pathway regulating ER-mitochondria contacts, and reveal that impairment of ER-mitochondria contacts may be a mechanism associated with the development of NASH and liver fibrosis.

Improved in situ characterization of protein complex dynamics at scale with thermal proximity co-aggregation.

Cellular activities are carried out vastly by protein complexes but large repertoire of protein complexes remains functionally uncharacterized which necessitate new strategies to delineate their roles in various cellular processes and diseases. Thermal proximity co-aggregation (TPCA) is readily deployable to characterize protein complex dynamics in situ and at scale. We develop a version termed Slim-TPCA that uses fewer temperatures increasing throughputs by over 3X, with new scoring metrics and statistical evaluation that result in minimal compromise in coverage and detect more relevant complexes. Less samples are needed, batch effects are minimized while statistical evaluation cost is reduced by two orders of magnitude. We applied Slim-TPCA to profile K562 cells under different duration of glucose deprivation. More protein complexes are found dissociated, in accordance with the expected downregulation of most cellular activities, that include 55S ribosome and respiratory complexes in mitochondria revealing the utility of TPCA to study protein complexes in organelles. Protein complexes in protein transport and degradation are found increasingly assembled unveiling their involvement in metabolic reprogramming during glucose deprivation. In summary, Slim-TPCA is an efficient strategy for characterization of protein complexes at scale across cellular conditions, and is available as Python package at https://pypi.org/project/Slim-TPCA/ .

Targeting autophagy in Alzheimer's disease: Animal models and mechanisms.

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder that leads to cognitive impairment and memory loss. Emerging evidence suggests that autophagy plays an important role in the pathogenesis of AD through the regulation of amyloid-beta (Aß) and tau metabolism, and that autophagy dysfunction exacerbates amyloidosis and tau pathology. Therefore, targeting autophagy may be an effective approach for the treatment of AD. Animal models are considered useful tools for investigating the pathogenic mechanisms and therapeutic strategies of diseases. This review aims to summarize the pathological alterations in autophagy in representative AD animal models and to present recent studies on newly discovered autophagy-stimulating interventions in animal AD models. Finally, the opportunities, difficulties, and future directions of autophagy targeting in AD therapy are discussed.

Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation.

Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage-mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3-associated phagocytosis (LAP), a non-canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G-protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL-induced efferocytosis, anti-colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3-associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.

Cell type-specific NRBF2 orchestrates autophagic flux and adult hippocampal neurogenesis in chronic stress-induced depression.

Dysfunctional autophagy and impairment of adult hippocampal neurogenesis (AHN) each contribute to the pathogenesis of major depressive disorder (MDD). However, whether dysfunctional autophagy is linked to aberrant AHN underlying MDD remains unclear. Here we demonstrate that the expression of nuclear receptor binding factor 2 (NRBF2), a component of autophagy-associated PIK3C3/VPS34-containing phosphatidylinositol 3-kinase complex, is attenuated in the dentate gyrus (DG) under chronic stress. NRBF2 deficiency inhibits the activity of the VPS34 complex and impairs autophagic flux in adult neural stem cells (aNSCs). Moreover, loss of NRBF2 disrupts the neurogenesis-related protein network and causes exhaustion of aNSC pool, leading to the depression-like phenotype. Strikingly, overexpressing NRBF2 in aNSCs of the DG is sufficient to rescue impaired AHN and depression-like phenotype of mice. Our findings reveal a significant role of NRBF2-dependent autophagy in preventing chronic stress-induced AHN impairment and suggest the therapeutic potential of targeting NRBF2 in MDD treatment.

Lycorine promotes IDH1 acetylation to induce mitochondrial dynamics imbalance in colorectal cancer cells.

