Dataset Information

Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile.

ABSTRACT: Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

SUBMITTER: de Wet S

PROVIDER: S-EPMC7827520 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile.

de Wet Sholto S Du Toit Andre A Loos Ben B

Cells 20210107 1

Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic inter ...[more]

PMID: 33430464

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Project description:Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.

Project description:BackgroundExosomes contribute to immunosuppression in head and neck squamous cell carcinoma (HNSCC), a tumor entity which lacks specific tumor biomarkers. Plasma-derived exosomes from HNSCC patients correlate with clinical parameters and have potential as liquid biopsy. Here, we investigate the cargo and functional profile of saliva-derived exosomes from HNSCC patients and their potential as non-invasive biomarkers for disease detection and immunomodulation.MethodsExosomes were isolated from saliva of HNSCC patients (n = 21) and healthy donors (HD, n = 12) by differential ultracentrifugation. Surface values of immune checkpoints and tumor associated antigens on saliva-derived exosomes were analyzed by bead-based flow cytometry using CD63 capture. Upon co-incubation with saliva-derived exosomes, activity and proliferation of T cells were assessed by flow cytometry (CD69 expression, CFSE assay). Adenosine levels were measured by mass spectrometry after incubation of saliva-derived exosomes with exogenous ATP. miRNA profiling of saliva-derived exosomes was performed using the nCounter® SPRINT system.ResultsSaliva-derived, CD63-captured exosomes from HNSCC patients carried high amounts of CD44v3, PDL1 and CD39. Compared to plasma, saliva was rich in tumor-derived, CD44v3+ exosomes and poor in hematopoietic cell-derived, CD45+ exosomes. CD8+ T cell activity was attenuated by saliva-derived exosomes from HNSCC patients, while proliferation of CD4+ T cells was not affected. Further, saliva-derived exosomes produced high levels of immunosuppressive adenosine. 62 HD- and 31 HNSCC-exclusive miRNAs were identified. Samples were grouped in "Healthy" and "Cancer" based on their saliva-derived exosomal miRNA profile, which was further found to be involved in RAS/MAPK, NF-κB complex, Smad2/3, and IFN-α signaling.ConclusionsSaliva-derived exosomes from HNSCC patients were enriched in tumor-derived exosomes whose cargo and functional profile reflected an immunosuppressive TME. Surface values of CD44v3, PDL1 and CD39 on CD63-captured exosomes, adenosine production and the miRNA cargo of saliva-derived exosomes emerged as discriminators of disease and emphasized their potential as liquid biomarkers specific for HNSCC.

Dataset Information

Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile.

Publications

Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile.

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