Abstract
The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4−/− mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1–5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.
Highlights
The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton
Fluorescence in situ hybridization (FISH) for Cdc42ep[4] mRNA highlighted the Purkinje cells (PCs) layer in the cerebellum (Fig. 1c, left), which is consistent with the data in the Allen Mouse Brain Atlas #71723875 (Allen Institute for Brain Science)
At a higher magnification with a lower gain level, CDC42EP4 immunoreactivity appeared as ‘hotspots’, which were interspersed along PC dendrites and tightly apposed to dendritic spines (Fig. 1d, right)
Summary
The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42 or the septin cytoskeleton. Previous studies demonstrated a physical interaction between GLAST and septins (a family of polymerizing GTPases that constitute the membrane skeleton) in vitro and in heterologous cells, and their partial co-localization in Bergmann glia[11,12]. Hypothetical septin dependence of the perisynaptic targeting and activity of GLAST has never been directly tested in vivo, partly because of the redundancy among the septin family (see Discussion) Another line of biochemical and cell biological studies showed that a family of CRIB-domain proteins CDC42EPs/BORGs binds to septin hetero-oligomers or CDC42 (a signalling small GTPase that controls cytoskeleton and cell morphogenesis) in a mutually exclusive manner. The unique and systematic approach reveals the requirement of CDC42EP4 in Bergmann glia for the septin-mediated perisynaptic localization of GLAST, and for the efficient buffering and clearance of glutamate from around synapses towards PCs
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