Age-matched Untreated Animals Research Articles

Mitochondrial Activity Identifies a Transcriptionally and Functionally Distinct Subset of Aged HSCs with Lineage-Balanced Output

During ageing, the haematopoietic stem cell (HSC) pool expands numerically, but declines functionally. This functional decline is characterised by myeloid skewing and decreased long-term reconstitution potential and clinically manifests as anaemia, immune compromise and increased risk of clonal malignancy. We postulate that HSCs do not synchronously functionally decline with age, but instead represent a spectrum in which physiologically aged HSCs become dominant. In this study, we aimed to reveal properties that might identify physiologically young HSCs during chronological ageing and to exploit these in an attempt to rescue haematopoietic ageing. Young adult ((y), 2-3 months) and aged (old (o), >18 months) mouse HSCs were profiled and we observed a significant decrease (30-50%) in mitochondrial membrane potential (MMP) in oHSCs. Interestingly, a small fraction (15%) of oHSCs maintained a similar MMP to the bulk (70%) of yHSCs. We explored, initially at the transcriptional level, whether these MMPhigh HSCs are distinct from MMPlow HSCs. Strikingly, RNA sequencing of MMPhigh and MMPlow young and aged HSCs revealed that samples cluster by MMP over age. MMPhigh young and aged HSCs were characterised by upregulation of lymphoid and erythroid lineage markers, as well as RNA processing, MYC and E2F pathways. MMPlow young and aged HSCs were transcriptionally associated with ageing, inflammation and myeloid bias. Based on these results, we hypothesised that enhancing MMP in oHSCs might restore lineage-balanced peripheral blood (PB) output, used as a measure of functional improvement. To achieve this, we chemically enhanced MMP in oHSCs in vivo, using the mitochondrially-targeted drug mitoquinol (MQ). Interestingly, our initial experiments show that a 5-day treatment with MQ significantly shifted the B-cell/myeloid ratio in PB from 0.6 (aged) to 1.5 (MQ), in the direction of the ratio observed in young mice (2.9). This ratio change was not due to numerical depletion of myeloid cells, but due to restoration of B-cell numbers, including IgM+ B-cells commonly reduced with age. To assess whether the effect of MQ was due to HSC-intrinsic changes and could be sustained over time, HSCs were isolated from MQ-treated or untreated aged mice and transplanted into lethally irradiated recipients. Our experiments to date show that HSCs isolated from MQ-treated aged mice show superior engraftment and faster and greater B-cell reconstitution than HSCs from age-matched untreated animals, and that these improvements are stable over the 16-week assay. Based on our sequencing results indicating that RNA processing, MYC and E2F pathways are associated with MMPhigh HSCs, and previous work reporting a relationship between MMP and rate of mRNA transcription (das Neves et al., PLoS Biol. 2010; Johnston et al., PLoS Comput. Biol. 2012), we explored the possibility that MMP might orchestrate the observed changes by altering transcriptional rate of HSCs. We tested transcription rate in HSCs in vivo and found that yHSCs display a particularly high rate of mRNA transcription. Rate of transcription was significantly reduced in oHSCs compared to yHSCs, equivalent to their quantitative reduction in MMP. We could demonstrate a direct correlation between MMP and transcription rate in HSCs, by showing that MMPhigh sorted HSCs were transcribing nearly twice as fast as MMPlow sorted HSCs. Furthermore, in vivo injection with mitochondrial uncoupler CCCP caused a similar reduction in transcription rate of HSCs (>50%) as did conventional RNA Pol-II inhibitors (DRB, Flavopiridol), hereby demonstrating that transcription rate directly depends on MMP. This work indicates that mitochondrial state can separate HSCs with distinct transcriptional profiles linked to different cell fates. We speculate that changes in transcriptional profile arise from MMP-driven regulation of transcriptional rate in HSCs. This would open up the possibility that pharmacological manipulation of mitochondrial activity can alter transcriptional programs of HSCs with consequences for functionality. Disclosures No relevant conflicts of interest to declare.

