Reduced lifespan in Atg5i mice

Previously, we have reported the development of a highly efficient dox-inducible shRNA mouse model targeting Atg5 (Atg5i)13 that phenocopies tissue-specific Atg5 knockout (KO) mice and enables dynamic control of autophagy (Supplementary Figs. 1 and 2). These mice lack brain expression of the shRNA and as such do not suffer from the lethal neurotoxic effects that characterise systemic autophagy knockout mice14,15, and enable us to perform longitudinal studies that were previously unachievable in vivo.

A common caveat of many mouse models is that genetic manipulations are often present during embryogenesis. Thus, any phenotypes that manifest are a combination of both developmental and tissue homoeostasis effects. To avoid the generation of these compound effects, Atg5i mice were aged until 8-weeks (young adults) before being transferred to a dox-containing diet and followed to assess overall survival. Atg5i mice on long-term dox (LT-Atg5i) had a median survival of ~6 months on dox (Male 185 days; Female 207 days on dox) with no apparent sex bias (Fig. 1a–c and Supplementary Fig. 3a).

Fig. 1: Autophagy inhibition decreases lifespan. LT-Atg5i mice on dox continuously from 2 months old display a reduced lifespan in comparison with LT-Control as shown in survival graphs for combined (p < 0.0001) (a), male (p < 0.0001) (b), female (p < 0.0001) (c) (Mantel–Cox test). Median survival (days on dox) and mice per group are indicated. During this period LT-Atg5i mice also display a reduced weight gain in both male (d) and female (e) cohorts. f LT-Atg5i mice also display an increased frequency of skin inflammation and eye infections in comparison with age-matched LT-Control mice. g Cardiac fibrosis was also evident in LT-Atg5i mice. Representative images of H&E and Massons Trichrome are shown. Scale bars, 100 μm. h Age-matched skinned mice. LT-Atg5i mice show kyphosis (yellow dotted line traces the arch of the spine). They often displayed premature greying (dotted rectangle). Arrows indicate the presence of inflammation. Full size image

In comparison with littermate controls, LT-Atg5i mice experienced a progressive deterioration, initially presenting with a reduction in coat condition within the first few weeks and a reduction in weight gain that became more pronounced over the life of the animal (Fig. 1d, e and Supplementary Fig. 3b). The majority of mice eventually succumbed to a general morbidity characterised by lethargy, piloerection, and a decrease in body condition, wherein they have to be sacrificed. As previously described with naturally aged colonies16, LT-Atg5i mice also appeared susceptible to eye infections and ulcerative dermatitis, the latter being primarily localised to the ears and neck and ranging from mild to severe (Fig. 1f and Supplementary Fig. 3c, respectively).

A singular cause of death in LT-Atg5i mice is difficult to determine and it is most likely of multifactorial aetiology across the cohort. At necropsy, all mice displayed hepatomegaly and splenomegaly in comparison to age and sex matched controls, consistent with phenotypes associated with tissue specific knockout mice17,18,19 (Supplementary Fig. 3d, e). Elevated serum Alanine Aminotransferase (ALT) and reduced levels of serum albumin were present throughout dox administration of Atg5i mice, yet were altered further at the time of death only in a subset of samples (Supplementary Fig. 3f, g, yellow circles). Consistent with this, an increase in serum bilirubin levels was only observed at the time of death within this same subset of mice (Supplementary Fig. 3h, yellow circle). These data suggest that severe liver failure occurs in only a fraction.

Interestingly serum creatinine levels, a marker of kidney function, also displayed an increase only in a different subset of LT-Atg5i mice at the time of death, although they were not generally elevated during dox administration (Supplementary Fig. 4a). Loss of autophagy also correlated with a general thickening of the basement membrane and the presence of sclerotic (Supplementary Fig. 4b) and enlarged glomeruli (Supplementary Fig. 4c, d) in comparison with age-matched tissue samples, indicative of degenerative kidney disorder. These data suggest that, similar to the liver, systemic autophagy defect causes age-associated degenerative alterations in kidney, yet only a distinct subset progresses to renal failure on death. In addition to this stochastic development of organ failure, LT-Atg5i mice universally presented with cardiomyopathy (Supplementary Fig. 4e). Histological examination highlighted the presence of enlarged, degenerate and vacuolated cardiomyocytes, in addition to the presence of cardiac fibrosis (Fig. 1g).

