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. 2011 Nov 8;124(19):2117-28.
doi: 10.1161/CIRCULATIONAHA.111.048934. Epub 2011 Oct 10.

COP9 signalosome regulates autophagosome maturation

Huabo Su  1 , Faqian Li, Mark J Ranek, Ning Wei, Xuejun Wang
Affiliations

COP9 signalosome regulates autophagosome maturation

Huabo Su et al. Circulation. .

Abstract

Background: Autophagy is essential to intracellular homeostasis and is involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8), and is known to regulate the ubiquitin-proteasome system. However, it is unknown whether CSN plays a role in autophagy.

Methods and results: Marked increases in the LC3-II and p62 proteins were observed on Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with green fluorescent protein-LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred before a global ubiquitin-proteasome system impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Downregulation of Rab7 was found to colocalize strikingly with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Long-term lysosomal inhibition with chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of nonselective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes.

Conclusions: Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway, but also selective autophagy. Likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation. Impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.

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Conflict of interest statement

Conflict of Interest Disclosures: None.

Figures

Figure 1
Figure 1. Increased abundance of autophagosomes in CR-Csn8KO hearts
In all figures, either Csn8KO or KO is used to indicate CR-Csn8KO. A, Representative western blot images for the indicated proteins in myocardium from mice of the indicated age. GAPDH was probed as loading control. B, A summary of the densitometric data of LC3-II from the experiments as illustrated in panel A. *p<0.05, #p<0.01 vs. CTL, Student’s t-test. C, Western blot analyses for the indicated proteins in the heart at 3 weeks (wk). D, Confocal micrographs of myocardial GFP-LC3 direct fluorescence from CTL/GFP-LC3 and CR-Csn8KO/GFP-LC3 mice at 3 weeks. Bar=10 μm. E and F, Western blot analyses of myocardial GFP-LC3 in mice as described in D. Representative images (E) and a summary of the densitometric quantification (F) are shown. n = 4 for each group; #p<0.01 vs. CTL. G, Electron micrographs of ventricular myocardium from 3-week-old mice. Representative images from CTL (a) and CR-Csn8KO (b~e) are shown. Panels c~e are magnified images respectively from the indicated areas of panel b, illustrating examples of autophagosomes (arrows). Bar=1 μm.
Figure 2
Figure 2. Autophagic flux assessments
A, and B, Time course of changes in p62 protein expression in CR-Csn8KO mouse hearts. Representative images (A) and a summary of densitometry data (B) of western blot analyses of p62 are shown. *p<0.05, #p<0.01 vs. CTL; Student’s t-test. C and D, Autophagic flux assays based on bafilomycin-A1 (BFA) induced changes in the endogenous LC3-II protein level. Three-week-old CTL and CR-Csn8KO mice were treated with BFA (3 μmol/kg, i.p.) or vehicles and sacrificed 1 hour after the BFA injection. LC3 protein levels in ventricular myocardium and the liver tissue were quantified using western blot analysis. Representative western blot images (C) and densitometric quantification (D) of LC3 proteins are presented. n=4 mice for each group. *p<0.05, #p<0.01, 2-way ANOVA followed by Holm-Sidak test; N.S., not significant. E and F, Probing myocardial UPS proteolytic function in CR-Csn8KO mice using a surrogate UPS substrate (GFPdgn). Through cross-breeding, transgenic GFPdgn was introduced into CR-Csn8KO and CTL mice, The resultant CTL/GFPdgn and CR-Csn8KO/GFPdgn mice at 2 and 3 weeks of age were examined for myocardial protein levels of GFPdgn. Representative images of western blot analyses for GFPdgn (E) and a summary of changes in GFPdgn protein levels (F) are shown. L.C., a non-specific band used as loading control. *p<0.05 vs. CTL.
Figure 3
Figure 3. Analyses of lysosomal genesis in mouse hearts
A, , Western blot analyses of indicated lysosomal proteins. B, Changes in cathepsin D activities. C~F, Confocal microscopic analyses of LAMP1 and cathepsin D (Cath D) positive vesicles. Perfusion fixed ventricular myocardial sections from the CTL and CR-Csn8KO mice were double-immunofluorescence stained for LAMP1 (red), a lysosomal membrane protein, and cathepsin-D (green), a lysosomal protease. Representative confocal micrographs are shown in panel C. The number densities of cathepsin D positive (Cath D+), LAMP1 positive (LAMP1+), and Cath D/LAMP1 double positive (Cath D+/LAMP1+) vesicles are summarized in panel D. The percentage of the Cath D/LAMPdouble positive vesicles (i.e., lysosomes) over the total number of Cath D+ vesicles or over the total number of LAMP1+ vesicles are respectively presented in panels E and F. *p<0.05 vs. CTL.
Figure 4
Figure 4. Analyses of the changes in autophagic vacuole dynamics caused by Csn8 deficiency, starvation, and proteasome inhibition
