Blood autophagy defect causes accelerated non-hematopoietic organ aging

Abstract

Autophagy has been well studied in regulating aging; however, the impact of autophagy in one organ on the aging of other organs has not been documented. In this study, we used a mouse model with deletion of an autophagy-essential gene Atg7 in hematopoietic system to evaluate the intrinsic role of hematopoietic autophagy on the aging of non-hematopoietic organs. We found that autophagy defect in hematopoietic system causes growth retardation and shortened lifespan, along with aging-like phenotypes including hypertrophic heart, lung and spleen, but atrophic thymus and reduced bone mineral density at organismal level. Hematopoietic autophagy defect also causes increased oxidative stress and mitochondrial mass or aging gene expression at cellular level in multiple non-hematopoietic organs. The organ aging in the Atg7-deleted mice was reversed by anatomic connection to wild-type mice with intact blood autophagy via parabiosis, but not by injection of blood cell-free plasma. Our finding thus highlights an essential role of hematopoietic autophagy for decelerating aging in non-hematopoietic organs.

Keywords: aging; autophagy; blood; non-hematopoietic organ.

Figure 1

Growth retardation and shortened lifespan of the mice with deletion of an autophagy-essential gene Atg7 in hematopoietic system. (A) Three genotypes for wild-type, heterozygote, and homozygote for Atg 7 deletion in hematopoietic system with representative images of the mice. The images were taken at age of 10 weeks. (B) PCR Genotyping analysis of the offsprings from Atg7^f/f mice crossing Vav-iCre mice to screen Atg7^f/f;Vav-iCre mice. The sequences for the primers used in PCR are given in the method section, and their PCR amplified bands representing specific genotypes were indicated in the agarose gel electrophoresis films. (C) Examination of Atg7 expression in wild-type and the Atg7-deleted mice. Upper panel, quantitative PCR analysis of Atg7 transcription normalized to Gapdh transcript in different organs; lower panel, western blotting analysis of autophagy-essential protein ATG7 and lipidation of LC3 in different organs. GAPDH used as a loading control. (D) Growth comparison between wild-type and Atg7-deleted mice. Wild-type mice progressively gain weight before age of 60 weeks (left panel), but Atg7-deleted mice cease weight gain at about age of 6 weeks (right panel). (E) Measurement of lifespan of wild-type and Atg7-deleted mice. (F) Immunohistological examination of heart, liver, lung and thymus from 10 weeks old wild-type and Atg7-deleted mice by HE staining.

Figure 2

Multiple aging-like organ abnormalities in the Atg7-deleted mice in hematopoietic system. (A) Alteration of heart in morphology and size in the Atg7-deleted mice. Left panel, representative image of heart unstained (upper) and HE stained (down); right panel, heart/body weight ratio (coefficients) of the Atg7-deleted and wild-type mice. (B) Lung coefficients of the Atg7-deleted and wild-type mice. (C) Spleen coefficients of the Atg7-deleted and wild-type mice. (D) Alteration of bone mineral density. Upper, micro-CT analysis of bones of in the Atg7-deleted mice and the same-age wild-type mice as well as old wild-type mice; down, HE staining of bones from 12-week wild-type, Atg7-deleted, and old wild-type mice.

Figure 3

Autophagy defect causes synchronous thymic atrophy and T cell (CD4⁺CD8⁺) reduction after mouse development is completed. (A) Alteration of thymus in size and weight. Left, a representative picture of thymus; right, thymus coefficient of Atg7-deleted mice as compared with the same-age wild-type mice and the old wild-type mice. (B) Measurement of thymus coefficients in time points indicated in the entire lifespan of the Atg7-deleted mice (organ/body, mg/g). (C) Scheme for analysis of T cells by flow cytometry. Shown are representative flow images for quantification of total blood cell (CD45⁺) and T cells (CD4⁺,CD8⁺) in total thymus cells. (D) Statistical analysis of the percentages of T cell populations in total thymus blood cells in the entire lifespan of the Atg7-deleted mice. (E) Statistical analysis of the percentages of NK cell populations in total thymus blood cells in the Atg7-deleted mice at age of 12 weeks.

Figure 4

Increased cellular aging markers in the non-hematopoietic organs of the mice with hematopoietic autophagy defect. (A) Flow cytometric analysis of ROS levels of the heart, lung and thymus cells with fluorescein DCFH-DA. Left and middle, gating strategy for the flow-cytometric assessment of non-hematopoietic cells (CD45^-Ter119^-); right, geometric mean fluorescence intensity (MFI) of DCFH-DA in the heart cells of wild-type mice and Atg7-deleted mice. (B) Flow cytometric analysis of mitochondrial mass levels of the heart, liver and spleen cells with florescent Mitotracker Deep Red. Left and middle, gating strategy for the flow-cytometric assessment of non-hematopoietic cells (CD45^-Ter119^-); right, geometric mean fluorescence intensity (MFI) of MitoTracker Deep Red in the heart or liver cells of wild-type and Atg7-deleted mice. (C) Flow cytometric analysis of DNA damage with γ-H2AX. Upper, gating strategy for non-hematopoietic cells (CD45^-Ter119^-) in the liver; lower, geometric mean fluorescence intensity (MFI) of γ-H2AX in the liver cells. (D) Analysis of apoptosis and necrosis in the lung cells of wild-type mice and Atg7-deleted mice by annexinV and PI double staining. Upper, representative flow cytometric measurement; lower, statistical results from cytometric analysis. (E) Quantitative RT-PCR analysis of four aging related genes (P15, P16, P19, P21) in the liver cells (blood cells removed by sorting against CD45⁺ or Ter119⁺) of young wild-type mice, Atg7-deleted mice and old wild-type mice.

Figure 5

Autophagy-intact blood cells not plasma reverse autophagy defect-caused aging of non-hematopoietic organs. (A) Upper, schematic depicting the parabiotic pairings; lower, graph representing the heart/lung/spleen weight after 4 weeks of parabiosis. (B) Upper, schematic illustrating plasma treatment; lower, graph representing the heart or lung or spleen/body weight ratio after plasma treatment. (C) Summary of the study. Autophagy defect in hematopoietic system accelerates multiple non-hematopoietic organ aging, ultimately leading to a much shortened lifespan of the mouse.