Impact of Cyfip1 Haploinsufficiency on White Matter Microstructure in a Rat Model

Background: The 15q11.2 BP1-BP2 deletion has been associated with learning and motor delays, autism and schizophrenia. This region includes a gene that codes for the cytoplasmic FMRP interacting protein 1 (CYFIP1). CYFIP1 haploinsufficiency is considered to be a likely significant contributor to the 15q11.2 BP1-BP2 deletion psychiatric phenotype due to its known involvement in a number of key brain plasticity-related functions. CYFIP1 gene plays a crucial role in actin remodeling, where dysregulations could result in changes in white matter microstructure by influencing axonal density, organisation, and myelination.

Objectives: Our aim is to identify possible cellular changes underlying white matter effects at 15q11.2 BP1-BP2 region.

Methods: We used a novel CRISPR/Cas9-engineered hemizygous-null Cyfip1 (Cyfip1^+/-) rat line. All the rats used in this experiments were males and 6-8 months old. Diffusion tensor imaging (DTI) data were collected in a cohort of 24 rats (wild-type (WT) n=12, Cyfip1^+/- n=12) and analyzed using Tract-Based Spatial Statistics (TBSS) available in FSL. To investigate the cellular nature of the DTI changes we used transmission electron microscopy (TEM) to measure the number of myelinated and unmyelinated axons, the axon diameter, and the myelin thickness and g-ratio (myelin thickness relative to axon diameter) of each myelinated axon. The experiment used a new cohort of rats (WT n=5, Cyfip1^+/- n=4). We then quantified the number of oligodendrocytes lineage and mature cells using immunofluorescence technique, where sections were stained for Olig2 and Cc1 proteins. This experiment used rats taken randomly from the same group of rats providing the DTI data (WT n=7 and Cyfip1^+/- n=7). Linear mixed effects (LME) models were used to analyze the effect of genotype on axon diameter, g-ratio and myelin thickness, considering variation across animals, whereas a two-tailed unpaired t-test was used to compare the number of axons and number of oligodendrocytes between groups.

Results: A main finding of DTI were significant decreases in fractional anisotropy (FA) in the Cyfip1^+/- rats that were most pronounced in the corpus callosum and external capsule. TEM showed that decreased FA was associated with thinning of the myelin sheath in the corpus callosum (LME: X²(1)=14.63, p<0.001), where no differences were found in the number of unmyelinated (t=0.39, df=5.15, p=0.71) and myelinated (t=-0.63, df=5.72, p=0.55) axons, or in the diameter of the axons (LME: X²(1)=0.05, p=0.83). Immunofluorescence showed a reduction in the number of mature oligodendrocytes (t=2.48, df=11.99, p<0.05) in the corpus callosum of the Cyfip1^+/- rats.

Conclusions: Altogether, our study suggest that haploinsufficiency in the Cyfip1 gene results in changes in white matter microstructure associated with thinning of the myelin sheath. The reduction in oligodendrocyte cells suggests that this might be caused by an insufficient supply of myelinating oligodendrocytes. Insufficient myelination can result in impaired cognitive processing, which can lead to deficits in tasks of higher cognitive demand and contribute to the cognitive deficits seen in 15q11.2 BP1-BP2 deletion carriers.