Cyfip1 Developmentally Regulates Presynaptic Function

Background:  Copy number variations in the gene encoding Cyfip1 are associated with several neurodevelopmental disorders and altered levels of Cyfip1 may correlate with increased disease severity. A common feature of several disorders is defective synapse development or function and work from our labs and others indicates that Cyfip1 plays a role in both normal synapse development and plasticity. Cyfip1 has independent actions as a regulator of protein translation and of actin polymerization, but how changes in Cyfip1 levels could contribute toward or exacerbate neurodevelopmental brain disorders is poorly understood.

Objectives: To test the impact of changing Cyfip1 levels on synapse development and function alone and via its interactions with Fmrp.  

Methods:  In order to test effects of Cyfip1 levels and genetic interaction between Cyfip1 and Fmr1, we crossed heterozygous mice and evaluated presynaptic and postsynaptic function in acute hippocampal slices at different ages from 4 different groups and in neuronal cultures. We applied a combination of FM4-64 imaging, biochemical, immunohistochemical and electrophysiological techniques in mouse tissue, principally focusing on hippocampal area CA1.

Results:  In neurons cultured from Cyfip1^+/- mice, developing synapses showed abnormal presynaptic properties. Assessments of vesicle recycling using FM4-64 uptake and release revealed an increase in recycling pool size and release kinetics than in neurons cultured from wild type littermates. Cyfip1^+/- knockdown in individual neurons indicates that this effect is cell autonomous and generated presynaptically with no detectable impact on the generation of postsynaptic densities (labeled with PSD95). The effect on presynaptic function in cultured neurons is reflected in abnormal paired-pulse ratio measurements of presynaptic function and mEPSCs in hippocampal slices from P10 animals—effects that are absent at mature synapses. At mature synapses, the major impact of Cyfip1 levels is reflected in altered plasticity and reflects at least in part that Cyfip1 partners with Fmrp and regulates protein translation. Our previous results show that mice lacking one functional copy of Cyfip1 exhibit enhanced hippocampal mGluR-LTD in 4-6 week old mice similar to Fmr1 knockout mice. In double mutant mice (Cyfip1^+/-; Fmr1^-/y) mGluR-LTD is enhanced similar to single mutants (a phenotype that is absent in young synapses). However, Fmrp and Cyfip1 levels are not regulated by each other. Double mutant mice also show significantly reduced  HFS (high frequency stimulus)-induced LTP and LFS (low FS)-induced GluN-LTD, which suggests an additive or interactive effect of Fmrp and Cyfip1. To confirm that decreased levels of Cyfip1 influence actin polymerization, we compared polymerized and soluble actin fractions isolated from hippocampal lysates in WT and Cyfip1 heterozygous mice, which showed and increase in the ratio of F- to G-actin in Cyfip1 heterozygous mice.

Conclusions:  These data demonstrate that Cyfip1 reduction has a strong effect on presynaptic function in young neurons, distinct from Fmrp action, but has an interactive effect with Fmrp in the regulation of postsynaptic function in mature synapses.  Altered levels of Cyfip1 may affect the mechanisms underlying a developmentally regulated switch from pre­ to postsynaptic phenotypes and contribute to increased disease severity.