Together, our data suggest that, despite the stereotypic premature death, LT-Atg5i mice suffered from a heterogeneous set of tissue degenerative disorders that appear to have contributed to an increase in mortality. Of note, there was no evidence of overt tumour development in these mice at the time of death.

Autophagy inhibition is associated with accelerated ageing

After 4 months of dox treatment, all LT-Atg5i mice displayed evidence of kyphosis that became progressively more pronounced as the animals aged until death, whilst 16/28 LT-Atg5i mice displayed evidence of premature greying to varying degrees (Fig. 1h). Furthermore, LT-Atg5i mice displayed evidence of extramedullary hematopoiesis (Fig. 2a) and immune aggregations, commonly seen in aged mouse colonies, were also found in the liver, lungs and kidneys but were generally absent in age matched controls, although the incidence of these increased in frequency with increasing age (Supplementary Fig. 5a–c).

Fig. 2: LT-Atg5i mice present with accelerated aging phenotypes. a Extramedullary haematopoiesis is present in the spleens of LT-Atg5i mice in comparison with age-matched controls. Scale bars, 100 μm. b 6-month-old LT-Atg5i mice (4 months dox treatment) display increased White Blood Cell counts (WBC). Meanwhile, irradiated wild-type mice in receipt of uninduced bone marrow from Ctrl or Atg5i mice display a reduced WBC count after 4 months of dox treatment (unpaired two-tailed Welches t test, n = 5–6 per group). c Composition of the peripheral immune system in LT-Atg5i mice is reminiscent of old control mice (n = 5–6 mice per group). d 6-month-old LT-Atg5i mice (4 months dox treatment) displayed increased serum levels of IL-6 and TNF (LT-Atg5i n = 5, LT-Ctrl n = 7; Mann Whitney Test). e Bone marrow transplantation of uninduced Ctrl and Atg5i bone marrow into irradiated wild-type recipient mice after 4 months of dox treatment Atg5i recipient mice display a myeloid skewing. LT-Atg5i mice display alterations in skeletal muscle after 4-month of dox treatment. LT-Atg5i mice display a significant difference in cross-sectional area (f) (n = 3 R-Ctrl and 3 R-Atg5i, unpaired two-tailed Welch’s t test) and minimum feret size (g) (n = 3 R-Ctrl and 3 R-Atg5i, unpaired two-tailed Welch’s t test). LT-Atg5i mice also display a decrease in Pax7 nuclear positivity per fibre (h), an increase in central nucleation (i), and positivity for the mitochondrial marker TOM20 (j), as determined by tissue immunofluorescence (unpaired two-tailed Welches t test; n = 3 R-Ctrl and 3 R-Atg5i). Error bars indicate standard deviations. *p < 0.05; **p < 0.01, ***p < 0.001. Full size image

As previously described in hematopoietic Atg5 KO mice, LT-Atg5i mice also displayed an increase in cellularity of the peripheral immune system18,20 (Fig. 2b, left) with a myeloid skewing (Fig. 2c) reminiscent of age-associated chronic inflammation. This ‘inflamm-ageing’ phenotype was further supported by an increase in serum TNF and IL-6 in LT-Atg5i mice in comparison with control (Fig. 2d). In addition, serum isolated from LT-Atg5i mice displayed positivity of antinuclear antibodies in 5/12 cases tested in comparison with 1/6 control mice, with the predominant staining pattern being homogeneous and speckled, implying a systemic autoimmune reaction in a subset of autophagy inhibited mice (Supplementary Fig. 5e).

To determine whether the immune phenotypes were driven by autophagy loss in the immune system or due to systemic autophagy loss, we transplanted bone marrow from untreated Control and Atg5i mice into irradiated wild-type C57BL/6 mice. Subsequent doxycycline treatment for 4 months recapitulated the myeloid skewing in peripheral blood in the mice with Atg5i bone marrow (Fig. 2e) but with an apparent decrease in the immune cellularity (Fig. 2b, right). Furthermore, in those mice, there appeared to be a reduction in the donor-derived component (i.e. Atg5i bone marrow-derived) of the peripheral blood (Supplementary Fig. 5g). Largely consistent with a previous study using Atg12 mutant mice18, combined these results suggest that the general WBC expansion is driven by systemic autophagy loss, while the myeloid skewing is immune cell intrinsic.