Effects of Losartan on Oxidative Stress, DNA Damage, and Metabolic Parameters in Whole Blood and Liver of Rhesus Monkeys (Macaca mulatta)

Angiotensin II (Ang II), the primary peptide of the Renin Angiotensin System (RAS), can induce the production of reactive oxygen species (ROS) and affect mitochondrial energetics, predisposing to age‐related disorders. Angiotensin converting enzyme inhibitors (ACE) and angiotensin receptor blockers (ARB) can attenuate ROS production, improve mitochondrial function and increase survival in aging rodents, however, the effects of losartan, an ARB, on age‐associated oxidative damage in rhesus monkeys have not been yet explored. We have previously shown that mitochondrial DNA (mtDNA) damage, depletion of mtDNA molecules and oxidative stress increase in liver and blood leukocytes of middle‐age and old rhesus monkeys. We hypothesize that losartan treatment prevents oxidative damage in liver and blood leukocytes from rhesus monkeys. To test our hypothesis we examined the effects of losartan on mtDNA, nuclear DNA (nDNA) and protein damage, mtDNA abundance in whole blood and liver and metabolic parameters in losartan‐treated and age‐matched untreated middle‐age rhesus monkeys. Middle‐age rhesus monkeys (8–19 years of age) were treated with losartan (30mg/day) for 12 months and samples were obtained at baseline, 3, 6, and 12 months after treatment. Young (3–6 years of age) untreated monkeys were also analyzed as a control for the effects of aging. Fasting insulin and fasting glucose were measured in the serum and mitochondrial DNA (mtDNA) damage and abundance, and nuclear DNA (nDNA) damage were assessed by quantitative PCR. Protein carbonylations were measured in peripheral blood mononuclear cells (PBMCs) and liver of rhesus monkeys using the FTC Fluorometric assay. Interestingly, we found that losartan significantly decreased fasting blood glucose levels in healthy middle‐age monkeys after 12 months of treatment when compared to age‐matched untreated animals. No effects were observed on systolic/diastolic blood pressure, fasting insulin, cholesterol, tryglycerides and glycated hemoglobin levels. In whole blood samples, middle‐age monkeys exhibit significant increased levels of nDNA lesions compared to young animals, and treatment with losartan decreased nDNA damage to levels similar to young animals. Moreover, we found a significant decrease in the levels of carbonylated proteins in PBMCs from middle‐age monkeys treated with losartan compared to untreated monkeys, indicative of lower levels of oxidative stress. No significant differences were observed in the levels of mtDNA copies between the groups. In liver, middle‐age monkeys exhibited decreased levels of mtDNA molecules and losartan had no effect on preventing the loss of mtDNA copies. In contrast to PBMCs, no significant differences were observed in nDNA lesions and protein carbonylations between groups. Surprisingly, losartan significantly increased the levels of mtDNA damage as compared to young animals. Collectively, our results suggest that losartan precludes nDNA (but not mtDNA) and protein oxidative damage in PBMCs (but not in liver) and improves glucose levels in healthy middle‐age rhesus monkeys.Support or Funding InformationP40RR003640R25 GM061838U54 MD007600This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Postnatal experience modulates functional properties of mouse olfactory sensory neurons

Early experience considerably modulates the organization and function of all sensory systems. In the mammalian olfactory system, deprivation of the sensory inputs via neonatal, unilateral naris closure has been shown to induce structural, molecular and functional changes from the olfactory epithelium to the olfactory bulb and cortex. However, it remains unknown how early experience shapes the functional properties of individual olfactory sensory neurons (OSNs), the primary odor detectors in the nose. To address this question, we examined the odorant response properties of mouse OSNs in both the closed and open nostril after 4 weeks of unilateral naris closure, with age-matched untreated animals as control. Using a patch-clamp technique on genetically tagged OSNs with defined odorant receptors (ORs), we found that sensory deprivation increased the sensitivity of MOR23 neurons in the closed side, whereas overexposure caused the opposite effect in the open side. We next analyzed the response properties, including rise time, decay time, and adaptation, induced by repeated stimulation in MOR23 and M71 neurons. Even though these two types of neuron showed distinct properties with regard to dynamic range and response kinetics, sensory deprivation significantly slowed down the decay phase of odorant-induced transduction events in both types. Using western blotting and antibody staining, we confirmed the upregulation of several signaling proteins in the closed side as compared with the open side. This study suggests that early experience modulates the functional properties of OSNs, probably by modifying the signal transduction cascade.

