P4‐218: q134r: novel small chemical compound with nfat inhibitory properties ameliorates synaptic deficits in a mouse model of alzheimer's disease

Calcineurin (CN) and the CN-dependent transcription factor, NFAT, exhibit increased activity at early stages of Alzheimer's disease (AD). In AD mouse models, commercial CN inhibitors typically exhibit anti-inflammatory, anti-amyloid, neuroprotective, and/or nootropic properties. Genetic inhibition of NFATs in AD mouse models has similar beneficial effects, suggesting that the reduction of NFAT activity alone is sufficient for clinical efficacy. Here, we tested the NFAT-inhibiting properties and synapto-modulatory effects of the novel chemical compound, Q134R, developed by Avidin Biotechnology. Q134R is safe and well-tolerated in humans and provides neuroprotection in experimental models. In the present study, CN and NFAT-inhibitory properties were tested in primary astrocytes, while target validity and efficacy were tested in APP/PS1 transgenic mice. Cell culture studies —NFAT activation in primary astrocytes was elicited by Ca2+-mobilizing agents or by inflammatory mediators (e.g. IL-1β) in the presence/absence of Q134R or the CN inhibitor, cyclosporine. To assess target specificity, the CN-dependent dephosphorylation of non-NFAT substrates was also tested under similar activating/inhibiting conditions. Intact mouse studies --- Starting at six-months-of-age, WT and APP/PS1 mice received vehicle or Q134R (4mg/kg) twice daily (via oral gavage) for three months. At endpoint, nuclear localization of NFAT4 in hippocampal astrocytes was assessed using confocal microscopy. Basal CA3-CA1 synaptic strength and long-term potentiation (LTP) were assessed in acutely-prepared brain slices using electrophysiology. In primary astrocytes, Q134R significantly inhibited NFAT activation in a concentration-dependent manner, but did not inhibit the CN-dependent dephosphorylation of non-NFAT substrates (e.g. Cx43). Similarly, Q134R had no effect on CN phosphatase activity in a cell-free assay. In intact mice, Q134R inhibited the nuclear translocation of NFAT4 in hippocampal astrocytes while causing an overall drop in GFAP volume. Q134R also significantly increased CA3-CA1 synaptic strength and LTP in APP/PS1 mice. In fact, synaptic indices in WT and Q134R-treated APP/PS1 mice were qualitatively and quantitatively similar. The results demonstrate that Q134R inhibits hyperactive NFAT signaling en route to protecting synaptic function during the progression of AD-like pathology. The findings offer important proof-of-concept support for the use of small chemical NFAT inhibitors, like Q134R, in the treatment of AD and related neurodegenerative disorders.