Abstract
Aging is the primary risk factor for many late-onset diseases including myeloid malignancies and immune deficiencies of the elderly. Aging of hematopoietic stem cells (HSCs) is thought to be in part responsible for these blood disorders. As quiescence maintains HSCs' health and function throughout life, loss of quiescence with age is likely implicated in defects of old HSCs including their myeloid-biased lineage commitment. Despite major progress, mechanisms that promote aging of HSCs remain to be elucidated. We have identified dynamic lysosomal modulations as a switch in the precise control of HSC quiescence vs. priming/activation in young HSCs. Here we find that lysosomal properties and functions are compromised in old HSCs. We find that lysosomes are relatively depleted in old HSCs, as evaluated by high resolution confocal microscopy analyses of immunofluorescence (IF) staining of lysosomal-associated membrane protein (LAMP)-1 and the more specific LAMP-2 markers. Notably, lysosomal properties are altered in old HSCs beyond what is known of their function in autophagy. We find increased recruitment of damage-associated proteins Galectin 1 and 2 to lysosomes in old relative to young highly purified LSKCD150+CD48- HSCs indicating increased lysosomal damage in old HSCs. In addition, lysosomal LC3 flux that is an indication of lysosomal activity was increased by two-fold in highly purified old relative to young HSCs. This was in line with old HSCs' alteration of expression of lysosomal enzymes Cytochalasin C and D and the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase). The increased lysosomal activity is supported by greatly acidified lysosomes as well as enhanced lysosomal degradation capacity of cargo in old relative to young HSCs. An 18-hour inhibition of lysosomal acidification by repressing v-ATPase' activity using specific and high affinity v-ATPase inhibitor, ex vivo, led to a dramatic enrichment of old HSCs in large lysosomes, restoration of their properties and reducing their Galectin protein burden similar to what was observed in young quiescent and healthy HSCs. This was specific to lysosomes in the endo-lysosomal pathway as the effect on the Golgi and endoplasmic reticulum compartments in HSCs was relatively negligeable. The v-ATPase inhibitory treatment increased substantially the fraction of non-dividing old HSCs in culture, and the frequency of LTC-ICs recovered in limiting dilution analysis of old HSCs. Importantly, a 4-day ex vivo inhibition of lysosomal activity increased old HSCs' in vivo competitive repopulation ability by over 8-fold over 21 weeks and balanced the production of lineages downstream of old HSCs. Remarkably, only v-ATPase inhibitor- but not vehicle-treated old HSCs sustained survival in lethally irradiated secondarily transplanted mice and maintained self-renewal. v-ATPase inhibition normalized the expression of TFEB, master regulator of lysosomal biogenesis, suppressed CDK6 nuclear expression, normalized metabolic properties of old HSCs, including mTOR activity and glucose uptake, normalized expression of histone marks H4K16ac, H3K27ac and H3K56ac in old HSCs, as well as signs of intrinsic inflammation. Overall, our work indicates that inhibiting lysosomal hyper-activation restores optimum mitochondrial sequestration in old HSCs' lysosomes, leading to reduced inflammation and restitution of stem cell function in old HSCs. These findings may be exploited for improving HSC function in the elderly.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
