Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons - PubMed (original) (raw)

Amyloid-beta peptide oligomers disrupt axonal transport through an NMDA receptor-dependent mechanism that is mediated by glycogen synthase kinase 3beta in primary cultured hippocampal neurons

Helena Decker et al. J Neurosci. 2010.

Abstract

Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (AbetaOs), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 microm AbetaOs. NMDA receptor (NMDAR) antagonists, including d-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for AbetaO action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3beta (GSK-3beta) and transfection of neurons with a kinase-dead form of GSK-3beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that AbetaOs do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that AbetaOs negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3beta and establish a new connection between toxic Abeta oligomers and AD pathology.

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Figures

Figure 1.

Figure 1.

AβOs block organelle flux in hippocampal neurons. A, Representative kymographs comparing axonal DCV transport in control (vehicle) and AβO-treated neurons (18 h, 0.5 μ

m

AβOs). Organelle flux is markedly reduced in the presence of AβOs (right kymograph). B, Effects of AβOs and other treatments on DCV flux. Disruption of transport can be partially prevented by memantine (Mem), MK-801, lithium, while full protection was obtained with

d

-AP5 and GSK-3β inhibitor VIII (GSK inh.). A minimum of 15 cells from at least 2 different cultures were analyzed per condition; **p < 0.0001 relative to vehicle-treated cultures, *p ranging from 0.007 to 0.05 relative to vehicle-treated cultures in different experimental conditions. #p ranging from 0.0001 to 0.01 relative to AβO-treated cultures in different experimental conditions. +p < 0.05 comparing Mem + AβOs and

d

-AP5 + AβOs; ++p < 0.005 comparing AβOs + LiCl and AβOs + GSK inh. (5 μ

m

). The first 4 bars are not significantly different between them (p > 0.05). Complete statistical evaluation is presented in supplemental Fig. S2, available at

www.jneurosci.org

as supplemental material.

Figure 2.

Figure 2.

Expression of kinase-dead GSK-3β (GSK-3β KD) (K85A) prevents AβO-induced transport defects. A, Expression of BDNF-RFP and HA-tagged GSK-3β KD in the same neuron; arrows indicate the axon. B, Expression of GSK-3β KD in neurons prevents AβO-induced transport defects. Conversely, expression of constitutively kinase-active GSK-3β (GSK-3β KA) (S9A) in the absence of AβOs disrupts 50% of DCV transport. Cells were fixed postimaging and stained with anti-HA to confirm the presence of GSK-3β (K85A or S9A). C, Summary of transport data. Vehicle/GSK-3β KD n = 12 kymographs (12 cells, 1930 vesicles); AβO/GSK-3β KD n = 19 kymographs (19 cells, 3468 vesicles); GSK-3β KA n = 13 kymographs (13 cells, 1320 vesicles). *p < 0.05; **p < 0.0001, statistically significant difference compared with vehicle KD; +p < 0.05; ++p < 0.0001, statistically significant difference compared with AβO KD. Scale bar, 25 μm.

Figure 3.

Figure 3.

Neuronal cytoskeleton integrity is unaffected by AβO treatment. A, Top, Immunoblots of tubulin from neurons extracted in MT buffer I. The ratio of soluble (S) to polymerized (P) tubulin in vehicle- and AβO-treated cells is unchanged. Bottom, Representative images of tubulin immunocytochemistry in neurons fixed in MT buffer II. B, Motor proteins implicated in the transport of DCVs and/or mitochondria display similar levels between control and AβO-treated neurons. For immunocytochemistry, a minimum of 24 cells per condition from at least 3 different cultures were analyzed; for immunoblots, extracts from three different cultures were analyzed. *p < 0.05; **p < 0.005, statistically significant differences from vehicle-treated neurons (100%). Scale bars, 25 μm.

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