Kynurenine Inhibits Autophagy and Promotes Senescence in Aged Bone Marrow Mesenchymal Stem Cells Through the Aryl Hydrocarbon Receptor Pathway

Osteoporosis is an age-related deterioration in bone health that is, at least in part, a stem cell disease. The different mechanisms and signaling pathways that change with age and contribute to the development of osteoporosis are being identified. One key upstream mechanism that appears to target a number of osteogenic pathways with age is kynurenine, a tryptophan metabolite and an endogenous Aryl hydrocarbon receptor (AhR) agonist. The AhR signaling pathway has been reported to promote aging phenotypes across species and in different tissues. We previously found that kynurenine accumulates with age in the plasma and various tissues including bone and induces bone loss and osteoporosis in mice. Bone marrow mesenchymal stem cells (BMSCs) are responsible for osteogenesis, adipogenesis, and overall bone regeneration. In the present study, we investigated the effect of kynurenine on BMSCs, with a focus on autophagy and senescence as two cellular processes that control BMSCs proliferation and differentiation capacity. We found that physiological levels of kynurenine (10 and 100 μM) disrupted autophagic flux as evidenced by the reduction of LC3B-II, and autophagolysosomal production, as well as a significant increase of p62 protein level. Additionally, Kynurenine also induced a senescent phenotype in BMSCs as shown by the increased expression of several senescence markers including senescence associated β-galactosidase in BMSCs. Additionally, western blotting reveals that levels of p21, another marker of senescence, also increased in kynurenine-treated BMSCs, while senescent-associated aggregation of nuclear H3K9me3 also showed a significant increase in response to kynurenine treatment. To validate that these effects are in fact due to AhR signaling pathway, we utilized two known AhR antagonists: CH-223191, and 3’,4’-Dimethoxyflavone to try to block AhR signaling and rescue kynurenine/AhR mediated effects. Indeed, AhR inhibition restored kynurenine-suppressed autophagy levels as shown by levels of LC3B-II, p62 and autophagolysosomal formation demonstrating a rescuing of autophagic flux. Furthermore, inhibition of AhR signaling prevented the kynurenine-induced increase in senescence associated β-galactosidase and p21 levels, as well as blocking aggregation of nuclear H3K9me3. Taken together, our results suggest that kynurenine inhibits autophagy and induces senescence in BMSCs via AhR signaling, and that this may be a novel target to prevent or reduce age-associated bone loss and osteoporosis

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis

The phosphorylation state of Ser-129 in human α-synuclein determines neurodegeneration in a rat model of Parkinson disease

Studies have shown that α-synuclein (α-syn) deposited in Lewy bodies in brain tissue from patients with Parkinson disease (PD) is extensively phosphorylated at Ser-129. We used recombinant Adeno-associated virus (rAAV) to overexpress human wild-type (wt) α-syn and two human α-syn mutants with site-directed replacement of Ser-129 to alanine (S129A) or to aspartate (S129D) in the nigrostriatal tract of the rat to investigate the effect of Ser-129 phosphorylation state on dopaminergic neuron pathology. Rats were injected with rAAV2/5 vectors in the substantia nigra pars compacta (SNc) on one side of the brain; the other side remained as a nontransduced control. The level of human wt or mutant α-syn expressed on the injected side was about four times the endogenous rat α-syn. There was a significant reduction of dopaminergic neurons in the SNc and dopamine (DA) and tyrosine hydroxylase (TH) levels in the striatum of all S129A-treated rats as early as 4 wk postinjection. Nigral DA pathology occurred more slowly in the wt-injected animals, but by 26 wk the wt α-syn group lost nigral TH neurons equivalent to the mutated S129A group at 8 wk. In stark contrast, we did not observe any pathological changes in S129D-treated animals. Therefore, the nonphosphorylated form of S129 exacerbates α-syn-induced nigral pathology, whereas Ser-129 phosphorylation eliminates α-syn-induced nigrostriatal degeneration. This suggests possible new therapeutic targets for Parkinson Disease. © 2008 by The National Academy of Sciences of the USA

MicroRNA-141-3p Negatively Modulates SDF-1 Expression in Age-Dependent Pathophysiology of Human and Murine Bone Marrow Stromal Cells

Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a cytokine secreted by cells including bone marrow stromal cells (BMSCs). SDF-1 plays a vital role in BMSC migration, survival, and differentiation. Our group previously reported the role of SDF-1 in osteogenic differentiation in vitro and bone formation in vivo; however, our understanding of the post-transcriptional regulatory mechanism of SDF-1 remains poor. MicroRNAs are small noncoding RNAs that post-transcriptionally regulate the messenger RNAs (mRNAs) of protein-coding genes. In this study, we aimed to investigate the impact of miR-141-3p on SDF-1 expression in BMSCs and its importance in the aging bone marrow (BM) microenvironment. Our data demonstrated that murine and human BMSCs expressed miR-141-3p that repressed SDF-1 gene expression at the functional level (luciferase reporter assay) by targeting the 3′-untranslated region of mRNA. We also found that transfection of miR-141-3p decreased osteogenic markers in human BMSCs. Our results demonstrate that miR-141-3p expression increases with age, while SDF-1 decreases in both the human and mouse BM niche. Taken together, these results support that miR-141-3p is a novel regulator of SDF-1 in bone cells and plays an important role in the age-dependent pathophysiology of murine and human BM niche

α-Synuclein Expression in Rat Substantia Nigra Suppresses Phospholipase D2 Toxicity and Nigral Neurodegeneration

We present genetic evidence that an in vivo role of α-synuclein (α-syn) is to inhibit phospholipase D2 (PLD2), an enzyme that is believed to participate in vesicle trafficking, membrane signaling, and both endo- and exocytosis. Overexpression of PLD2 in rat substantia nigra pars compacta (SNc) caused severe neurodegeneration of dopamine (DA) neurons, loss of striatal DA, and an associated ipsilateral amphetamine-induced rotational asymmetry. Coexpression of human wild type α-syn suppressed PLD2 neurodegeneration, DA loss, and amphetamine-induced rotational asymmetry. However, an α-syn mutant defective for inhibition of PLD2 in vitro also failed to inhibit PLD toxicity in vivo. Further, reduction of PLD2 activity in SNc, either by siRNA knockdown of PLD2 or overexpression of α-syn, both produced an unusual contralateral amphetamine-induced rotational asymmetry, opposite to that seen with overexpression of PLD2, suggesting that PLD2 and α-syn were both involved in DA release or reuptake. Finally, α-syn coimmunoprecipitated with PLD2 from extracts prepared from striatal tissues. Taken together, our data demonstrate that α-syn is an inhibitor of PLD2 in vivo, and confirm earlier reports that α-syn inhibits PLD2 in vitro. Our data also demonstrate that it is possible to use viral-mediated gene transfer to study gene interactions in vivo