Faculty Bibliography

Osteoarthritis (OA) is themost common form of arthritis, affecting nearly 10%of the US population. There is no therapy to prevent the progression of or reverse OA pathology. Endogenous adenosine 2A receptor (A2AR) stimulation is crucial for chondrocyte viability and cartilage homeostasis, as its downstream signalingmediates inflammation.We have published thatmice lacking the A2AR or ecto-5'nucleotidase (CD73) develop spontaneous OA, suggesting that diminished extracellular adenosine levels promote the development of OA. Since human OA chondrocytes have been found to diminish mitochondrial content, we proposed to test the hypothesis that OA pathogenesis deregulates A2AR signaling at least in part by affecting the cell's capacity for ATP production via reduced content or functionality of mitochondria. Primary neonatal WT and A2ARKO chondrocytes were subjected to Seahorse Mito Stress Kit Assays, which revealed reduced oxygen consumption rates (OCR) at baseline and after mitochondrial uncoupling (corresponding to reduced coupling capacity and ATP production). RNA sequencing DESEQ analysis showed increased metalloproteinases expression along with OA-associated pro-inflammatory pathways. We also saw upregulation of genes involved with aging and the production of nitric oxide (NO) and reactive oxygen species (ROS). Histologic staining for 8hydroxyguanosine (8OH-G) residues as a marker for ROS is also markedly increased in A2ARKO mice as early as 8 weeks. A human chondrocyte cell line, T/C28-a2, was used to determine the effect of IL-1beta-induced inflammation and A2AR stimulation in vitro.Mitochondrial health and functionality was assessed by mean pixel intensity (MPI) of a fluorescent probe for monitoring mitochondrial membrane potential, TMRM, and by measuring OCR. IL-1beta (5ng/mL) incubation for 3 hours followed by A2AR ligation (CGS21680 (CGS; 1uM)) increases mitochondrial membrane potential compared to control, IL-1beta alone and CGS alone as measured by TMRM staining. IL-1beta incubation for 4 hours with a last hour of A2AR stimulation increases basal OCR and maximal respiratory rate. IL-1beta+CGS treated cells had significantly increased ATP production than the control, IL-1beta and CGS treated cells as measured by one-way ANOVA. A2AR ligation improves mitochondrial functionality during inflammation and OA progression. Lack of A2AR signaling not only promotes inflammation and catabolism of cartilage matrix, it also damages the cell's mitochondrial metabolic capacity

