bims-lypmec 2025-01-19 papers

J Cell Biol. 2025 Feb 03. pii: e202310150. [Epub ahead of print]224(2):

A STING-CASM-GABARAP pathway activates LRRK2 at lysosomes.

Amanda Bentley-DeSousa, Agnes Roczniak-Ferguson, Shawn M Ferguson.

Mutations that increase LRRK2 kinase activity have been linked to Parkinson's disease and Crohn's disease. LRRK2 is also activated by lysosome damage. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through stimulator of interferon genes (STING) as an activator of LRRK2 via the conjugation of ATG8 to single membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM results in the lipidation of multiple ATG8 protein family members, we establish that LRRK2 lysosome recruitment and kinase activation are highly dependent on interactions with the GABARAP member of this family. Collectively, these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation of LRRK2 via this pathway may be of relevance in both Parkinson's and Crohn's diseases.

DOI: https://doi.org/10.1083/jcb.202310150

Aging Cell. 2025 Jan 16. e14487

V-ATPase Disassembly at the Yeast Lysosome-Like Vacuole Is a Phenotypic Driver of Lysosome Dysfunction in Replicative Aging.

Fiza Hashmi, Patricia M Kane.

Declines in lysosomal acidification and function with aging are observed in organisms ranging from yeast to humans. V-ATPases play a central role in organelle acidification, and V-ATPase activity is regulated by reversible disassembly in many different settings. Using the yeast Saccharomyces cerevisiae as a replicative aging model, we demonstrate that V-ATPases disassemble into their V1 and V0 subcomplexes in aging cells, with release of V1 subunit C (Vma5) from the lysosome-like vacuole into the cytosol. Disassembly is observed after > 5 cell divisions and results in overall vacuole alkalinization. Caloric restriction, an established mechanism for reversing many age-related outcomes, prevents V-ATPase disassembly in older cells and preserves vacuolar pH homeostasis. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors: the Regulator of Acidification of Vacuoles and Endosomes (RAVE) complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1∆ mutant shortens replicative lifespan; Oxr1 promotes disassembly and an oxr1∆ mutation extends the lifespan. Importantly, the level of Rav2, a subunit of the RAVE complex, declines in aged cells, and Rav2 overexpression delays V-ATPase disassembly with age. These data indicate that reduced V-ATPase assembly contributes to the loss of lysosomal acidification with age, which affects replicative lifespan.

Keywords: Saccharomyces cerevisiae ; aging; caloric restriction; lysosomes; proton pumps

DOI: https://doi.org/10.1111/acel.14487

Int J Mol Sci. 2024 Dec 24. pii: 17. [Epub ahead of print]26(1):

Tracking Chaperone-Mediated Autophagy Flux with a pH-Resistant Fluorescent Reporter.

Ruotong Qi, Xingyi Chen, Zihan Li, Zheng Wang, Zhuohui Xiao, Xinyue Li, Yuanyuan Han, Hongfei Zheng, Yanjun Wu, Yi Xu.

Chaperone-mediated autophagy (CMA) is a selective autophagic pathway responsible for degrading cytoplasmic proteins within lysosomes. Monitoring CMA flux is essential for understanding its functions and molecular mechanisms but remains technically complex and challenging. In this study, we developed a pH-resistant probe, KFERQ-Gamillus, by screening various green fluorescent proteins. This probe is activated under conditions known to induce CMA, such as serum starvation, and relies on LAMP2A and the KFERQ motif for lysosomal localization and degradation, demonstrating its specificity for the CMA pathway. It enables the detection of CMA activity in living cells through both microscopy and image-based flow cytometry. Additionally, we created a dual-reporter system, KFERQ-Gamillus-Halo, by integrating KFERQ-Gamillus with the Halo-tag system. This probe not only distinguishes between protein synthesis and degradation but also facilitates the detection of intracellular CMA flux via immunoblotting and the rapid assessment of CMA activity using flow cytometry. Together, the KFERQ-Gamillus-Halo probe provides quantitative and time-resolved monitoring for CMA activity and flux in living cells. This tool holds promising potential for high-throughput screening and biomedical research related to CMA.

Keywords: Gamillus; chaperone-mediated autophagy; flux; halo

DOI: https://doi.org/10.3390/ijms26010017

Heart Lung Circ. 2025 Jan 15. pii: S1443-9506(24)01923-1. [Epub ahead of print]

Adeno-Associated Viruses as Gene Delivery Tools for Diabetic Heart Disease and Failure: Key Considerations for Clinicians and Preclinical Researchers.

Kate L Weeks, Bianca C Bernardo.

Diabetes is becoming more common worldwide, and people with diabetes are twice as likely to experience heart problems compared to those without diabetes. These cardiovascular complications are the foremost cause of mortality among people with diabetes. A specific form of heart failure known as "diabetic cardiomyopathy" can develop in individuals with diabetes. There are no treatments specifically approved for diabetic cardiomyopathy. Ongoing research is exploring innovative treatments, including the development of gene therapy (e.g., adeno-associated viral vectors) techniques designed to target specific molecular pathways affected in the disease. Here, we discuss the progress, challenges, and experimental considerations of gene therapy for the diabetic heart.

Keywords: AAV; Diabetic cardiomyopathy; Gene therapy; Heart failure

DOI: https://doi.org/10.1016/j.hlc.2024.11.021