Isocitrate dehydrogenase (IDH) 1 and 2, as essential enzymes in energy metabolism, contribute to the survival and drug resistance of a variety of solid tumors, especially for colorectal cancer (CRC). However, the underlying molecular mechanism still remains unclear. In this study, IDH1 was identified as a crucial cellular target of a natural-derived anti-CRC small molecule lycorine, using the unbiased thermal proteome profiling (TPP) strategy. We found that lycorine directly targeted a unique C-terminal domain of IDH1, and disrupted IDH1 interaction with deacetylase sirtuin 1 (SIRT1), thereby significantly promoting IDH1 acetylation modification. Then, lycorine noticeably triggered oxidative stress in CRC cells to cause mitochondrial membranes injury, and subsequently facilitated mitochondrial fission. Specific knockdown of IDH1 or SIRT1 markedly aggrieved lycorine-mediated oxidative stress and mitochondrial fragmentation in CRC cells. Furthermore, the combination of lycorine and sirtuins blocker nicotinamide (NAM) exhibited a synergic therapeutic effect in CRC cells. Collectively, our results reveal that IDH1 may serve as a promising therapeutic target for CRC via pharmacologically driving oxidative stress-dependent mitochondrial dynamics imbalance.

Corynoxine B targets at HMGB1/2 to enhance autophagy for α-synuclein clearance in fly and rodent models of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disease. It is featured by abnormal alpha-synuclein (α-syn) aggregation in dopaminergic neurons in the substantia nigra. Macroautophagy (autophagy) is an evolutionarily conserved cellular process for degradation of cellular contents, including protein aggregates, to maintain cellular homeostasis. Corynoxine B (Cory B), a natural alkaloid isolated from Uncaria rhynchophylla (Miq.) Jacks., has been reported to promote the clearance of α-syn in cell models by inducing autophagy. However, the molecular mechanism by which Cory B induces autophagy is not known, and the α-syn-lowering activity of Cory B has not been verified in animal models. Here, we report that Cory B enhanced the activity of Beclin 1/VPS34 complex and increased autophagy by promoting the interaction between Beclin 1 and HMGB1/2. Depletion of HMGB1/2 impaired Cory B-induced autophagy. We showed for the first time that, similar to HMGB1, HMGB2 is also required for autophagy and depletion of HMGB2 decreased autophagy levels and phosphatidylinositol 3-kinase III activity both under basal and stimulated conditions. By applying cellular thermal shift assay, surface plasmon resonance, and molecular docking, we confirmed that Cory B directly binds to HMGB1/2 near the C106 site. Furthermore, in vivo studies with a wild-type α-syn transgenic drosophila model of PD and an A53T α-syn transgenic mouse model of PD, Cory B enhanced autophagy, promoted α-syn clearance and improved behavioral abnormalities. Taken together, the results of this study reveal that Cory B enhances phosphatidylinositol 3-kinase III activity/autophagy by binding to HMGB1/2 and that this enhancement is neuroprotective against PD.

Chemical mitophagy modulators: Drug development strategies and novel regulatory mechanisms.

Maintaining mitochondrial homeostasis is a potential therapeutic strategy for various diseases, including neurodegenerative diseases, cardiovascular diseases, metabolic disorders, and cancer. Selective degradation of mitochondria by autophagy (mitophagy) is a fundamental mitochondrial quality control mechanism conserved from yeast to humans. Indeed, small-molecule modulators of mitophagy are valuable pharmaceutical tools that can be used to dissect complex biological processes and turn them into potential drugs. In the past few years, pharmacological regulation of mitophagy has shown promising therapeutic efficacy in various disease models. However, with the increasing number of chemical mitophagy modulator studies, frequent methodological flaws can be observed, leading some studies to draw unreliable or misleading conclusions. This review attempts (a) to summarize the molecular mechanisms of mitophagy; (b) to propose a Mitophagy Modulator Characterization System (MMCS); (c) to perform a comprehensive analysis of methods used to characterize mitophagy modulators, covering publications over the past 20 years; (d) to provide novel targets for pharmacological intervention of mitophagy. We believe this review will provide a panorama of current research on chemical mitophagy modulators and promote the development of safe and robust mitophagy modulators with therapeutic potential by introducing high methodological standards.