Background/Purpose: Osteoarthritis (OA) is the most common form of arthritis, affecting nearly 10% of the US population. With age and injury, chondrocytes have diminished mitochondrial content and mitochondrial production of ATP contributing to OA pathogenesis. We have previously reported that chondrocytes release ATP, which is converted extracellularly to adenosine and maintains chondrocyte homeostasis via endogenous stimulation of the A2AR. Injured/ inflamed chondrocytes have lower ATP levels and release less ATP resulting in diminished extracellular adenosine and A2AR stimulation. Mice and humans lacking the capacity to convert extracellular ATP to adenosine (ecto-5'nucleotidase deficient) develop spontaneous OA as do mice lacking A2AR (A2ARKO). We therefore studied the effect of A2AR stimulation on mitochondrial health and function in chondrocytes from WTand A2ARKO mice and in a human chondrocytic cell line.
Method(s): A human chondrocyte cell line, T/C28-a2, or neonatal chondrocytes isolated from WTand A2ARKO mice (C57Bl6 background) were grown in culture, treated with IL-1beta (5ng/ml) or medium (4h, 37oC) and during the last hour of incubation, with either medium or the selective A2AR agonist (CGS21680, 1mM). Mitochondrial function was then analyzed by Seahorse Mito Stress Kits using the Seahorse apparatus. Mitochondrial health was assessed by analyzing mean pixel intensity (MPI) of tetramethylrhodamine (TMRM) live cell staining which correlates with reduced collapsibility of mitochondrial membrane potential. Mitochondrial content and ROS burden were assessed in live cell confocal imaging (MitoTracker; MitoSox) and by immunohistochemistry (anti-ATPase Ab; anti-8hydroxyguanosine Ab, 8OHg) in paraffinembedded tissue from WTand A2ARKO mice.
Result(s): The mitochondrial membrane potential and mitochondrial content were reduced in A2ARKO chondrocytes compared to WT. In WT chondrocytes mitochondrial content increased after IL-1beta treatment and A2AR stimulation increased mitochondrial membrane potential as well. Histologic staining of knee cartilage for 8OHg, a marker of ROSinduced oxidation in mitochondria, of age-matched WTand A2ARKO mice revealed increased ROS burden in OA (A2AR KO) cartilage. In T/C28-a2 cells, neither IL-1beta nor CGS21680 affected basal O2 consumption rates (OCR), maximal respiratory rate or ATP production but IL-1beta-treated T/C28-a2 cells stimulated by CGS21680 increased all three measures of mitochondrial function (p<0.03, one-way ANOVA). Membrane potential (measured by TMRM MPI) decreased in T/ C28-a2 cells after IL-1beta or CGS treatment alone, but IL-1beta + CGS21680 treatment significantly increased MPI, indicating enhanced mitochondrial health. Mitochondrial content is not significantly modulated by IL-1beta, CGS or IL-1beta+CGS in vitro, but IL-1beta treatment significantly increased ROS burden (p<0.0001) and IL-1beta+CGS did not affect ROS-burden in T/C28-a2 cells.
Conclusion(s): Mitochondrial function and biomass decline with age and after injury and diminished mitochondrial function contributes to the development of OA. A2AR stimulation enhances the function of mitochondria in inflamed chondrocytes and contributes to the maintenance of healthy chondrocytes and cartilage

Mitochondrial permeability transition (PT) is a phenomenon of an increase of the inner membrane permeability in response to an excessive matrix calcium accumulation. PTP is caused by the opening of the large weakly selective channel. Molecular composition and regulation of permeability transition pore (PTP) are not well understood. Here we used isolated mitochondria to investigate dependence of PTP activation on the osmotic pressure. We found that in low osmotic strength solution calcium-induced PTP is significantly inhibited. We propose that this effect is linked to the changes in the curvature of the mitochondrial inner membrane. This interpretation is consistent with the idea about the importance of ATP synthase dimerization in modulation of the PTP activity.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease.

Mounting evidence suggests that mitochondrial dysfunction plays a causal role in the etiology and progression of Alzheimer's disease (AD). We recently showed that the carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) prevents amyloid β (Aβ)-mediated onset of apoptosis in the mouse brain. In this study, we used MTZ and, for the first time, the analog CAI acetazolamide (ATZ) in neuronal and cerebral vascular cells challenged with Aβ, to clarify their protective effects and mitochondrial molecular mechanism of action. The CAIs selectively inhibited mitochondrial dysfunction pathways induced by Aβ, without affecting metabolic function. ATZ was effective at concentrations 10 times lower than MTZ. Both MTZ and ATZ prevented mitochondrial membrane depolarization and H₂ O₂ generation, with no effects on intracellular pH or ATP production. Importantly, the drugs did not primarily affect calcium homeostasis. This work suggests a new role for carbonic anhydrases (CAs) in the Aβ-induced mitochondrial toxicity associated with AD and cerebral amyloid angiopathy (CAA), and paves the way to AD clinical trials for CAIs, FDA-approved drugs with a well-known profile of brain delivery.

