Endophilin2 Interacts with GluA1 to Mediate AMPA Receptor Endocytosis Induced by Oligomeric Amyloid- β

Abstract

Amyloid-β (Aβ) plays an important role in Alzheimer's disease (AD), as oligomeric Aβ induces loss of postsynaptic AMPA receptors (AMPARs) leading to cognitive deficits. The loss of postsynaptic AMPARs is mediated through the clathrin-dependent endocytosis pathway, in which endophilin2 is one of the important regulatory proteins. Endophilin2, which is enriched in both the pre- and postsynaptic membrane, has previously been reported to be important for recycling of synaptic vesicles at the presynaptic membrane. However, the role of endophilin2 in oligomeric Aβ-induced postsynaptic AMPAR endocytosis is not well understood. In this study, we show that endophilin2 does not affect constitutive AMPAR endocytosis. Endophilin2 knockdown, but not overexpression, resisted oligomeric Aβ-induced AMPAR dysfunction. Moreover, endophilin2 colocalized and interacted with GluA1, a subunit of AMPAR, to regulate oligomeric Aβ-induced AMPAR endocytosis. Thus, we have determined a role of endophilin2 in oligomeric Aβ-induced postsynaptic AMPAR dysfunction, indicating possible directions for preventing the loss of AMPARs in cognitive impairment and providing evidence for the clinical treatment of AD.

Figure 1

The electrophysiology of neurons with endophilin2 knockdown or overexpression. (a) Image of a neuron obtained from patch recording. Scale bar, 50 μm. (b-c) Bar plots of the mean values of resting membrane potentials and capacitance of patched neurons in the four groups. (d-e) mEPSC tracings are shown in neurons transfected with NC, Endo2 siRNA, GFP, and Endo2-GFP. (f) Histogram plots of mEPSC frequency and amplitude in neurons transfected with NC and Endo2 siRNA. n = 16 cells, 3 cultures from 6 SD rats. (g) Histogram plots of mEPSC frequency and amplitude in neurons transfected with GFP and Endo2-GFP. n = 16 cells, 3 cultures from 6 SD rats.

Figure 2

The electrophysiology of endophilin2 knockdown neurons processed by oligomeric Aβ. (a) mEPSC tracings are shown in control and oligomeric Aβ-treated neurons transfected with NC and Endo2 siRNA. (b) Histogram plots and scatterplots of mEPSC frequency in control and oligomeric Aβ-processed neurons transfected with NC and Endo2 siRNA, n = 17 cells in NC + Tris and Endo2 SiRNA + Aβ groups, n = 16 cells in NC + Aβ and Endo2 SiRNA + Tris groups, 4 cultures from 8 SD rats, ^∗P < 0.05, ^∗∗P < 0.005, and ^#P < 0.005. (c) Histogram plots and scatterplots of mEPSC amplitude in control and in oligomeric Aβ-processed neurons transfected with NC and Endo2 siRNA, n = 17 cells in NC + Tris and Endo2 SiRNA + Aβ groups, n = 16 cells in NC + Aβ and Endo2 SiRNA + Tris groups, 4 cultures from 8 SD rats, ^∗P < 0.05, ^∗∗P < 0.005, and ^#P < 0.005.

Figure 3

The electrophysiology of endophilin2 overexpression in neurons processed by oligomeric Aβ. (a) mEPSC tracings are shown in control and oligomeric Aβ-processed neurons transfected with GFP and Endo2-GFP. (b) Histogram plots and scatterplots of mEPSC frequency in control and oligomeric Aβ-processed neurons transfected with GFP and Endo2-GFP, n = 16 cells, 4 cultures from 8 SD rats, ^∗∗P < 0.005. (c) Histogram plots and scatterplots of mEPSC amplitude in control and oligomeric Aβ-processed neurons transfected with GFP and Endo2-GFP, n = 16 cells, 4 cultures from 8 SD rats, ^∗P < 0.05, ^∗∗P < 0.005.

Figure 4

Endophilin2 localizes to postsynaptic sites in mature neurons. DIV14 hippocampal neurons expressing the green fluorescent protein (GFP) as a volume marker were immunostained with antibodies to endophilin2 (red) and PSD95 (blue). Merge 1 shows the endophilin2 colocalization with PSD95 and merge 2 the three proteins. Arrows indicate the colocalization part as indicated. The images shown are representative confocal microscopy images. Scale bar, 5 μm.

Figure 5

Endophilin2 colocalizes with GluA1. (a) GST-endophilin2 binds to GluA1 not GluA2. GST-tagged endophilin2 fragments were incubated with 1-month-old Sprague Dawley rat brain lysates. Input and bound proteins were analyzed by immunoblotting with antibodies against GluA1 and GluA2. (b) Lysates from 1-month-old Sprague Dawley rat brain were subjected to coimmunoprecipitation with endophilin2 antibody and then subjected to Western blot analysis with GluA1 antibody (left). GST-tagged endophilin2 fragments were incubated with the lysates of HEK293 cells with GluA1-C-flag overexpression (right). (c) Immunostaining of DIV14 hippocampal cultured neurons shows endogenous endophilin2 localized with GluA1. Scale bars are 20 μm and 5 μm in the magnified dendrite. For pulldown assay, we used rat brain lysates, n = 4 from 4 SD rats.

Figure 6

The expression levels of surface GluA1 in endophilin2 knockdown neurons treated by oligomeric Aβ. (a) Hippocampal cultured neurons transfected with NC and Endo2 siRNA immunostained with surface GluA1. Scale bar is 20 μm. (b) The fluorescence intensity of surface GluA1 in cytoplasm transfected with Endo2 siRNA or NC was normalized to neighboring untransfected neurons. n = 10 cells, 3 cultures from 6 SD rats, ^∗∗P < 0.005.