Abstract
ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates various neuronal events including formation of the axon, dendrites and dendritic spines, and synaptic plasticity through actin cytoskeleton remodeling and endosomal trafficking. EFA6C, also known as Psd2, is a guanine nucleotide exchange factor for Arf6 that is preferentially expressed in the cerebellar cortex of adult mice, particularly in Purkinje cells. However, the roles of EFA6C in cerebellar development and functions remain unknown. In this study, we generated global EFA6C knockout (KO) mice using the CRISPR/Cas9 system and investigated their cerebellar phenotypes by histological and behavioral analyses. Histological analyses revealed that EFA6C KO mice exhibited normal gross anatomy of the cerebellar cortex, in terms of the thickness and cellularity of each layer, morphology of Purkinje cells, and distribution patterns of parallel fibers, climbing fibers, and inhibitory synapses. Electron microscopic observation of the cerebellar molecular layer revealed that the density of asymmetric synapses of Purkinje cells was significantly lower in EFA6C KO mice compared with wild-type control mice. However, behavioral analyses using accelerating rotarod and horizontal optokinetic response tests failed to detect any differences in motor coordination, learning or adaptation between the control and EFA6C KO mice. These results suggest that EFA6C plays ancillary roles in cerebellar development and motor functions.
Highlights
ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates actin cytoskeleton remodeling and vesicular transport between the plasma membrane and endosomes [1,2,3]
Among the ten founder mice carrying indels in the EFA6C gene, PCR and sequencing analyses revealed that six founder mice (5 males and 1 females) carried bi-allelic mutations around the target sequence, two mice (2 females) carried a mono-allelic mutation and the remaining two male mice were mosaic carrying more than two mutant alleles (S1 Fig)
Both anti-EFA6C antibodies detected an immunoreactive band around 110 kDa, which was the same electrophoretic mobility as FLAG-EFA6C, in the cerebellar lysates of wild-type and heterozygous mice, with the expression level correlated with the allele number (EFA6C/α-tubulin: WT, 1.0 ± 0.05; Hetero (+/-), 0.62 ± 0.04; KO, 0.02 ± 0.004; EFA6C /α-tubulin: WT, 1.0 ± 0.26; Hetero (+/-), 0.49 ± 0.02; KO, 0.03 ± 0.01, n = 3 of each genotype), whereas the band was undetectable in the those of EFA6C KO mice (Fig 1D)
Summary
ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates actin cytoskeleton remodeling and vesicular transport between the plasma membrane and endosomes [1,2,3]. The EFA6/PSD family comprises EFA6A/PSD1 [16, 17], EFA6B/PSD4 [18], EFA6C/PSD2 [19] and EFA6D/PSD3 [20], which are generated from distinct genes and function primarily as an Arf6-specific GEF [16, 21, 22] They are structurally characterized by a conserved domain organization consisting of a central catalytic Sec domain, an adjacent pleckstrin homology (PH) domain responsible for interaction with the plasma membrane and F-actin [18, 23], and a C-terminal region containing a coiled coil motif that mediates protein-protein interaction and GEF-independent actin cytoskeleton remodeling [16, 17, 24, 25]. The physiological significance of individual EFA6 members in the brain is still unknown at the whole animal level
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