Skeletal muscle exhibits an age-related decline and autophagy has been reported to be required for the maintenance of Pax7 positive satellite cells (myogenic precursors)21. In accordance, LT-Atg5i mice displayed evidence of skeletal muscle degeneration with the presence of smaller fibres, a reduction in the population of Pax7 positive satellite cells, and an increase in central nucleation in comparison with age-matched littermate control mice (Fig. 2f–i, Supplementary Fig. 6a, b). Central nucleation represents muscle fibre regeneration after acute muscle injury but an increase in basal frequency of centrally nucleated myofibres is also a sign of sarcopenia at geriatric age both in mice and human22. In addition, LT-Atg5i muscle fibres displayed increased staining positivity for the mitochondrial marker Tom20 indicative of increased mitochondrial mass and a reduction in autophagy mediated turnover (Fig. 2j).

The accumulation of senescent cells is considered a key marker of chronological ageing. Autophagy has been reported to have context dependent and sometimes opposing roles during cellular senescence: typically basal autophagy is considered to promote fitness and its loss may promote senescence, whereas in oncogene-induced senescence, autophagy may be important for the establishment of senescent phenotypes23,24,25,26. To determine if the systemic loss of basal autophagy is sufficient to drive the establishment of cellular senescence in vivo, we performed western blotting across a number of tissues from 4-month dox treated LT-Atg5i mice and found an increased staining pattern for key senescence markers (i.e. p16, p21, and p53) (Fig. 3a–c and Supplementary Fig. 6c). In addition, whole mount senescence-associated beta-galactosidase staining from 6-month treated livers highlighted a marked increase in staining patterns in comparison with LT-Control mice (Fig. 3d). Histologically, nuclear accumulation of p21 was also evident, particularly in hepatocytes with enlarged morphology (Fig. 3d). Furthermore LT-Atg5i mice display a significant increase in both the abundance and frequency of telomere-associated γ-H2AX foci (TAF) in liver, lung and heart tissue (Fig. 3e, f and Supplementary Fig. 6d, e). TAF represent persistent damage in telomeric regions, independent of length, that are resistant to repair machinery and have been shown to correlate with senescence, increasing age and mitochondrial dysfunction27,28,29. The increase in TAF abundance therefore reinforces the notion that mice exhibit age acceleration upon systemic autophagy reduction.

Fig. 3: Autophagy inhibition drives senescence in vivo. Markers of senescence can also be seen across multiple tissues in our LT-Atg5i cohorts treated with dox for 4 months including in kidney (a), heart (b), and liver (c). LT-Atg5i livers stain positively for senescence associated β-galactosidase and p21 unlike age-matched control mice (d) (scale bar, 25 μm). e 6-month doxycycline treated LT-Atg5i livers display an increase in the frequency and abundance of γ-H2AX at telomeres, a marker associated with increasing chronological age (unpaired two-tailed t test; n = 5). f A representative example image shown. Arrowheads point to TAF that are magnified on the right of the image. Scale bar, 10 μm. Error bars indicate standard deviation ***p < 0.001. For a–c source data are provided as a Source Data file. Full size image

Of note, similar gross phenotypic results were also seen in mice with a second hairpin targeting Atg5 (LT-Atg5i_2). LT-Atg5i_2 mice display evidence of premature ageing-like phenotypes (Supplementary Fig. 7a–c), however the appearance of these phenotypes was delayed in comparison with LT-Atg5i mice, seemingly due to a hypomorphic phenotype. Accordingly, these mice displayed the accumulation of p62/Sqstm1 and LC3 in multiple tissues but at lower levels in comparison with LT-Atg5i mice, and did not display phenotypes associated with complete Atg5 knockout mice, including hepatomegaly and splenomegaly (Supplementary Fig. 7d–f). These findings in particular are important as they establish that the reduction in longevity and presence of ageing phenotypes is not dependent on the hepatomegaly and splenomegaly phenotypes encountered in the original LT-Atg5i mouse strain with the highest degree of autophagy inhibition.

Combined these data support a role for basal autophagy in maintaining tissue and organismal homoeostasis and provide evidence that causally links autophagy inhibition to the induction of ageing-like phenotypes in mammals.