Targeting macrophage autophagy for inflammation resolution and tissue repair in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic, non-specific, recurrent inflammatory disease, majorly affecting the gastrointestinal tract. Due to its unclear pathogenesis, the current therapeutic strategy for IBD is focused on symptoms alleviation. Autophagy is a lysosome-mediated catabolic process for maintaining cellular homeostasis. Genome-wide association studies and subsequent functional studies have highlighted the critical role of autophagy in IBD via a number of mechanisms, including modulating macrophage function. Macrophages are the gatekeepers of intestinal immune homeostasis, especially involved in regulating inflammation remission and tissue repair. Interestingly, many autophagic proteins and IBD-related genes have been revealed to regulate macrophage function, suggesting that macrophage autophagy is a potentially important process implicated in IBD regulation. Here, we have summarized current understanding of macrophage autophagy function in pathogen and apoptotic cell clearance, inflammation remission and tissue repair regulation in IBD, and discuss how this knowledge can be used as a strategy for IBD treatment.

Disease-Modifying Activity of Huperzine A on Alzheimer's Disease: Evidence from Preclinical Studies on Rodent Models.

(1) Background: Huperzine A, a natural cholinesterase (AChE) inhibitor isolated from the Chinese herb Huperzia Serrata, has been used as a dietary supplement in the United States and a drug in China for therapeutic intervention on Alzheimer's disease (AD). This review aims to determine whether Huperzine A exerts disease-modifying activity through systematic analysis of preclinical studies on rodent AD models. (2) Methods: Sixteen preclinical studies were included based on specific criteria, and the methodological qualities were analyzed by SYRCLE's risk of bias tool. Some outcomes were meta-analyzed: latencies and time spent in quadrant of Morris water maze, soluble amyloid-ß (Aß) level measured by ELISA in the cortex and hippocampus, Aß plaque numbers measured by immunohistochemistry in hippocampus, choline acetyltransferase (ChAT) activity, and AChE activity. Finally, the mechanisms of Huperzine A on AD models were summarized. (3) Conclusions: The outcomes showed that Huperzine A displayed AChE inhibition, ChAT activity enhancement, memory improvement, and Aß decreasing activity, indicating the disease-modifying effect of Huperzine A. However, due to the uneven methodological quality, the results need to be rationally viewed, and extensively repeated.

TRPV1 Modulator Ameliorates Alzheimer-Like Amyloid-ß Neuropathology via Akt/Gsk3ß-Mediated Nrf2 Activation in the Neuro-2a/APP Cell Model.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder for which there is no effective therapeutic strategy. PcActx peptide from the transcriptome of zoantharian Palythoa caribaeorum has recently been identified and verified as a novel antagonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). In the present study, we further investigated the neuroprotective potential of PcActx peptide and its underlying mechanism of action, in an N2a/APP cell model of AD. Both Western blot and RT-PCR analysis revealed that PcActx peptide markedly inhibited the production of amyloid-related proteins and the expression of BACE1, PSEN1, and PSEN2. Moreover, PcActx peptide notably attenuated the capsaicin-stimulated calcium response and prevented the phosphorylation of CaMKII and CaMKIV (calcium-mediated proteins) in N2a/APP cells. Further investigation indicated that PcActx peptide significantly suppressed ROS generation through Nrf2 activation, followed by enhanced NQO1 and HO-1 levels. In addition, PcActx peptide remarkably improved Akt phosphorylation at Ser 473 (active) and Gsk3ß phosphorylation at Ser 9 (inactive), while pharmacological inhibition of the Akt/Gsk3ß pathway significantly attenuated PcActx-induced Nrf2 activation and amyloid downregulation. In conclusion, PcActx peptide functions as a TRPV1 modulator of intercellular calcium homeostasis, prevents AD-like amyloid neuropathology via Akt/Gsk3ß-mediated Nrf2 activation, and shows promise as an alternative therapeutic agent for AD.

Presenilin-1 F105C mutation leads to tau accumulation in human neurons via the Akt/mTORC1 signaling pathway.

