Mitochondrial DNA heteroplasmy rises in substantial nigra of aged PINK1 KO mice

Mutations in PINK1 and Parkin result in early-onset autosomal recessive Parkinson’s disease (PD). PINK1/Parkin pathway maintain mitochondrial function by mediating the clearance of damaged mitochondria. However, the role of PINK1/Parkin in maintaining the balance of mtDNA heteroplasmy is still unknown. Here, we isolated mitochondrial DNA (mtDNA) from cortex, striatum and substantia nigra of wildtype (WT), PINK1 knockout (PINK1 KO) and Parkin knockout (Parkin KO) mice to analyze mtDNA heteroplasmy induced by PINK1/Parkin deficiency or aging. Our results showed that the Single Nucleotide Variants (SNVs) of late-onset somatic variants mainly increased with aging. Conversely, the early-onset somatic variants exhibited significant increase in the cortex and substantia nigra of PINK1 KO mice than WT mice of the same age. Increased average variant allele frequency was observed in aged PINK1 KO mice and in substantial nigra of aged Parkin KO mice than in WT mice. Cumulative variant allele frequency in the substantia nigra of PINK1 KO mice was significantly higher than that in WT mice, further supporting the pivotal role of PINK1 in mtDNA maintenance.This study presented a new evidence for PINK1 and Parkin in participating in mitochondrial quality control and provided clues for further revealing the role of PINK1 and Parkin in the pathogenesis of PD.     

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.     

Senolytic treatment reduces cell senescence and necroptosis in Sod1 knockout mice that is associated with reduced inflammation and hepatocellular carcinoma

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn‐Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence‐associated secretory phenotype factors (IL‐6, IL‐1β, CXCL‐1, and GDF‐15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin‐1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age‐associated pathology in the Sod1KO mice.     

Physiological and metabolic characteristics of novel double‐mutant female mice with targeted disruption of both growth hormone‐releasing hormone and growth hormone receptor

Mice with disruptions of growth hormone‐releasing hormone (GHRH) or growth hormone receptor (GHR) exhibit similar phenotypes of prolonged lifespan and delayed age‐related diseases. However, these two models respond differently to calorie restriction indicating that they might carry different and/or independent mechanisms for improved longevity and healthspan. In order to elucidate these mechanisms, we generated GHRH and GHR double‐knockout mice (D‐KO). In the present study, we focused specifically on the characteristics of female D‐KO mice. The D‐KO mice have reduced body weight and enhanced insulin sensitivity compared to wild‐type (WT) controls. Growth retardation in D‐KO mice is accompanied by decreased GH expression in pituitary, decreased circulating IGF‐1, increased high‐molecular‐weight (HMW) adiponectin, and leptin hormones compared to WT controls. Generalized linear model‐based regression analysis, which controls for body weight differences between D‐KO and WT groups, shows that D‐KO mice have decreased lean mass, bone mineral density, and bone mineral content, but increased adiposity. Indirect calorimetry markers including oxygen consumption, carbon dioxide production, and energy expenditure were significantly lower in D‐KO mice relative to the controls. In comparison with WT mice, the D‐KO mice displayed reduced respiratory exchange ratio (RER) values only during the light cycle, suggesting a circadian‐related metabolic shift toward fat utilization. Interestingly, to date survival data suggest extended lifespan in D‐KO female mice.     

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.     

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS‐induced cell apoptosis

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self‐renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP^−/− mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5‐FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP^−/− HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP^−/− HSCs exhibited an increased resistance to IR‐induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc ^−/−) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.     

Reduced repression of cytokine signaling ameliorates age‐induced decline in hematopoietic stem cell function

Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self‐propagation of repopulating‐defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK^?/? HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK^?/? HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK^?/? HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK^?/? HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age‐related HSC decline.     

Scavenging mitochondrial hydrogen peroxide by peroxiredoxin 3 overexpression attenuates contractile dysfunction and muscle atrophy in a murine model of accelerated sarcopenia

Age‐related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle‐specific overexpression of the mitochondrial H₂O₂ scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H₂O₂ can prevent or delay the redox‐dependent sarcopenia. Basal rates of H₂O₂ generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross‐sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild‐type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle‐specific PRDX3 overexpression reduces mitochondrial H₂O₂ generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox‐dependent sarcopenia.     

Coordination of mitochondrial and lysosomal homeostasis mitigates inflammation and muscle atrophy during aging

Sarcopenia is one of the main factors contributing to the disability of aged people. Among the possible molecular determinants of sarcopenia, increasing evidences suggest that chronic inflammation contributes to its development. However, a key unresolved question is the nature of the factors that drive inflammation during aging and that participate in the development of sarcopenia. In this regard, mitochondrial dysfunction and alterations in mitophagy induce inflammatory responses in a wide range of cells and tissues. However, whether accumulation of damaged mitochondria (MIT) in muscle could trigger inflammation in the context of aging is still unknown. Here, we demonstrate that BCL2 interacting protein 3 (BNIP3) plays a key role in the control of mitochondrial and lysosomal homeostasis, and mitigates muscle inflammation and atrophy during aging. We show that muscle BNIP3 expression increases during aging in mice and in some humans. BNIP3 deficiency alters mitochondrial function, decreases mitophagic flux and, surprisingly, induces lysosomal dysfunction, leading to an upregulation of Toll‐like receptor 9 (TLR9)‐dependent inflammation and activation of the NLRP3 (nucleotide‐binding oligomerization domain (NOD)‐, leucine‐rich repeat (LRR)‐, and pyrin domain‐containing protein 3) inflammasome in muscle cells and mouse muscle. Importantly, downregulation of muscle BNIP3 in aged mice exacerbates inflammation and muscle atrophy, and high BNIP3 expression in aged human subjects associates with a low inflammatory profile, suggesting a protective role for BNIP3 against age‐induced muscle inflammation in mice and humans. Taken together, our data allow us to propose a new adaptive mechanism involving the mitophagy protein BNIP3, which links mitochondrial and lysosomal homeostasis with inflammation and is key to maintaining muscle health during aging.     

Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.     

Defective dimerization of FoF1‐ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging

Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1‐ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy‐dissipating channel involved in cell death. We investigated whether aging alters FoF1‐ATP synthase self‐assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1‐ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1‐ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes’ susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1‐ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1‐ATP synthase glycation in H9c2 myoblasts recapitulated the age‐related defective FoF1‐ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1‐ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.     

Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment

Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.     

Sex‐ and strain‐specific effects of mitochondrial uncoupling on age‐related metabolic diseases in high‐fat diet‐fed mice

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti‐aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled‐release mitochondrial protonophore (CRMP) that is functionally liver‐directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver‐directed fashion could reduce oxidative damage and improve age‐related metabolic disease and lifespan in diet‐induced obese mice. Oral administration of CRMP (20 mg/[kg‐day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74‐week‐old) high‐fat diet (HFD)‐fed C57BL/6J male mice, independently of changes in body weight, whole‐body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long‐term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94–104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex‐specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof‐of‐concept data to support further studies investigating the use of liver‐directed mitochondrial uncouplers to promote healthy aging in humans.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

Whole‐body senescent cell clearance alleviates age‐related brain inflammation and cognitive impairment in mice

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16^Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16^Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16^Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions'' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.