Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions

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.

EFA6A, an Exchange Factor for Arf6, RegulatesNGF‐DependentTrkARecycling From Early Endosomes and Neurite Outgrowth inPC12Cells

“…After the PCR fragments were subcloned into pGEM-Teasy (Promega) and sequenced, the inserts were digested with BstXI and XbaI, and subcloned into the BstXI/XbaI-digested pRc/ CMV-HA-TrkA vector. Expression vectors (pCAGGS) encoding Arf6-FLAG and FLAG-EFA6C were described previously [16,57]. Expression vectors (pCAGGS) encoding Arf6 (T27N)-FLAG and Arf6 (Q67L)-FLAG were created using the insert from the plasmids used in the previous yeast two-hybrid assay [58].…”

The small GTPase ARF6 regulates GABAergic synapse development

“…EFA6A, an ARF6-specific GEF, is highly expressed in brains and is critical for dendritic spine development and maintenance [3,12]. Deletion of another EFA6 isoform, EFA6C/Psd2, in mice reduces synaptic density in Purkinje neurons of the cerebellum [13]. Still another ARF6-specific GEF, BRAG1 (synonymous with IQSEC2 [14];), interacts with PSD-95 and some PDZ domain-containing scaffolds through its Cterminal PDZ domain-binding sequence and binds to IRSp53 (also known as BAIAP2) through its proline-rich sequence to form multiprotein complexes at excitatory synapses of postsynaptic neurons [15][16][17].…”