Insulin receptor dysfunction impairs cellular clearance of neurotoxic oligomeric a{beta}

Abstract

Accumulation of amyloid beta (Abeta) oligomers in the brain is toxic to synapses and may play an important role in memory loss in Alzheimer disease. However, how these toxins are built up in the brain is not understood. In this study we investigate whether impairments of insulin and insulin-like growth factor-1 (IGF-1) receptors play a role in aggregation of Abeta. Using primary neuronal culture and immortal cell line models, we show that expression of normal insulin or IGF-1 receptors confers cells with abilities to reduce exogenously applied Abeta oligomers (also known as ADDLs) to monomers. In contrast, transfection of malfunctioning human insulin receptor mutants, identified originally from patient with insulin resistance syndrome, or inhibition of insulin and IGF-1 receptors via pharmacological reagents increases ADDL levels by exacerbating their aggregation. In healthy cells, activation of insulin and IGF-1 receptor reduces the extracellular ADDLs applied to cells via seemingly the insulin-degrading enzyme activity. Although insulin triggers ADDL internalization, IGF-1 appears to keep ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDL-induced surface insulin receptor loss. Our results suggest that dysfunctions of brain insulin and IGF-1 receptors contribute to Abeta aggregation and subsequent synaptic loss.

FIGURE 1.

Insulin promotes redistribution of ADDLs. A, cultured rat primary cortical neurons (14 days in vitro) were treated with 100 nm ADDLs for 30 min in the presence or absence of 100 nm insulin, which was added to neurons either simultaneously with ADDLs (30 min) or 5 min prior to termination of the reaction. To inhibit IR tyrosine kinase activity neurons were treated with AG1024 prior to ADDL and insulin addition. At the end of the reaction, ADDL levels from the extracellular medium from each treatment were measured on dot blots with specific ADDL antibody NU2. B, time course of ADDL degradation in rat cortical neurons (14 days in vitro) with and without insulin. B-1, ADDL levels from the medium; B-2, ADDL levels from the cell lysate; C, NIH3T3 cells with (3T3-IR (+)) and without (3T3-IR (−)) overexpression of IR treated with ADDLs as described in A. **, p < 0.001.

FIGURE 2.

IR mutations prevent ADDL degradation. The human full-length wild-type IR (hIR-wt), or two IR mutations (IR-1153IIe and IR1030Ala) were transient transfected to primary hippocampal neurons or NIH3T3 cells. Cells were then treated with biotin-labeled ADDLs (bADDLs) in the presence or absence of insulin. The media were collected and concentrated. The cell lysates were prepared with a lysates buffer. Both the extracellular and the cell-attached bADDLs were measured with dot blots using NU1. A, the insulin-stimulated IR tyrosine phosphorylation from NIH3T3 cells transfected with huIR-wt and mutated human IR. Only the huIR-wt showed positive insulin-induced tyrosine phosphorylation on the β-subunit. B, bADDL digestions in the medium were inhibited in the IR mutants-transfected NIH 3T3 cells. C-1, bADDL digestions in the medium were inhibited in the IR mutants-transfected hippocampal neurons. C-2, insulin-induced translocation of ADDLs were not seen in the IR mutant-transfected hippocampal cells. **, p < 0.001, n = 3. C-3, transfected neurons were treated with bADDLs. The concentrated medium from different conditions was pulled down by streptavidin covalently immobilized to agarose resin and detected on Western blots with 6E10. bADDL species were increased in the medium of primary neurons transfected with IR mutants.

FIGURE 3.

IGF-1 stimulates the degradation of extracellular ADDLs. Cultured rat primary cortical neurons (A) or NIH3T3 cells overexpressing IGF-1R (B) were treated with 100 nm ADDLs for 30 min in the presence or absence of 100 nm IGF-1. AG1024 was used to inhibit IGF-1R tyrosine kinase activity. Upon termination of the reaction, ADDL levels from the extracellular medium from each treatment were measured on dot blots as described in Fig. 1. 3T3-IGF-1R: NIH3T3 cells overexpressing IGF-1R. **, p < 0.001, n = 4.

FIGURE 4.

AG1024 increases the aggregation of extracellular ADDLs. Cultured rat primary cortical neurons were treated with ADDLs, insulin and IGF-1 with or without preincubation with AG1024. In addition, a negative IGF-1R antibody was added to neurons for 15 min to inhibit the IGF-1R activity. A, the extracellular medium from each condition was concentrated. B, the cell lysates from the same conditions were immunoprecipitated (IP) with the 6E10 antibody. C, NIH3T3 cells with or without expression of IR or IGF-1R treated in similar conditions and the medium processed as in A. All samples were resolved on SDS-PAGE. The species of ADDLs were then assessed on Western blots with 6E10.

FIGURE 5.