ATP and norepinephrine contributions to sympathetic vasoconstriction of tail artery are altered in streptozotocin-diabetic rats

Sympathetic vasoconstriction is susceptible to diabetes, but contributions made by purinergic neurotransmission in this state have not been investigated. We aimed to evaluate sympathetic vasoconstriction contributions by ATP and norepinephrine in the tail artery from streptozotocin-diabetic rats by using isometric vascular rings. Tail arteries were isolated from rats made diabetic 3 mo earlier with streptozotocin (diabetic group), age-matched nondiabetic rats (nondiabetic injected), age-matched untreated animals (noninjected normal), and age-matched untreated animals in high glucose control Krebs solution (high glucose control). Responses to KCl (60 mM) or nerve stimulus trains of 1-100 impulses were identical in all groups. Electrical stimulation produced progressively greater contractions with increasing impulse numbers. These were partially reduced by suramin (100 microM, P2 antagonist), NF-279 (1 microM, P2X blocker), and phentolamine (2 microM, alpha-blocker). For purinergic antagonists, blockade was greater in diabetic vessels compared with that in others. No differential effect could be detected for phentolamine between groups. Bath-applied ATP (1 nM-1 mM) and norepinephrine (0.1 nM-100 microM) showed increased potency with diabetic group vessels. Desipramine (1 microM, norepinephrine reuptake inhibitor) potentiated neurally evoked responses in all groups equally and increased sensitivity to exogenous norepinephrine in a similar fashion. Histochemical labeling of sympathetic nerves with neuronal marker protein PGP-9.5 and a sympathetic nerve-specific antibody for tyrosine hydroxylase showed no reduction in diabetic innervation density. We demonstrate, for the first time, changes in contributions of ATP and norepinephrine in sympathetic responses of rat tail artery in diabetes, which cannot be accounted for by axonal degeneration or by changes in norepinephrine reuptake.

Increased levels of cytosolic thioredoxin reductase activity and mRNA in rat liver nodules

Background/Aims : Thioredoxin reductase, a redox active enzyme, is induced in several tumors. This study focuses on the presence of and subcellular localisation of thioredoxin reductase in a tumor model where neoplastic lesions are selected by their resistance to the toxic effects of the promotor. Methods : Liver nodules produced by intermittent feeding of 2-acetylaminofluorene to male Wistar rats were analyzed for thioredoxin reductase (TrxR) activity and mRNA. Results : This activity was increased 3.5-fold in the cytosol but decreased 60% in the mitochondrial fraction compared to the liver of age-matched untreated animals. Only traces of activity were observed in the microsomal, plasma membrane and nuclear fractions from normal liver or nodules. The level of TrxR mRNA was 3-fold higher in nodules than in normal rat liver. Furthermore, the total level of SH groups in homogenates was 2-fold higher in the case of the nodules. Conclusions : These findings indicate that the thioredoxin system makes an important contribution to the resistant phenotype of the neoplastic liver cell, which conveys a growth advantage of significance for tumor progression.

Topical Tretinoin Increases the Tropoelastin and Fibronectin Content of Photoaged Hairless Mouse Skin

Topical tretinoin treatment of photoaged hairless mice has been shown in previous studies to stimulate formation of a subepidermal zone of new connective tissue characterized by enhanced collagen synthesis. The aims of this study were to localize and/or quantify elastin, fibronectin, and glycosaminoglycans in the same model. Hairless mice (Skh-1) were irradiated thrice weekly for 10 weeks with gradually increasing doses of ultraviolet (up to 4.5 minimal erythema doses per exposure) from Westinghouse FS-40 bulbs. Mice were then treated five times a week with either 0.05% tretinoin, the ethanol:propylene glycol vehicle, or nothing for another 10 weeks. Controls included mice sacrificed after 10 weeks of ultraviolet treatment and age-matched untreated animals. The distribution of elastin and fibronectin was examined by immunofluorescence microscopy, which revealed fine fibrils in the subepidermal zone in tretinoin-treated skin. A quantitative slot-blot immunobinding assay showed that tretinoin induced a threefold higher amount of tropoelastin compared with controls. Insoluble elastin content (desmosine levels) was similar in all groups. Although fibronectin content was increased by ultraviolet radiation, tretinoin treatment induced the largest increase. In contrast, the amount of glycosaminoglycans, although increased by UVB radiation, was reduced by tretinoin treatment.

