(A) Schematic of GRASP1 protein with a GRIP1
binding domain (GBD) and locations of GRASP1
mutations found in severe ID patients.
(B) Sequence alignments of GRASP1 flanking the ID mutations.
(C) Representative images of rat hippocampal neurons and dendritic segments
transfected with GFP and a control vector (Vector ctrl), shRNA for GRASP1
protein knockdown (KD), or KD plus
either GRASP1 WT, S73N, or R822Q with mock treatment. A highlighted dendritic
region (red box) is shown at higher magnification with separate channels to show
GFP, surface GluA2 (sGluA2) and total GluA2/3 (tGluA2/3) staining.
(D) Same as (C) except that neurons were AMPA-treated to induce AMPAR
recycling.
(E) Quantifications of (C). Integrated intensity of sGluA2, normalized to
tGluA2/3 signal, is comparable between all groups. (% Vector control:
Vector ctrl= 99.9 ± 4.3; KD= 102.8 ± 4.5;
KD+WT= 100.1 ± 4.8; KD+S73N= 98.2
± 5.4; KD+R822Q= 110.0 ± 4.6; n=
84–96 dendrites from 21–24 neurons/group; p>0.05;
one-way ANOVA).
(F) Quantifications of (D). Integrated intensity of s/tGluA2(3) were normalized
to vector ctrl at basal state to quantify AMPA-induced AMPAR recycling.
(% Vector ctrl at basal: Vector ctrl= 86.4 ± 4.2 and
KD= 74.0 ± 3.3, p= 0.04; KD+WT= 92.1
± 4.8 and KD, p=0.002; KD+WT and
KD+S73N= 77.4 ± 4.2, p=0.015; KD+WT and
KD+R822Q= 70.3 ± 4.1, p=0.0002; n=
88–100 dendrites from 22–25 neurons/group; one-way ANOVA).