Autophagy restoration partially reverses ageing phenotypes

We next sought to determine whether autophagy restoration alone is able to reverse the ageing-like phenotypes by removing dox from the diet. 8-week old Atg5i and control mice treated with dox for 4 months, the point at which they universally presented with kyphosis, were switched back to a diet absent of dox leading to a restoration in Atg5 levels and autophagy (termed R-Atg5i cohort) (Fig. 4a, b and Supplementary Fig. 8a)13. Interestingly, while p16 levels reduced in the livers R-Atg5i mice, they still appeared elevated in comparison with age-matched control mice 4-months post dox removal (Fig. 4b). This is in contrast to the kidney that exhibited only a mild increase in p16 that was mostly reversed upon autophagy restoration. While further systematic analyses would be required, the data suggest a differential susceptibility to autophagy inhibition across organs.

Fig. 4: Restoration of autophagy partially restores health-span. a Schematic of R-Atg5i study. Briefly 2-month old mice are given dox to induce Atg5 downregulation for 4 months at which point they exhibit ageing-like phenotypes. Dox is then removed and autophagy restored. b Liver and kidney tissues from R-Atg5i mice with autophagy restored for 4 months display evidence of Atg5 protein and autophagic flux restoration, yet the liver still stains positively for the marker of senescence p16. c Atg5i mice on dox for 4 months and 6 months display increase frailty scores in comparison with controls (ARU, arbitrary units). While R-Atg5i mice where autophagy has been restored for 4 months, display a recovery (Two-way ANOVA with Tukey’s correction for all comparisons, n = 3–16). d Whole blood cell counts from R-Atg5i mice display no difference in comparison with age matched R-Control mice (unpaired two-tailed t test; n = 11 per group). e Inflammatory serum cytokines IL-6 and TNF are equivalent in R-Atg5i and R-Control mice 2-months post dox removal (Mann Whitney test; n = 3 R-Ctrl and 4 R-Atg5i). f Red blood cell distribution width (RDW) is unaltered in LT-Ctrl and LT-Atg5i cohorts (unpaired two-tailed t test; n = 3 per group), yet appears increased in autophagy-restored cohorts in comparison with age-matched littermate control mice (4 months dox, 8 months restoration) (unpaired two-tailed t test; n = 14 per group). Error bars indicate standard deviation; NS denotes not significant. *p < 0.05; **p < 0.01, ***p < 0.001. For b source data are provided as a Source Data file. Full size image

An increase in chronological age is generally associated with the deviations in multiple health parameters that when measured can be combined into a clinical ‘frailty-score’30. As expected, R-Atg5i mice displayed an initial increase in their frailty scores during autophagy inhibition in comparison with littermate controls, yet once mice have been switched back to a diet absent of dox, the frailty scores displayed a significant decrease over the next 4 months (Fig. 4c, Supplementary Movie 1). In contrast, LT-Atg5i mice treated on dox for 6 months (median survival is around ~6 months on dox) continued to display a significant difference in their frailty scores, while almost all LT-Atg5i mice had already succumbed by 8-months (Fig. 4c). A similar increase in frailty was also noted in the LT-Atg5i_2 cohorts (Supplementary Fig. 7b). The penetrant kyphosis phenotype was largely irreversible, however 3/26 R-Atg5i mice did show evidence of recovery from kyphosis, while no mice displayed a reversal of the greying phenotypes. As such, while autophagy inhibition in vivo appears to promote frailty, autophagy restoration is seemingly able to substantially reverse this effect.

Remarkably the profound immune-associated phenotypes that we observed in autophagy-deficient LT-Atg5i mice were reversed in R-Atg5i mice. Serum markers of inflammation and white blood cell counts were indistinguishable between R-Atg5i and R-Control mice (Fig. 4d, e and Supplementary Fig. 8b). However, it should be noted that, in aged R-Atg5i mice removed from dox for 8 months (14 months old), there was a trend towards a larger red blood cell distribution width (RDW), which has previously been linked to a range of diseases and an increased risk of acute myeloid leukaemia (AML) (Fig. 4f)31. In addition, R-Atg5i livers displayed a complete reversal of hepatomegaly and serum ALT levels (Supplementary Fig. 8c, d). The kidneys of R-Atg5i mice appeared to recover from autophagy inhibition and lacked evidence of sclerotic and enlarged glomeruli (Supplementary Fig. 8e–g). Consistently, serum albumin levels displayed evidence of normalisation, although there was still a trend for reduced levels in R-Atg5i mice at the time point tested, suggesting that liver and/or kidney functions are largely recovered, if not completely (Supplementary Fig. 8h).

