Very low density lipoprotein receptor regulates dendritic spine formation in a RasGRF1/CaMKII dependent manner - PubMed (original) (raw)

Very low density lipoprotein receptor regulates dendritic spine formation in a RasGRF1/CaMKII dependent manner

Amanda Marie DiBattista et al. Biochim Biophys Acta. 2015 May.

Abstract

Very Low Density Lipoprotein Receptor (VLDLR) is an apolipoprotein E receptor involved in synaptic plasticity, learning, and memory. However, it is unknown how VLDLR can regulate synaptic and cognitive function. In the present study, we found that VLDLR is present at the synapse both pre- and post-synaptically. Overexpression of VLDLR significantly increases, while knockdown of VLDLR decreases, dendritic spine number in primary hippocampal cultures. Additionally, knockdown of VLDLR significantly decreases synaptophysin puncta number while differentially regulating cell surface and total levels of glutamate receptor subunits. To identify the mechanism by which VLDLR induces these synaptic effects, we investigated whether VLDLR affects dendritic spine formation through the Ras signaling pathway, which is involved in spinogenesis and neurodegeneration. Interestingly, we found that VLDLR interacts with RasGRF1, a Ras effector, and knockdown of RasGRF1 blocks the effect of VLDLR on spinogenesis. Moreover, we found that VLDLR did not rescue the deficits induced by the absence of Ras signaling proteins CaMKIIα or CaMKIIβ. Taken together, our results suggest that VLDLR requires RasGRF1/CaMKII to alter dendritic spine formation.

Keywords: Alzheimer's disease; ApoE receptor; CaMKII; Dendritic spine; Ras; VLDLR.

Copyright © 2015 Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1

VLDLR is expressed at synapses. (A) Primary hippocampal neurons (DIV 21) were co-immunostained with Synaptophysin (red) and VLDLR (green, IIII antibody, upper panels) or PSD-95 (red) and VLDLR (green, IIII antibody, lower panels). (B) Immunoblot following synaptosomal fractionation for VLDLR, CAMKIIα, CAMKIIβ, RasGRF1, Synaptophysin and PSD-95 in pre-synaptic vesicles (SV) and post-synaptic density (PSD) fractions. (D) Immunoblot comparing VLDLR levels in wild-type (WT) and VLDLR knockout (KO) tissue using VLDLR antibodies α-IIII (left), α-5F3 (middle), α-74 (right), and β-Actin as a loading control.

Fig. 2

Fig. 2

VLDLR promotes dendritic spine density in primary hippocampal neurons. (A, C) Primary hippocampal neurons were transfected with GFP + Vector (n=12), GFP + VLDLR (n=10), GFP + PLL (control vector for shRNA, n=5), or GFP + VLDLR shRNA (n=4), for 3 days. Cells (DIV 14) were then fixed, immunostained for GFP, and dendritic spines were counted on primary dendrites. (B, D) Quantification of A and C. (E, G) Hippocampal neurons (DIV18) were transfected with GFP + Vector (n=10), GFP + VLDLR (n= 8), GFP + PLL (n=8), or GFP + VLDLR shRNA (n=8) for 3 days. Cells (DIV 21) were then fixed, immunostained with GFP, and dendritic spines were counted on primary dendrites. (F, H) Quantification of E and G (*p<0.05, ***p<0.001). (I) COS7 cells were co-transfected with rodent VLDLR and VLDLR shRNA #1 or VLDLR shRNA #3 or control PLL vector. VLDLR in cell lysates was measured with antibody IIII. (J) A representative image of hippocampal neurons (DIV 21) transfected with empty vector or VLDLR shRNA(#3) immunostained for VLDLR. (K) Schematic of the different deletion constructs for VLDLR. (L) Primary hippocampal neurons (DIV18) were transfected with GFP + Vector (n=8), GFP + VLDLR construct #1 (lacking the ligand binding domain of VLDLR, n=8), GFP + VLDLR construct #2 (lacking the extracellular domain of VLDLR, n=5), or GFP + VLDLR construct #3 (full length VLDLR, n=9). Dendritic spines were analyzed and quantified (**p <0.01). Error bars represented as S.E.M.

