Cyfip1 Regulates Presynaptic F-Actin Polymerization and Synaptic Vesicle Release during Development.

Background:  Copy number variations in CYFIP1 have been associated with multiple developmental brain disorders including autism spectrum disorders and schizophrenia. In Angelman and Prader-Willi syndromes chromosome deletions including CYFIP1 have been associated with more severe symptoms than deletions sparing it. CYFIP1 encodes a cytoplasmic protein, Cyfip1 (cytoplasmic FMRP interacting protein), which has two distinct functions:

i) Suppressing mRNA translation initiation.

ii) Promoting the generation of branched F-actin filaments at the plasma membrane as a component of the WAVE regulatory complex (WRC).

Objectives:  While regulation of protein translation and actin polymerization are highly independent processes, they are both essential for the generation of normal synapses. Based on this we asked whether Cyfip1 coordinately regulates both processes when synapses are first forming.

Methods:  To examine the role of Cyfip1 in synapse development, we assayed several parameters of synapse composition and function in hippocampus or in hippocampal neurons cultured from mice haploinsufficient for Cyfip1 and compared the data to wild type littermates (all on a C57BL/6J background). Mechanistic studies were carried out in neurons cultured from rat hippocampus in which Cyfip1 levels were reduced using shRNA and then rescued with full length Cyfip1 resistant to the effects of the knockdown or Cyfip1 mutant constructs. Presynaptic function was assessed in field recordings of Schaeffer collateral synapses in CA1 or in cultured neurons using synapto-pHluorins or FM-dye uptake and release. Distribution of synaptic markers and F-actin were evaluated in fixed sections or in cultured neurons using immunostaining or tagged Phalloidin, respectively, using masks generated in Image J or SynD for image analysis. Differences between groups were compared using Prism. We are currently testing whether F-actin is driven into distinct or alternate patterns when Cyfip1 levels are reduced using super-resolution microscopy. 

Results:  Our data show that reducing levels of Cyfip1 serves to increase presynaptic terminal size and increase release probability in field recordings in mice hippocampal slices. shRNA knockdown of Cyfip1 that is restricted to presynaptic neurons recapitulates these effects in contrast to postsynaptic knockdown, which had no effect. The effects of Cyfip1 knockdown can be rescued by co-expression with a full length Cyfip1 protein or by a Cyfip1 mutant lacking the ability to suppress protein translation. A mutant unable to interact with WRC was unable to rescue the effects on synapses. The data observed also shows that in neurons having reduced levels of Cyfip1, F-actin intensity is greater at presynaptic terminals, but unchanged elsewhere in the neurons.

Conclusions:   Our data support that in developing synapses Cyfip1 regulates synaptic vesicle cluster size and release probability via its participation in the WRC. Reduced Cyfip1 levels lead to a selective increase in the levels of presynaptic F-actin suggesting the recruitment of an alternate or compensatory actin polymerization pathway. Hence, pathway dependent small molecule inhibitors of actin polymerization may ultimately have therapeutic potential.