Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling

Abstract

Alzheimer's Disease (AD) is characterized by cerebral accumulation of beta-amyloid peptides (Abeta), which are proteolytically derived from beta-amyloid precursor protein (betaAPP). betaAPP metabolism is highly regulated via various signal transduction systems, e.g., several serine/threonine kinases and phosphatases. Several growth factors known to act via receptor tyrosine kinases also have been demonstrated to regulate sbetaAPP secretion. Among these receptors, insulin and insulin-like growth factor-1 receptors are highly expressed in brain, especially in hippocampus and cortex. Emerging evidence indicates that insulin has important functions in brain regions involved in learning and memory. Here we present evidence that insulin significantly reduces intracellular accumulation of Abeta and that it does so by accelerating betaAPP/Abeta trafficking from the trans-Golgi network, a major cellular site for Abeta generation, to the plasma membrane. Furthermore, insulin increases the extracellular level of Abeta both by promoting its secretion and by inhibiting its degradation via insulin-degrading enzyme. The action of insulin on betaAPP metabolism is mediated via a receptor tyrosine kinase/mitogen-activated protein (MAP) kinase kinase pathway. The results suggest cell biological and signal transduction mechanisms by which insulin modulates betaAPP and Abeta trafficking in neuronal cultures.

Figures

Fig. 1.

Effect of insulin on the extracellular levels of Aβ and sβAPPα in murine neuroblastoma cells (N2a) and primary rat cortical neuronal cultures. Cells were pulse-labeled for 20 min with [35S]methionine and incubated in serum-free medium in the absence or presence of various concentrations of insulin for 4 hr. a, Representative autoradiographic analysis of extracellular Aβ (top) and sβAPPα (bottom) after incubation with or without 300 nm insulin. b, Extracellular levels of Aβ from N2a cells as a function of insulin concentration.c, Extracellular levels of sβAPPα from N2a cells as a function of insulin concentration. For b andc the data represent means ± SD;n = 3. d, e, IP–MS analysis of extracellular Aβ from primary neurons (d) and N2a cells (e) after incubation in the absence or presence of 1 μm insulin for 4 hr.

Fig. 2.

Time course of Aβ (a) and sβAPPα (b) extracellular levels from N2a cells in the absence or presence of insulin. Cells were pulse-labeled with [35S]methionine for 20 min and incubated in serum-free medium in the absence or presence of 1 μminsulin for different intervals. Data represent means ± SD;n = 3. Inset in ashows a representative autoradiographic analysis of extracellular Aβ after periods of chase from 30 min to 4 hr.

Fig. 3.

Insulin inhibits Aβ degradation via IDE and stimulates the secretion of Aβ. a, IDE released in the medium from cultured neurons as a function of time. Primary cortical neurons were pulse-labeled for 20 min and chased for different intervals up to 16 hr. IDE was immunoprecipitated with 28H1 monoclonal anti-IDE antibody and analyzed on SDS-PAGE. b, c, N2a cells were incubated for 4 hr with [35S]methionine to produce labeled Aβ and sβAPPα. Serum-free conditioned medium was collected from cultured primary neurons, which had been incubated for 16 hr at 37°C. Then this medium was mixed with the medium containing labeled Aβ or sβAPPα and incubated for a further 16 hr in the absence or presence of the indicated substances (see Materials and Methods). b, Insulin inhibits Aβ degradation. Shown is an autoradiographic analysis of labeled Aβ (top) and sβAPPα (bottom). Media were collected before or after in vitro incubation in the absence or presence of 1 μm insulin, immunoprecipitated with anti-Aβ (4G8) or anti-sβAPP (22C11) antibodies, and analyzed on SDS-PAGE. Insulin caused a marked inhibition of Aβ1–40/42 degradation. c, IDE-mediated Aβ degradation was inhibited by 1 μm insulin (Ins), 1 mm 1,10-phenantroline (Phen), or immunodepletion of IDE with a monoclonal anti-IDE antibody (9B12; see Materials and Methods). Data represent means ± SD; n = 3. *p < 0.01 with respect to the sample with no incubation; **p < 0.01 with respect to control sample.d, Insulin stimulates Aβ secretion. N2a cells were pulse-labeled for 20 min and chased for various times in the absence or presence of 1 μm insulin, 1 mm1,10-phenantroline (Phen), or a combination of the two compounds. Data represent means ± SD; n = 3.