Fig. 3

Fig. 3

Knockdown of VLDLR significantly decreases synaptophysin puncta number. (A–B) Mouse brain lysates were immunoprecipitated with IgG or VLDLR (IIII) followed by an immunoblot with synaptophysin (A) and PSD-95 (B). (C) Primary hippocampal neurons (DIV12) were transfected with GFP + PLL or GFP + VLDLR shRNA for 3 days. Cells were then fixed and immunostained with Synaptophysin (left) or PSD-95 (right). (D) Quantification of synaptophysin puncta number in C (PLL n=6 neurons, VLDLR shRNA n=7 neurons, *p<0.05). (E) Quantification of PSD-95 puncta number in C (PLL n=6 neurons, VLDLR shRNA n=8 neurons) (F) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 3 days. Cells were then fixed and immunostained with Synaptophysin (left) or PSD-95 (right). (G) Quantification of synaptophysin puncta number in F (PLL n=5 neurons, VLDLR shRNA n=7 neurons, *p<0.05). (H) Quantification of PSD-95 puncta number in F (PLL n=8 neurons, VLDLR shRNA n=8 neurons).

Fig. 4

Fig. 4

Knockdown of VLDLR decreases GluN1 levels. (A–C) Mouse brain lysates were immunoprecipitated with IgG or VLDLR (IIII) followed by immunoblot with GluN1 (A), GluN2A (B), and GluN2B (C). Arrows indicate specific bands at the expected molecular weight. (D) Hippocampal neurons (DIV18) were transfected with GFP + Vector (n= 11 neurons) or GFP + VLDLR (n=11 neurons) for 3 days. Cells were then fixed and immunostained for GluN1. Representative images for each condition are shown. (E) Quantification of GluN1 integrated intensity in D. (F) Hippocampal neurons (DIV18) were transfected with GFP + PLL (n=7 neurons) or GFP+VLDLR shRNA (n=8 neurons) for 3 days. Cells were then fixed and immunostained for GluN1. Representative images for each condition are shown. (G) Quantification of GluN1 integrated intensity in F (**p<0.01). (H–K) Hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 3 days. Cells were fixed and immunostained for GluN2A or GluN2B. (I) Quantification of GluN2A integrated intensity in H (n=7–16 neurons/group). (K) Quantification of GluN2B integrated intensity in J (n=8–18 neurons/group).

Fig. 5

Fig. 5

Knockdown of VLDLR decreases cell surface GluA1 levels. (A, B) Mouse brain lysates were immunoprecipitated with IgG or VLDLR (IIII) followed by immunoblot with GluA1 (A) and GluA2 (B). Arrows indicate specific bands at the expected molecular weight. (C–F) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 3 days. Cells were then fixed in a non-permeabilizing and permeabilizing condition to detect for cell surface (C–D, PLL n=10 neurons, VLDLR shRNA n=8 neurons) and total levels (E–F, PLL n=9 neurons, VLDLR shRNA n=9 neurons) of GluA1. (*p<0.05) (G–J) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 3 days. Cells were then fixed in a non-permeabilizing and permeabilizing condition to detect for cell surface levels of GluA2 (G–H, PLL n=10 neurons, VLDLR shRNA n=12 neurons) and total levels of GluA2 (I–J, PLL n=9 neurons, VLDLR shRNA n=9 neurons).

Fig. 6

Fig. 6

VLDLR requires RasGRF1 to mediate dendritic spine density (A) Mouse brain lysates were immunoprecipitated with IgG or VLDLR (IIII) followed by immunoblot with RasGRF1. Arrow indicates specific band at the expected molecular weight. (B) COS7 cells were transfected with VLDLR + Vector or VLDLR + RasGRF1, and cell surface biotinylation was performed to measure cell surface levels of VLDLR (n=3/group). (C) Primary hippocampal neurons (DIV18) were transfected with GFP + Vector or GFP+ VLDLR for 72 hours. Cells were then fixed and immunostained for RasGRF1. Representative images from each condition are shown. (D) Quantification of RasGRF1 integrated intensity from C (n=7–10 neurons/group). (E) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 72 hours and cells were then fixed and immunostained for RasGRF1. (F) Quantification of RasGRF1 integrated intensity from E (n=5–11 neurons/group). (G) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL+ empty vector (n=9 neurons), GFP + VLDLR + vector (n=10 neurons), GFP + RasGRF1 shRNA + vector (n=11 neurons), or GFP + RasGRF1 shRNA + VLDLR (n=10 neurons) for 72 hours. After 72 hours, cells were fixed and immunostained for GFP and dendritic spine density was measured. (H) Dendritic spines visualized by GFP immunostaining under the conditions in G were quantified (*p<0.05). (I) A GST-pulldown assay was conducted to measure the levels of active Ras from wild-type and VLDLR knockout brains (n=3/group).