Carnitine palmitoyl transferase-I activity in the aging mouse heart

We investigated the influence of age on carnitine palmitoyl transferase-I (CPT-I, EC 2.3.1.21) activity in the mouse heart. There was an age-associated decrease in CPT-I activity from 2 to 26 months ( P = 0.006). We studied the effect of oxygen-derived radicals on CPT-I activity. Mitochondria from 2-month-old mouse hearts exposed to different concentrations of hydrogen peroxide (H 2O 2) showed a dose-related decrease in CPT-I activity ( P < 0.002). To determine the possible reversibility of the age change in CPT-I activity, we studied the effect of oral administration of propionyl-L-carnitine (PLC). Oral pretreatment of middle-aged (18-month-old) mice with PLC resulted in a 37% increase of basal CPT-I activity ( P < 0.05) compared to age-matched untreated animals, and restored it to a level similar to that of 2-month-old mice. Pretreatment of senescent (26-month-old) mice with PLC, however, showed no significant change in basal CPT-I activity. It is possible that the age-related decrease in CPT-I activity may result from an in vivo accumulation of oxygen-derived radical damage. It appears that the age change in CPT-I activity in 18- but not in the 26-month-old mice is reversible with PLC.

Age-related changes in brain microanatomy: Sensitivity to treatment with the dihydropyridine calcium channel blocker darodipine (PY 108-068)

The influence of aging and of treatment with the dihydropyridine Ca 2+ antagonist darodipine (PY 108-068) on the age-related microanatomical changes of rat brain were studied in male Wistar rats treated from the 18th to the 24th month of age with an oral dose of 5 mg/kg/day of darodipine. Twelve-month-old untreated rats were used as an adult reference group. A decreased number of nerve cells and of alkaline phosphatase-positive capillaries and an increased lipofuscin deposition were observed in the frontal and occipital cortex, in the hippocampus, and in the cerebellar cortex of rats of 24 months in comparison with 12-month-old animals. The number of nerve cells was higher in the occipital cortex and in the hippocampus, but not in the frontal cortex and in the cerebellar cortex, of darodipine-treated rats in comparison with age-matched untreated animals. Lipofuscin deposition is reduced in all the brain areas investigated. The density of alkaline phosphatase-reactive capillaries is also increased in the frontal and occipital cortex and in the hippocampus of aged rats treated with darodipine. The above results suggest that treatment with darodipine is able to counter some microanatomical changes occurring in the brain of aged rats and involving not only microvascular parameters. The occipital (visual) cortex and the hippocampus were the cerebral areas more sensitive to treatment with darodipine. The possible relevance of these findings is discussed.

Streptozotocin-induced liver tumors in the Syrian hamster.

Streptozotocin was administered in a dose of 50 mg/kg body weight intraperitoneally on three consecutive days to 35 8-week old male Syrian golden hamsters. At sacrifice 16-36 weeks later, macroscopic liver tumors had developed in 27 of 29 surviving animals. The majority of animals had multiple hepatic nodules. Some tumors were already large (greater than 1 cm) 16 weeks after streptozotocin. By histologic criteria, five animals (17%) had hepatocellular carcinoma, 23 (79%) had adenomas with varying degrees of atypia, and one (4%) had a normal liver. No metastases to organs outside the liver were noted. No other primary tumors were observed. Sixteen of 29 animals had ascites. Twenty-five age-matched untreated animals showed no evidence of liver tumors. Although sporadic liver tumors due to streptozotocin have been reported in rats and mice, the high incidence of tumors and short latency period to tumor induction seem to be unique to the Syrian hamster. The development of liver carcinoma without the necessity for long-term administration of the carcinogen and without adjunctive procedures such as partial hepatectomy represents a potential improvement over other animal models of hepatic neoplasia.