Fig. 4.

Insulin reduces intracellular levels of Aβ in N2a cells. Cells were treated for 4 or 16 hr with or without 1 μm insulin and lysed in SDS. a, b, Intracellular Aβ was detected by immunoprecipitation with 4G8, followed by SDS-PAGE and Western blotting, using 6E10 monoclonal antibody, which recognizes only Aβ1–40/42. a, Representative autoradiographic analysis of intracellular Aβ after 16 hr of treatment in the absence or presence of 1 μminsulin. b, Quantitative analysis of intracellular Aβ after treatment with insulin for 4 or 16 hr. Data represent means ± SD; n = 5. *p < 0.05 versus control. c, IP–MS analysis of intracellular Aβ40/42 levels after 4 hr of treatment with 1 μm insulin.d, e, Western blot analysis for full-length βAPP (d) and the PS-1 N-terminal fragment (e) after 16 hr of treatment with 1 μm insulin.

Fig. 5.

Insulin influences Aβ and βAPP trafficking in N2a cells. Cells were treated for 16 hr in the absence or presence of 1 μm insulin, homogenized, and fractionated on an equilibrium flotation sucrose gradient (see Materials and Methods).a, Representative autoradiographic analysis and quantitative analysis (b) of Aβ subcellular distribution after insulin treatment. c, Representative autoradiographic analysis and quantitative analysis (d) of intracellular βAPP subcellular distribution after insulin treatment. e, Markers for subcellular compartments. Proteins from each fraction were precipitated by trichloroacetic acid and analyzed by Western blot, using the antibodies anti-calnexin (ER), anti-γ-adaptin (TGN), or anti-ARF3 (post-TGN vesicles, cytosol). Surface βAPP (plasma membrane) was determined as described (see Materials and Methods). Fraction 1 = 0.25 m sucrose solution (loading, cytosol). Fractions 2–5 correspond, respectively, to interfaces between 0.25/0.8 m (post-TGN vesicles), 0.8/1.16m (Golgi/TGN), 1.16/1.3 m, and 1.3m/2 m (heavy membranes such as ER, plasma membranes) sucrose solutions. f, N2a cells were treated for 4 hr in the absence or presence of 1 μminsulin. Surface proteins were labeled with biotin. Biotinylated βAPP was analyzed by immunoprecipitation with 369 antibody and Western blot with HRP-conjugated streptavidin.

Fig. 6.

Lack of effect of insulin on βAPP trafficking from ER to Golgi. N2a cells were pulse-labeled for 5 min with [35S]methionine and chased in serum-free medium in the absence or presence of 1 μm insulin for 5–45 min. βAPP was immunoprecipitated by using 369 antibody; one-half of the sample was digested by endoglycosidase-H (Endo H). The arrowhead indicates the endoglycosidase-H-resistant βAPP species.

Fig. 7.

The effect of insulin requires tyrosine kinase activity and is mediated via the MEK/MAP kinase cascade.a, Western blot analysis for insulin receptor (IR) in N2a cells and primary neurons. b, N2a cells were incubated for 16 hr in serum-free medium in the absence or presence of 1 μm insulin and/or 25 μmtyrphostin-25. Data represent means ± SD; n = 3. *p < 0.05 with respect to no addition; **p < 0.05 with respect to treatment with insulin alone; †Not significant with respect to tyrphostin-25 alone. c, N2a cells were incubated for 4 hr in serum-free medium in the absence or presence of 1 μminsulin, 25 μmU73122, 500 nm wortmannin, and/or 10 μm PD98059. Data represent means ± SD;n = 3. *p < 0.05 with respect to no addition; **p < 0.05 with respect to treatment with insulin alone.