The mitochondrial calcium uniporter (MCU) mediates high-capacity mitochondrial calcium uptake that stimulates energy production. However, excessive MCU activity can cause ischemic heart injury. To examine if the MCU is also involved in hypoxic/ischemic (HI) brain injury, we have generated conditional MCU knockout mice by tamoxifen (TMX) administration to adult MCU-floxed (MCU^fl/fl) mice expressing a construct encoding Thy1-cre/ERT2-eYFP. Relative to TMX/Thy1-cre/ERT2-eYFP controls, HI-induced sensorimotor deficits, forebrain neuron loss and mitochondrial damage were decreased for conditional MCU knockout mice. MCU knockdown by siRNA-induced silencing in cortical neuron cultures also reduced cell death and mitochondrial respiratory deficits following oxygen-glucose deprivation. Furthermore, MCU silencing did not produce metabolic abnormalities in cortical neurons observed previously for global MCU nulls that increased reliance on glycolysis for energy production. Based on these findings, we propose that brain-penetrant MCU inhibitors have strong potential to be well-tolerated and highly-efficacious neuroprotectants for the acute management of ischemic stroke.

The effects of global mitochondrial calcium (Ca2+) uniporter (MCU) deficiency on hypoxic-ischemic (HI) brain injury, neuronal Ca2+ handling, bioenergetics and hypoxic preconditioning (HPC) were examined. Forebrain mitochondria isolated from global MCU nulls displayed markedly reduced Ca2+ uptake and Ca2+-induced opening of the membrane permeability transition pore. Despite evidence that these effects should be neuroprotective, global MCU nulls and wild-type (WT) mice suffered comparable HI brain damage. Energetic stress enhanced glycolysis and depressed Complex I activity in global MCU null, relative to WT, cortical neurons. HI reduced forebrain NADH levels more in global MCU nulls than WT mice suggesting that increased glycolytic consumption of NADH suppressed Complex I activity. Compared to WT neurons, pyruvate dehydrogenase (PDH) was hyper-phosphorylated in MCU nulls at several sites that lower the supply of substrates for the tricarboxylic acid cycle. Elevation of cytosolic Ca2+ with glutamate or ionomycin decreased PDH phosphorylation in MCU null neurons suggesting the use of alternative mitochondrial Ca2+ transport. Under basal conditions, global MCU nulls showed similar increases of Ca2+ handling genes in the hippocampus as WT mice subjected to HPC. We propose that long-term adaptations, common to HPC, in global MCU nulls compromise resistance to HI brain injury and disrupt HPC.

Mitochondrial dysfunction has been recognized as a prominent feature of the diminishing cell function in aging bone. Studies of the long-lived growth hormone receptor knockout (GHRKO) mice, which show compromised skeletal growth, have suggested increased mitochondrial biogenesis and function in tissues, such as kidney, heart, and skin fibroblasts. The roles of GHR in maintaining mitochondrial function in osteocytes, the predominant population of bone cells, were not studied. Our goal was to understand the mechanisms by which GHRKO affects mitochondrial volume and function in the adult and aged bones. Using primary osteocyte cultures from 8 weeks old control and GHRKO mice we found that unlike in kidney and heart, in bone tissue of GHRKO mice despite ~40% decreases in osteocyte volume, mitochondrial volume remained the same at the level of ~20% of cell volume. Mitochondrial membrane potential (MMP) is a critical functional measure of mitochondria. Using tetramethylrhodamine ethyl ester, a potentiometric fluorescent probe, we found ~10% decreases in MMP in osteocytes from GHRKO when compared to osteocytes from control mice, suggesting decreased mitochondrial function with ablation of GHR. To further investigate the changes in mitochondrial function we measured the redox state of the mitochondrial NAD+/NADH pair. We found that GHRKO osteocytes show reduced NADH redox index compared to controls, indicating that the overall levels of NADH in the cell reduced. This suggests reduced TCA cycle activity. The impaired mitochondrial function was further confirmed by assay of cellular respiration. We found that GHRKO osteocytes show reduced oxygen consumption rate as compared to controls, and reduced mitochondrial reserve capacity. This data together with decreased MMP and NADH levels, establish that mitochondrial function is compromised in the absence of GHR in osteocytes