Mesenchymal stem cell energy deficit and oxidative stress contribute to osteopenia in the Pahenu2 classical PKU mouse

Osteopenia occurs in a subset of phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) patients. While osteopenia is not fully penetrant in patients, the Pah^enu2 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pah^enu2 Phe management, with a Phe-fee amino acid defined diet, does not improve bone density as histomorphometry metrics remain indistinguishable from untreated animals. Previously, we demonstrated Pah^enu2 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pah^enu2 MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pah^enu2 MSCs identified mitochondrial stress by increased basal respiration with concurrently reduced maximal respiration and respiratory reserve. Proton leak secondary to mitochondrial complex 1 dysfunction is a recognized superoxide source. Respirometry applied to Pah^enu2 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pah^enu2 MSCs treated with the antioxidant resveratrol demonstrated increased mitochondrial mass by MitoTracker green labeling. In hyperphenylalaninemic conditions, resveratrol increased in situ alkaline phosphatase activity suggesting partial recovery of Pah^enu2 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pah^enu2 MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pah^enu2 MSC differentiation in the osteoblast lineage to subsequently increase bone density.