Age-associated mitochondrial complex I deficiency is linked to increased stem cell proliferation rates in the mouse colon

Abstract

One of the hallmarks of aging is an accumulation of cells with defects in oxidative phosphorylation (OXPHOS) due to mutations of mitochondrial DNA (mtDNA). Rapidly dividing tissues maintained by stem cells, such as the colonic epithelium, are particularly susceptible to accumulation of OXPHOS defects over time; however, the effects on the stem cells are unknown. We have crossed a mouse model in which intestinal stem cells are labelled with EGFP (Lgr5-EGFP-IRES-creERT2) with a model of accelerated mtDNA mutagenesis (PolgA^mut/mut ) to investigate the effect of OXPHOS dysfunction on colonic stem cell proliferation. We show that a reduction in complex I protein levels is associated with an increased rate of stem cell cycle re-entry. These changes in stem cell homeostasis could have significant implications for age-associated intestinal pathogenesis.

Keywords: aging; colon; complex I; mitochondria; stem cells.

FIGURE 1

Mitochondrial OXPHOS labelling in colonic crypts of 12‐month‐old PolgA ^+/+, PolgA ^+/mut and PolgA ^{m ut/mut} mice. (a) Immunofluorescent panel showing levels of mitochondrial NDUFB8 (CI, Alexa Fluor 647), MTCO1 (CIV, Alexa Fluor‐546) and TOMM20 (Alexa Fluor‐488). Yellow arrows: OXPHOS normal; red arrows: CIV deficiency; green arrows: CI deficiency; and white arrows: CI + CIV deficiency. Cell nuclei are labelled with DAPI (blue). Scale bars = 50 µm (b–c) Dot plots showing z‐scores of NDUFB8 and MTCO1 relative to TOMM20. Crypts with z‐score <−4.5 (dashed line) were defined as "deficient." Error bars show mean ± SD. Numbers of crypts quantified per mouse were as follows: PolgA ^+/+: (l‐r) n = 223, 259, 306 and 332; PolgA ^+/mut: (l‐r) n = 201, 200, 200 and 200; PolgA ^mut/mut: (l‐r) n = 253, 191, 257 and 222. (d) The percentage of crypts per genotype with the 4 OXPHOS categories, bars show mean, and error bars are SEM. (f) Immunofluorescent images showing VDAC1 (Alexa Fluor 488) levels. Scale bars = 50 µm (g) Dot plots showing z‐scores of VDAC1 levels. n = 150 crypts were quantified per mouse. Error bars show mean ± SD

FIGURE 2

Quantification of thymidine analogue labelling in LGR5^High stem cells in colonic crypts of 12‐month‐old PolgA ^+/+, PolgA ^{+/ mut} and PolgA ^{mut / mut} mice. (a) Immunofluorescent panel showing LGR5 (Alexa Fluor 488), CldU (Alexa Fluor 546), IdU (Alexa Fluor 647) and Ki‐67 (Alexa Fluor 750) labelling in PolgA ^+/+ and PolgA ^mut/mut mice. Scale bars = 20 µm. (b–f) Quantification of total cells, CldU, IdU, CldU + IdU and Ki‐67 labelling per crypt. (g–i) Proportion of LGR5^High cells per crypt incorporating CldU, IdU and CldU + IdU. (b–i) Each dot represents a single crypt and is colour‐coded by mouse ID, n = 4 mice per group. Numbers of crypts quantified per mouse were as follows: PolgA ^+/+: n = 64, n = 81, n = 60 and n = 65; PolgA ^mut/mut: n = 49, n = 24, n = 41 and n = 20. (j) Immunofluorescent panel showing MTCO1 (Alexa Fluor 546) and NDUFB8 (Alexa Fluor 647), labelling in section 1 and LGR5, CldU, IdU and Ki‐67 labelling in the adjacent section. Scale bars = 20 µm. Crypts were grouped by OXPHOS status. (k–o) Quantification of total cells, CldU, IdU, CldU + IdU and Ki‐67 labelling per crypt. (p–r) The proportion of LGR5^High cells per crypt incorporating CldU, IdU and CldU + IdU. (k–r) Each dot represents a single crypt and is colour‐coded by mouse ID (n = 4). Numbers of crypts per category: +, n = 100; CI, n = 117; CIV, n = 35; and CI + CIV, n = 34. (b–i) and (k–r) Negative binomial or Poisson GLMM, *p < 0.05, **p < 0.001. and ***p < 0.0001