17877
IGF-1 and its Analogs: Restoration of Biological Deficits in mouse models of Fragile X and Rett Syndromes
Objectives: We conducted experiments to characterize the effects of NNZ-2566 in vitro and in vivo in a model of Fragile X Syndrome (fmr1 knockout) at doses comparable to those being utilized in Phase II clinical trials of Rett Syndrome (ClinicalTrials.gov ID: NCT00299312) and Fragile X Syndrome (ClinicalTrials.gov ID: NCT01894958).
Methods: Fmr1 KO and wild-type mice (C57BL/6J background) were dosed with either vehicle or NNZ-2566 (100 mg/kg i.p.) 1/day, starting at 14 weeks of age, for 28 days. Behavioral, anatomic and molecular effects were assessed following treatment. Western blot analysis was conducted on extracellular-signal-regulated kinase (ERK), and Akt from wild-type and fmr1 KO mouse brain (obtained ex vivo, following 28 day treatment with NNZ-2566 or vehicle). Dissociated hippocampal cells were plated in 15 mm multi-well vessels and a plating medium of MEM-Eagle’s salts (glutamine free) was supplemented with 10% FBS. After 3 days (culture conditions: 37 °C in humidified 5% CO2), GFP was applied to monitor dendritic spine morphogenesis during culture. Cells were treated with NNZ-2566 (50 nM).
Results: NNZ-2566 normalized differences between fmr1 KO and wild-type mice in all behavioral outcomes assessed and normalized macro-orchidism in the fmr1 KO mice. Dendritic spines usually form between 7 and 14 days in vitro (DIV). By 14 DIV most dendritic protrusions are spines; however, their maturation continues until 21 DIV. Fmr1 KO significantly increased spine density. Increased spine density was reversed by in vitro treatment with NNZ-2566. ERK is a MAPK signal transduction protein, responsible for growth factor transduction, proliferation and cytokine response to stress and apoptosis. Akt is a key component in the PI3K/Akt/mTOR signalling pathway and regulates cellular survival and metabolism by binding and regulating downstream effectors. Fmr1 KO exhibited increased ERK and Akt phosphorylation. This effect was reversed by NNZ-2566. Nrf2 (nuclear erythroid 2-related factor 2) is a leucine-zipper transcription factor, which binds to the antioxidant response elements (ARE) thereby regulating the expression of genes involved in cellular antioxidant and anti-inflammatory defense and mitochondrial protection. Lack of Fmr1 protein leads to an inhibitory effect on Nrf2 by E-cadherin. E-cadherin inhibits the relocation of Nrf2 to the nucleus and prevents Nrf2-dependent gene induction in the brain of Fmr1 KO mice. In vivo treatment with NNZ-2566 exhibits a knockdown effect on E-cadherin normalizing the inducible activity of Nrf2.
Conclusions: NNZ-2566 reverses key molecular and cellular features and normalizes behavioral and anatomic aspects of the of the fmr1 KO phenotype.