Mesenchymal stem cell energy deficit and oxidative stress contribute to osteopenia in the Pah 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 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pah 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 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pah MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pah 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 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pah 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 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pah MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pah MSC differentiation in the osteoblast lineage to subsequently increase bone density.