BACKGROUND: The mammalian target of rapamycin (mTOR) plays a critical role in controlling cellular homeostasis, and its dysregulation has been implicated in Alzheimer's disease (AD). Presenilin-1 (PS1) mutations account for the most common causes of familial Alzheimer's disease (FAD); however, whether PS1 mutation causes mTOR dysregulation in human neurons remains a key unresolved issue. METHODS: We generated heterozygotes and homozygotes of PS1 F105C knock-in mutation in human induced pluripotent stem cells (iPSCs) via CRISPR/Cas9/piggyback-based gene editing and differentiated them into human neurons. Secreted Aß and tau accumulation were determined by ELISA assay, immunofluorescence staining, and western blotting analysis. mTOR signaling was evaluated by western blotting analysis, immunofluorescence staining, and co-immunoprecipitation. Autophagy/lysosome activities were determined by LC3-based assay, LysoTracker Red staining, and DQ-Red BSA staining. RESULTS: Through comparison among these isogenic neurons, PS1 F105C mutant neurons exhibited elevated Aß and tau accumulation. In addition, we found that the response of mTORC1 to starvation decreases in PS1 F105C mutant neurons. The Akt/mTORC1/p70S6K signaling pathway remained active upon EBSS starvation, leading to the co-localization of the vast majority of mTOR with lysosomes. Consistently, PS1 F105C neurons displayed a significant decline in starvation-induced autophagy. Notably, Torin1, a mTOR inhibitor, could efficiently reduce prominent tau pathology that occurred in PS1 F105C neurons. CONCLUSION: We demonstrate that Chinese PS1 F105C mutation causes dysregulation of mTORC1 signaling, contributing to tau accumulation in human neurons. This study on inherited FAD PS1 mutation provides unprecedented insights into our understanding of the molecular mechanisms of AD. It supports that pharmaceutical blocking of mTOR is a promising therapeutic strategy for the treatment of AD.

Pharmacological modulation of autophagy for Alzheimer's disease therapy: Opportunities and obstacles.

Alzheimer's disease (AD) is a prevalent and deleterious neurodegenerative disorder characterized by an irreversible and progressive impairment of cognitive abilities as well as the formation of amyloid ß (Aß) plaques and neurofibrillary tangles (NFTs) in the brain. By far, the precise mechanisms of AD are not fully understood and no interventions are available to effectively slow down progression of the disease. Autophagy is a conserved degradation pathway that is crucial to maintain cellular homeostasis by targeting damaged organelles, pathogens, and disease-prone protein aggregates to lysosome for degradation. Emerging evidence suggests dysfunctional autophagy clearance pathway as a potential cellular mechanism underlying the pathogenesis of AD in affected neurons. Here we summarize the current evidence for autophagy dysfunction in the pathophysiology of AD and discuss the role of autophagy in the regulation of AD-related protein degradation and neuroinflammation in neurons and glial cells. Finally, we review the autophagy modulators reported in the treatment of AD models and discuss the obstacles and opportunities for potential clinical application of the novel autophagy activators for AD therapy.

Chaperone-Mediated Autophagy in Neurodegenerative Diseases: Molecular Mechanisms and Pharmacological Opportunities.

Chaperone-mediated autophagy (CMA) is a protein degradation mechanism through lysosomes. By targeting the KFERQ motif of the substrate, CMA is responsible for the degradation of about 30% of cytosolic proteins, including a series of proteins associated with neurodegenerative diseases (NDs). The fact that decreased activity of CMA is observed in NDs, and ND-associated mutant proteins, including alpha-synuclein and Tau, directly impair CMA activity reveals a possible vicious cycle of CMA impairment and pathogenic protein accumulation in ND development. Given the intrinsic connection between CMA dysfunction and ND, enhancement of CMA has been regarded as a strategy to counteract ND. Indeed, genetic and pharmacological approaches to modulate CMA have been shown to promote the degradation of ND-associated proteins and alleviate ND phenotypes in multiple ND models. This review summarizes the current knowledge on the mechanism of CMA with a focus on its relationship with NDs and discusses the therapeutic potential of CMA modulation for ND.