A, , Confocal microscopic analyses of the abundance of autophagic vacuoles, lysosomes, and autolysosomes in mouse hearts. The GFP-LC3 transgene was introduced to the CR-Csn8KO and CTL background through cross-breeding and the resultant CTL/GFP-LC3 and CR-Csn8KO/GFP-LC3 mice at 3-weeks were examined at the baseline. Cryosections of ventricular myocardium were immunostained for LAMP1 (red) to identify lysosomes. Representative duo-color images of GFP-LC3 direct fluorescence (green) and LAMP1 immunofluorescence (red) are shown. The insets are enlarged images of asterisk-marked areas. Arrowheads point to GFP- and LAMP1- co-localized dots. Bar=10 μm. B~D, Box plots summarizing changes in the density of autophagic vacuoles (B), lysosomes (C), and autolysosomes (D). E and F, Box plots summarizing changes in the relative density of autolysosomes among total number of lysosomal vacuoles (E) or among total number of autophagic vacuoles (F) in the heart of CR-Csn8KO, Starved, or MG262-treated mice. Mouse grouping was the same as described in Figure 4A. The numbers of LAMP1 positive (LAMP1 dots, all lysosomes), GFP-LC3 positive (GFP dots, all autophagic vacuoles), and GFP::LAMP1 double positive (GFP::LAMP1 dots, autolysosomes) puncta were analyzed using the images collected as described in panel A (see Methods for details). *#p<0.01; N.S., not significant; the Mann-Whitney U test.
Figure 5
Figure 5. Impaired autophagosome maturation causes cardiomyocyte necrosis in mice
A, and B, Cardiomyocytes with severe accumulation of autophagosomes more likely undergo necrosis (arrowhead). GFP-LC3::CR-Csn8KO and GFP-LC3::CTL mice at 3 weeks were administered with Evan’s blue dye (EBD, 100 mg/kg, i.p.) 18 hours before the heart was flushed with saline and fixed with 4% paraformaldehyde via retrograde perfusion through the abdominal aorta. Cryosections of ventricular myocardium were stained with Alexa-568 conjugated Phalloidin to identify cardiomyocytes. The sections were imaged with fluorescence confocal microscopy for GFP-LC3 (green), EBD (red), and F-actin (blue). Representative confocal micrographs (A) and a comparison of the prevalence of EBD positive cells in CR-Csn8KO hearts between the cardiomyocytes containing 16 or fewer GFP-LC3 puncta per 100 μm2 and those showing >16 puncta per 100 μm2 (B) are presented. Scale bar=20 μm; **p<0.0001, Fisher’s exact test. C and D, chronic blockade of autophagic flux causes cardiomyocytes necrosis (arrows) in wild type mice. Adult mice were injected with Chloroquine (CQ, 10 mg/kg, daily) or saline for 3 weeks. At the end of the experiment, mice were injected with EBD and ventricular myocardium samples were processed to detect cardiomyocyte EBD uptake (C) as described in panel A. The sections were counterstained with wheat germ agglutinin (WGA, green) and DAPI (blue) to reveal cell membrane and the nuclei. An adjacent set of sections were immunostained for CD45 (red) and the nuclei were stained with DAPI (blue) for quantification of the percentage of CD45 positive nuclei among all nuclei in the field (D). Bar=20μm; n=3 mice/group; *p<0.05.
Figure 6
Figure 6. Down-regulation of Rab7 in Csn8 deficient mouse hearts
A, and B, Representative images (A) and a summary of densitometry data (B) of western blot analysis of Rab5, Rab7, and Rab11 in mouse hearts at 3 weeks. n=4 mice per group; *p<0.05 vs. CTL. C, Representative confocal micrographs to illustrate the co-localization between Rab7 down-regulation and autophagosome accumulation at the individual cardiomyocyte level in CR-Csn8KO hearts. Scale bar=10 μm.
Figure 7
Figure 7. Rab7 knockdown (Rab7KD) impairs autophagic flux in cultured cardiomyocytes
NRVMs were transfected with siRNAs against either luciferase (siLuci) or Rab7 (siRab7) for 48 hours before subsequent treatments. A~D, Rab7KD impaired baseline autophagic flux. Representative images (A, C) and the summary of densitometry data (B, D) from western blot analyses for the indicated proteins are shown. E~H, Rab7KD attenuated glucose deprivation (GD) or proteasome inhibition induced increases in autophagic flux. To induce non-selective autophagy cells were subjected to GD for 4 hours. To activate selective autophagy, cells were treated proteasome inhibitor bortezomib (BZM, 20nM) for 12 hours. To assess autophagic flux cells were treated with BFA (100nM) or vehicle 3 hours before harvesting the cells. Representative images (E, G) and summaries of densitometry data (F, H) from western blot analyses are shown. *p<0.05 vs. siLuci.
Figure 8
Figure 8. Rab7 down-regulation disrupts autophagosome maturation and increases proteasome inhibition-induced cell death in cultured neonatal rat ventricular myocytes (NRVMs)
A, and B, Changes in autolysosome formation as probed with tf-LC3. Cultured NRVMs were infected with Ad-tf-LC3 24hrs after transfection of siRab7 or siLuci (as control); 24hrs later, the cells were treated with bortezomib (BZM, 20nM) for 12hrs or subjected to glucose deprivation (GD) for 4hrs before the cells were imaged live for the GFP (green) and mRFP (red) signals from tf-LC3. Representative epi-fluorescent images (A) and a summary (B) of the changes of autophagic vacuole abundance with indicated treatments. Insets are the enlarged images of the indicated area. Arrowheads, red-only puncta (autolysosome); arrows, green and red superimposed puncta (autophagosomes); scale bar=50μm; *p<0.05; #p<0.01. C~E, Rab7 knockdown exacerbates BZM-induced cell death. BZM (10nM) treatment was initiated 48hrs after the siRNA transfection. At the indicated time points of BZM treatment, culture media were sampled for measuring lactate dehydrogenase (LDH) activities (C). At 24hrs of BZM treatment, propidium iodide (PI) was applied to the cell culture. Ten minutes later, the unbound PI was thoroughly removed and the cells were then fixed in 4% paraformaldehyde, counter-stained with DAPI, and imaged for PI and DAPI staining. Representative epi-fluorescence micrographs are shown in panel D. The percent of PI-positive (red) nuclei among all DAPI-stained nuclei (blue) was determined from 3 independent repeats (E). Scale bar=100 μm; *p<0.01 vs. siLuci; #p< 0.01 vs. siLuci+BZM.
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