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PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy.

ABSTRACT: Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12-ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.

SUBMITTER: Dias MV

PROVIDER: S-EPMC5103360 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy.

Dias Marcos V S MV Teixeira Bianca L BL Rodrigues Bruna R BR Sinigaglia-Coimbra Rita R Porto-Carreiro Isabel I Roffé Martín M Hajj Glaucia N M GN Martins Vilma R VR

Autophagy 20160914 11

Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-typ ...[more]

PMID: 27629560

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Project description:CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression.Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3'-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside.

Project description:Appropriate glucagon secretion from pancreatic alpha cells in response to hypoglycemia is an important component of maintaining glucose homeostasis. Dysregulated glucagon secretion leads to the delayed recovery from a hypoglycemic attack in type 1 diabetes patients which can be lethal. Although elucidating the precise mechanism of glucagon secretion in hypoglycemia is warranted, the underlying mechanism remains poorly understood.The present study provides evidence of the role of autophagy in glucagon secretion in hypoglycemia by demonstrating that autophagy regulates adrenergic stimulation of glucagon secretion downstream of beta2 adrenergic receptor. First, from the analyses of T1D human islets and the published database of scRNA-seq of T1D human alpha cells, we described autophagy pathways altered in T1D alpha cells. Second, we generated alpha cell-specific Atg7KO mice (alphaAtg7KO) and clarified that the lack of autophagy in alpha cells impairs the reactive glucagon secretion in acute hypoglycemia. Third, to interrogate the molecular mechanism of autophagy-mediated glucagon regulation, we analyzed top genes downregulated inT1D and T2D alpha cells and found the expression of beta2 adrenergic receptor showed significant down-regulation in T1D alpha cells. We confirmed the decreased expression of beta2 adrenergic receptor in alpha cells of the T1D pancreas section, the islets of alphaAtg7KO mice, and a murine alpha cell line with stable knockdown of Atg7. Furthermore, in vivo and ex vivo studies of alphaAtg7KO mice exhibited that lack of autophagy led to the loss of stimulatory effect of beta2-adrenergic signaling on glucagon secretion. Finally, we established the Atg7 knockdown model in sorted human alpha cells and confirmed the effect of autophagy on the expression of the beta2 adrenergic receptor. Together, our study provides novel insights into the regulatory role of autophagy in glucagon secretion in response to hypoglycemia and provides therapeutic options to achieve stable glycemic control in T1D patients.

Dataset Information

PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy.

Publications

PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy.

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