IGF-1 and its Analogs: Restoration of Biological Deficits in mouse models of Fragile X and Rett Syndromes

Friday, May 16, 2014: 1:30 PM
Marquis D (Marriott Marquis Atlanta)
L. Glass1, F. J. Altimiras2, M. Snape3, J. Horrigan1 and P. Cogram4, (1)Neuren Pharmaceuticals, Bethseda, MD, (2)Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciencies, Faculty of Medicine, University of Chile, Santiago, Chile, (3)Autism Therapeutics Ltd, Wonersh, United Kingdom, (4)Oxidative stress, Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciencies, Faculty of Medicine, University of Chile, Santiago, Chile
Background:  IGF-1 and its n-terminal tripeptide ([1-3]IGF-1) are widely distributed in the CNS and exert a range of effects on neurons in the normal state (Corvin et al, 2012; Ramsey et al, 2005).  These neurotrophic factors also have been reported to effect synaptic function and other processes in certain pathological states including neurodevelopmental disorders (Riikonen et al, 2003; Tropea et al, 2009; Landi et al, 2011; Bozdagi et al, 2013) and brain injury (Lu et al, 2009; Rubovitch et al, 2010; Madathil et al, 2010). NNZ-2566 is a synthetic analog of (1-3)IGF-1 with longer half-life and oral availability.

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.