The Mecp2 knockout mouse, strain B6.129P2(C)-Mecp2tm1.1Bird/J (003890), shares many of the disease phenotypes observed in RTT patients. Like the human gene, Mecp2 is located on the X chromosome, so male Mecp2 -/Y mice are complete nulls. Mecp2 -/Y males have numerous neurological abnormalities resulting in hypoactivity and abnormal gate by 3-8 weeks of age. They also manifest weight loss, shivering, and premature death by 50-60 days of age. Affected human males typically do not survive infancy due to a broad range of brain dysfunctions.
Female Mecp2 heterozygous (-/+) mice have less severe phenotypes and live longer than males, largely due to mosaic loss of gene function following random X inactivation. The heterozygous females show hind limb clasping when picked up (normal mice spread their legs), along with diminished mobility by 6 months and abnormal breathing by 9 months. Human female patients have a number of neurological issues that include irregular breathing, impaired learning, decline in motor skills, and dysfunctional social behaviors similar to those observed in autism.
Krishnan et al. made the astute observation that both mouse models of RTT and human RTT patients show a number of metabolic changes, too. These changes include elevated cholesterol in the brain, glucose intolerance, and impaired insulin signaling. The insulin signaling pathway relies on tyrosine phosphorylation of several intracellular proteins, and inhibiting this phosphorylation decreases glucose receptor translocation to the cell surface and prevents glucose uptake from the blood. When the authors examined changes in gene expression in the brains of Mecp2 -/Y male mice, they discovered significantly elevated Ptp1b (Ptpn1) mRNA. Interestingly, fibroblasts from human patients also showed elevated expression of this tyrosine phosphatase. The authors hypothesized that MECP2 acts directly to increase PTP1B expression, causing reduced tyrosine phosphorylation. This was confirmed by using MECP2 to drive expression from a plasmid containing the PTP1B promoter linked to a luciferase reporter in cell cultures. They also examined tyrosine phosphorylation of several proteins in the insulin signaling pathway and confirmed loss or diminished phosphorylation in Mecp2 mutant mice compared to wild type controls.
Given the strong evidence for PTP1B playing a role in RTT, the authors treated Mecp2 -/Y male mice with small molecule inhibitors of PTP1B. The mice showed improved glucose intolerance and weight gain, increased survival, and restored phosphorylation of proteins in the insulin signaling pathway. Female Mecp2 -/+ mice also showed improved glucose intolerance and enhanced insulin signaling, as well as improved motor function, measured by decreased hind limb clasping and longer latency to fall in rotarod trials. These experiments serve as an important step in identifying therapeutic targets for RTT, and show for the first time that neurological defects associated with this disease may be reversible.
While looking for small molecules that increased tyrosine phosphorylation and improved glucose intolerance in Mecp2-deficient mice, the authors also tested several molecules already known to clinically improve glucose homeostasis. These drugs included metformin, rosiglitazone, and phenformin. When tested in Mecp2 -/Y males, glucose tolerance in the mice improved, but not life span. These observations suggested that upregulated PTP1B expression was altering the phosphorylation of additional pathways in RTT in addition to those important in glucose metabolism.
BDNF plays an important role in neurological development, synaptic transmission, and cognitive function. Functional signaling through its receptor, TRKB, relies on tyrosine phosphorylation. When Krishnan et al. examined BDNF expression in the Mecp2 mutant mice they found that the mutant mice maintained 60-70% of BDNF expression compared to wild-type. TRKB tyrosine phosphorylation of TRKB in female Mecp2 -/+ mice, however, was significantly decreased, but could be restored following treatment with the PTP1B small molecule inhibitor. Further, biochemical analysis confirmed that TRKB is a direct substrate of PTP1B. The research reported by Krishnan et al. provides both a better understanding of the mechanisms that underlie RTT and insights into new, potential strategies for treating this devastating disease.