Insulin induces ADDL internalization in NIH3T3-IR(+) cells. A, NIH3T3-IR (−) cells were treated with bADDLs or vehicle followed by double immunostaining. These cells show negative IR and bADDL staining. B, confocal microscopy of NIH3T3-IR (+) cells treated with VEH and ADDLs in the presence or absence of insulin. The parent NIH3T3 cells showed little ADDL binding (1), whereas clear ADDL binding was observed in the IR-expressing cells (2). Insulin stimulated internalization of both IR and ADDLs to the nuclear envelop and nucleus (3 and 4, pointed by arrows). C, comparison of ADDL uptake in NIH3T3-IR (+) and NIH3T3-IGF-1R (+) Cells. Cells were treated with VEH and ADDLs in the presence of insulin, or AG1024. The immunostaining for both ADDLs and IR was observed with an epifluorescence microscope. While insulin stimulates uptake of ADDLs to the nucleus (arrows) of the IR-expressing cells (4, upper panel), it retains ADDLs on the membrane surface of the IGF-1R-expressing cells (4, lower panel). In the presence of AG1024, the ADDL internalization in both type of cells was blocked (5).

FIGURE 6.

Insulin-induced ADDL internalization in neurons is accompanied by reduction in dendritic binding. Cultured hippocampal neurons were treated with 200 nm ADDLs for 60 min, with 100 nm insulin added either simultaneously, or 10 min prior to termination of the reaction. ADDL binding was detected with NU-1(A, arrows point to neuronal boundaries), or 6E10 (B-1). Neurons were stained with anti-GAP43 antibody or cholera toxin B. B-2, NU-1 detected dendritic ADDL binding from 10–20 neurons from each treatment was quantified with ImageJ and analyzed in one-way analysis of variance. Veh, vehicle-treated; ChTX, cholera toxin B. *, p < 0.05; **, p < 0.001. C, IR immunoreactivity in dendritic process of rat hippocampal neurons. D, IR is expressed in both neuronal and non-neuronal cells. D-1, IR was seen in the interior of dendrites bound with bADDLs. D-2, IR was lost in the bADDL-bound dendrites. E, internalization of ADDLs and bADDLs in astrocytes is enhanced by insulin.

FIGURE 7.

Insulin prevents ADDL-induced spine loss. A, drebrin labeling of mature hippocampal cells treated with vehicle (VEH, equivalent volume to 500 nm ADDLs) or soluble Aβ oligomers (ADDLs, 500 nm) in combination or not with 0. 1 μm IGF-1 (IGF-1 0.1), 1 mm IGF-1 (IGF-1 1), and 1 μm insulin (INS 1) for 24 h. Images of ADDL-bound neurons were captured on a confocal microscope and quantified with Metamorph. A-1, representative images of drebrin immunolabeling obtained in the different treatment groups. A-2, bar graph illustrating the dendritic spine density obtained from drebrin immunofluorescence of confocal image sets as shown in A-1. Data represent the number of drebrin positive puncta after threshold applied and integrated analysis morphometry of selected dendritic regions and are expressed as spine density per length of dendrites with Vehicle serving as control, data are expressed as % over control. Dendrites of neurons treated with ADDLs for 24 h (ADDL) exhibit decreased drebrin immunofluorescence when compared with neurons treated with vehicle for 24 h (Veh). Neurons treated with ADDLs in the presence of 1 μm IGF-1 (AD-IGF-1 1) and 1 μm insulin (AD-INS 1) exhibit drebrin staining comparable to the VEH-treated neurons, demonstrating that IGF-1 and insulin have a protective effect on ADDL-induced drebrin loss. IGF-1 (VEH-IGF-1) and insulin alone (VEH-INS) did not show much effect on dendritic spine density. Differences between VEH versus ADDL as well as differences between ADDL versus ADDL plus IGF-1, or ADDL plus INS are highly significant (***, p < 0.001). B, ADDL-induced loss of surface IR was prevented by insulin and IGF-1: Rat hippocampal neurons were treated with 500 nm ADDLs for 24 h in the presence or absence of 1 μm IGF-1 or insulin. Cells were fixed but without permeabilization. The surface IR was detected with an anti-IRα antibody. IGF-1 and insulin demonstrated complete protection of the surface IR against ADDLs. C, surface biotinylation of IR confirming the protective effects of IGF-1 and IR: Rat hippocampal neurons were treated with 500 nm ADDLs for 1 h. The surface IR was labeled with biotin before isolation with streptavidin immobilized to agarose beads. The isolated surface IR and the total IR from the cell lysates were resolved on SDS-PAGE and detected with an anti-IRα antibody on Western blots. ADDL caused a significant reduction in the surface IR. Insulin and IGF-1 treatment prevent this loss. **, p < 0.01.

FIGURE 8.

Effects of IDE and neprilysin on extra- and intracellular ADDL degradation. Cortical neurons were preincubated with phosphoramidon (0.1 μm) and phenanthroline (2 μm) at 37 °C for 30 min. ADDLs (100 nm) was added to neurons in the presence or absence of 100 nm of insulin or 100 nm IGF-1. After 1-h treatment, the medium and lysates were collected and measured with 20C2–6E10 ELISA. A, extracellular ADDL levels from the medium; B, ADDL levels from the cell lysates. ***, p < 0.001; **, p < 0.01; *, p < 0.05.