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

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.