Similarly, the protein aggregation marker p62/SQSTM1 in the liver appeared much reduced in R-Atg5i mice in comparison to the LT-Atg5i mice, yet a small but substantial number of cells still exhibited a marked accumulation of p62 aggregation in R-Atg5i mice that had been off dox for 4 months (Fig. 5a). In addition, R-Atg5i livers were also found to contain the presence of ceroid-laden macrophages and lipofuscin positivity, pigments known to increase with age and not seen in age-matched controls mice (Fig. 5b). Importantly, and in accordance with this partial restoration phenotype, molecular markers of ageing such as TAF also remained significantly elevated in R-Atg5i mice (Fig. 5c). This is consistent with the persistent nature of telomeric DNA damage, which is reported to be irreparable27,32. Together with other senescence markers (Fig. 4b), these data suggest that a portion of the cellular damage caused by a chronic block in autophagy is irreversible.

Fig. 5: Restoration of autophagy does not reverse all markers of aging. a p62/Sqstm1 staining of R-Atg5i liver highlights the incomplete removal of aggregates 4 months after autophagy restoration. Scale bars, 100 μm. b The same livers have a higher incidence of age associated pigmentation in comparison with age-matched control mice. (yellow arrow). c TAF frequency and abundance also remains elevated in R-Atg5i mice (unpaired two-tailed t test; n = 4 R-Ctrl and 3 R-Atg5i). Skeletal muscle analysis from 4 months dox treated and 2 months restored R-Atg5i mice. R-Atg5i muscle fibres continue to display significant alterations in minimum feret size (d) (n = 3 R-Ctrl and 4 R-Atg5i, Mann Whitney test) and cross-sectional area (e) (n = 3 R-Ctrl and 4 R-Atg5i, Mann Whitney test). Whilst Pax7 nuclear positivity per fibre still displays no evidence of recovery (f), both central nucleation (g) and positivity for the mitochondrial marker Tom20 (h) exhibit levels similar to R-Ctrl mice. (f–h, unpaired two-tailed Welches t test; n = 3 R-Ctrl and 4 R-Atg5i). Error bars indicate standard deviations. *p < 0.05; **p < 0.01, ***p < 0.001. Full size image

Analysis of skeletal muscle from R-Atg5i mice, with autophagy restoration, suggests that muscle fibre size, morphology, and satellite cell frequency display no sign of recovery 2 months post dox removal (Fig. 5d–f and Supplementary Fig. 6b). However, central nucleation frequency was dramatically reduced and comparable with control (Fig. 5g). As expected with Atg5 restoration, Tom20 positivity appeared similar to control levels (Fig. 5h). In addition, the cardiac fibrosis observed LT-Atg5i mice appears to still be present 4 months post dox removal in R-Atg5i cohorts (Supplementary Fig. 9c). Together these data suggest that autophagy restoration may have tissue and pathology specific limitations in the capacity to recover from the tissue and cellular damage induced upon its inhibition. Crucially, whilst some tissues, such as the liver, appear to recover, they are still exhibit age-associated pathologies at the molecular level.

Accelerated tumour development in R-Atg5i mice

As R-Atg5i mice displayed some evidence of organismal rejuvenation and an increase in overall health, we sought to determine if autophagy restoration is able to reinstate natural longevity to the level seen in littermate control mice, or whether the damage accumulation impacting on lifespan was irreversible. Remarkably, the life-span of R-Atg5i mice was significantly extended in comparison with LT-Atg5i mice (median survival 493 days versus 185 days since treatment began, respectively), while it was still significantly shorter than the R-Control cohorts (Fig. 6a). In marked contrast to LT-Atg5i mice, the cause of death was predominantly associated with the development of tumours with an increased frequency and at earlier timepoints (Fig. 6b, c). These tumours display no evidence of continued autophagy inhibition via immunohistochemical (IHC) analysis (Fig. 6d). Of note a whole-body mosaic Atg5 knockout mouse model has been previously reported to only develop liver adenomas but without any malignant tumours33. Together, our data suggest that a temporary period of autophagy inhibition may be enough to induce irreversible cellular damage, which might facilitate tumour development cooperatively with the restoration of autophagy.