Post-weaning social isolation increases ΔFosB/FosB protein expression in sex-specific patterns in the prelimbic/infralimbic cortex and hippocampus in mice.

Social isolation is a growing public health concern across the lifespan. Specifically, isolation early in life, during critical periods of brain development, increases the risk of psychiatric disorders later in life. Previous studies of isolation models in mice have shown distinct neurological abnormalities in various regions of the brain, but the mechanism linking the experience of isolation to these phenotypes is unclear. In this study, we show that ΔFosB, a long-lived transcription factor associated with neuronal activity, chronic stress, and drug-induced neuroplasticity, is upregulated in the prelimbic/infralimbic (PL/IL) region of the cortex and hippocampus of adult C57BL/6J mice transiently isolated for two weeks post-weaning. Additionally, a related transcription factor, FosB, is also increased in the PL/IL in socially isolated females.In contrast, both ΔFosB and FosB are increased in male mice isolated for six weeks from weaning until tissue collection. These results show that short-term isolation during the critical post-weaning period has long-lasting and sex-dependent effects on gene expression in brain and that FosB/ΔFosB expression provides a potential mechanistic link between post-weaning social isolation and associated neurological abnormalities.

Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol O-Methyltransferase.

A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies, levels of unbound drug exposure are seen in the brain, which exceed their respective IC50s, leading to changes in the levels of dopamine metabolites in a manner consistent with COMT inhibition.

Active mitochondria support osteogenic differentiation by stimulating ß-catenin acetylation.

Bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) can differentiate into osteoblasts (OBs), adipocytes, or chondrocytes. As BMSCs undergo OB differentiation, they up-regulate mitochondrial oxidative phosphorylation (OxPhos). Here, we investigated the mechanism(s) connecting mitochondrial OxPhos to OB differentiation. First, we found that treating BMSC-like C3H10T1/2 cells with an OxPhos inhibitor reduces their osteogenic potential. Interestingly, ATP levels were not reduced, as glycolysis compensated for the decreased OxPhos. Thus, mitochondria support OB differentiation not only by supplying ATP, but also by other mechanisms. To uncover these mechanisms, we stimulated OxPhos in C3H10T1/2 cells by replacing media glucose with galactose and observed that this substitution increases both OxPhos and osteogenesis even in the absence of osteoinducers. ß-Catenin, an important signaling pathway in osteogenesis, was found to be responsive to OxPhos stimulation. ß-Catenin activity is maintained by acetylation, and mitochondria generate the acetyl donor acetyl-CoA, which upon entering the Krebs cycle is converted to citrate capable of exiting mitochondria. Cytosolic citrate is converted back to acetyl-CoA by ATP citrate lyase (ACLY). We found that inhibiting ACLY with SB204990 (SB) reverses the galactose-induced ß-catenin activity and OB differentiation. This suggested that acetylation is involved in ß-catenin activation after forced OxPhos stimulation, and using immunoprecipitation, we indeed detected SB-sensitive ß-catenin acetylation. Both ß-catenin acetylation and activity increased during osteoinduction coincident with OxPhos activation. These findings suggest that active mitochondria support OB differentiation by promoting ß-catenin acetylation and thus activity.

Cyclophilin D Knock-Out Mice Show Enhanced Resistance to Osteoporosis and to Metabolic Changes Observed in Aging Bone.

Pathogenic factors associated with aging, such as oxidative stress and hormone depletion converge on mitochondria and impair their function via opening of the mitochondrial permeability transition pore (MPTP). The MPTP is a large non-selective pore regulated by cyclophilin D (CypD) that disrupts mitochondrial membrane integrity. MPTP involvement has been firmly established in degenerative processes in heart, brain, and muscle. Bone has high energy demands and is therefore expected to be highly sensitive to mitochondrial dysfunction. Despite this, the role of mitochondria and the MPTP in bone maintenance and bone pathology has not been elucidated. Our goal was to determine whether mitochondria are impaired in aging bone and to see if protecting mitochondria from MPTP opening via CypD deletion protects against bone loss. We found that bone mass, strength, and formation progressively decline over the course of 18 months in C57BL/6J mice. Using metabolomics and electron microscopy, we determined that oxidative metabolism is impaired in aging bone leading to a glycolytic shift, imbalance in nucleotides, and decreased NAD+/NADH ratio. Mitochondria in osteocytes appear swollen which is a major marker of MPTP opening. CypD deletion by CypD knockout mouse model (CypD KO) protects against bone loss in 13- and 18-month-old mice and prevents decline in bone formation and mitochondrial changes observed in wild type C57BL/6J mice. Together, these data demonstrate that mitochondria are impaired in aging bone and that CypD deletion protects against this impairment to prevent bone loss. This implicates CypD-regulated MPTP and mitochondrial dysfunction in the impairment of bone cells and in aging-related bone loss. Our findings suggest mitochondrial metabolism as a new target for bone therapeutics and inhibition of CypD as a novel strategy against bone loss.

Energy Metabolism in Mesenchymal Stem Cells During Osteogenic Differentiation.

There is emerging interest in stem cell energy metabolism and its effect on differentiation. Bioenergetic changes in differentiating bone marrow mesenchymal stem cells (MSCs) are poorly understood and were the focus of our study. Using bioenergetic profiling and transcriptomics, we have established that MSCs activate the mitochondrial process of oxidative phosphorylation (OxPhos) during osteogenic differentiation, but they maintain levels of glycolysis similar to undifferentiated cells. Consistent with their glycolytic phenotype, undifferentiated MSCs have high levels of hypoxia-inducible factor 1 (HIF-1). Osteogenically induced MSCs downregulate HIF-1 and this downregulation is required for activation of OxPhos. In summary, our work provides important insights on MSC bioenergetics and proposes a HIF-based mechanism of regulation of mitochondrial OxPhos in MSCs.