Fig. 7

Fig. 7

VLDLR interacts with CaMKIIα and requires both CaMKIIα and CaMKIIβ to alter dendritic spine formation (A) Primary cortical neurons and mouse brain lysates were immunoprecipitated with IgG or VLDLR (IIII) followed by immunoblot with CaMKIIα, p-CaMKIIα, or CaMKIIβ. Arrows indicate specific bands at the expected molecular weights. (B) COS7 cells were transfected with VLDLR + Vector, VLDLR + CaMKIIα WT, VLDLR + CaMKIIα T286D (non-autophosphorylatable form), or VLDLR + CaMKIIα K42R (kinase dead form), and cell surface biotinylation was performed to measure surface levels of VLDLR (n=2/group). (C) Primary hippocampal neurons (DIV18) were transfected with GFP + Vector or GFP + VLDLR for 72 hours. Cells were then immunostained for CaMKIIα. (D) Quantification of CaMKIIα levels in C (n=7–13 neurons/group). (E) Primary hippocampal neurons (DIV18) were transfected with GFP+PLL or GFP+VLDLR shRNA for 72 hours. Cells were then immunostained for CaMKIIα. (F) Quantification of CaMKIIα levels in E (n=7 neurons/group, *p<0.05). (G) Primary hippocampal neurons (DIV18) were transfected with GFP + Vector +PLL (n=12 neurons), GFP + VLDLR + Vector (n=11 neurons), GFP + CaMKIIα shRNA +Vector (n=7 neurons), or GFP + VLDLR + CaMKIIα shRNA (n=11 neurons) for 72 hours. Cells were then immunostained for GFP to visualize dendritic spines. Representative images are shown. (H) Quantification of dendritic spine density under the conditions in G (*p<0.05). (I) Primary hippocampal neurons (DIV18) were transfected with GFP + PLL or GFP + VLDLR shRNA for 72 hours. Cells were then immunostained for CaMKIIβ. Quantification of CaMKIIβ levels (n= 12–17 neurons/group, *p<0.05). (J) Primary hippocampal neurons (DIV18) were transfected with GFP + Vector + PLL (n=12 neurons), GFP + VLDLR + Vector (n=11 neurons), GFP + CaMKIIβ shRNA +Vector (n=10 neurons), or GFP + VLDLR + CaMKIIβ shRNA (n=10 neurons) for 72 hours. Cells were then immunostained for GFP to visualize dendritic spines and quantification of dendritic spine density (*p<0.05).

Fig. 8

Fig. 8

Reelin alters the interaction between VLDLR and its new interaction partners. (A) Primary hippocampal neurons (DIV21) were treated with control or Reelin (50 ng/ml). Following 1 hour treatment, cell lysates were immunoprecipitated with IIII or IgG and probed for GluA1 (upper panel), RasGRF1 (middle panel), CamKIIα (lower panel). As an additional negative control, brain lysate from VLDLR knock out (−/−) were also immunoprecipitated with IIII, and loaded in the first lane of the western. (B–C) A working model for how VLDLR can promote dendritic spine formation. VLDLR (light gray rectangle) can be expressed pre- or post-synaptically. The ligand binding domain of VLDLR is necessary for the VLDLR-mediated increase in dendritic spines. A recent study demonstrated that the ligands for VLDLR can promote homo- and hetero-clustering with itself or another receptor, respectively. Therefore, it is possible that these ligands can help promote the (B) homo- or (C) hetero- clustering of VLDLR cis- or trans-synaptically. This clustering would serve as an amplification of receptor signaling, which downstream impacts the interaction of CaMKII and RasGRF1 to VLDLR, which subsequently leads to the increase in dendritic spine number.

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