A persistent stress response to impeded axonal transport leads to accumulation of amyloid-β in the endoplasmic reticulum, and is a probable cause of sporadic Alzheimer's disease

Abstract

Background and objective: Could a normal--but persistent--stress response to impeded axonal transport lead to late-onset Alzheimer's disease (AD)? Our results offer an affirmative answer, suggesting a mechanism for the abnormal production of amyloid-β (Aβ), triggered by the slowed axonal transport at old age. We hypothesize that Aβ precursor protein (APP) is a sensor at the endoplasmic reticulum (ER) that detects, and signals to the nucleus, abnormalities in axonal transport. When persistently activated, due to chronically slowed-down transport, this signaling pathway leads to accumulation of Aβ within the ER.

Methods and results: We tested this hypothesis with the neuronal cell line CAD. We show that, normally, a fraction of APP is transported into neurites by recruiting kinesin-1 via the adaptor protein, Fe65. Under conditions that block kinesin-1-dependent transport, APP, Fe65 and kinesin-1 accumulate in the soma, and form a complex at the ER. This complex recruits active c-Jun N-terminal kinase (JNK), which phosphorylates APP at Thr(668). This phosphorylation increases the cleavage of APP by the amyloidogenic pathway, which generates Aβ within the ER lumen, and releases Fe65 into the cytoplasm. Part of the released Fe65 translocates into the nucleus, likely to initiate a gene transcription response to arrested transport. Prolonged arrest of kinesin-1-dependent transport could thus lead to accumulation and oligomerization of Aβ in the ER.

Conclusion: These results support a model where the APP:Fe65 complex is a sensor at the ER for detecting the increased level of kinesin-1 caused by halted transport, which signals to the nucleus, while concomitantly generating an oligomerization-prone pool of Aβ in the ER. Our hypothesis could thus explain a pathogenic mechanism in AD.

Fig. 1

The molecular mechanism of ER stress response triggered by arrested axonal transport. a The complex APP:Fe65:kinesin-1, normally targeted to neurites, accumulates at the ER when transport is arrested. b Recruitment of active JNK kinase complex by kinesin-1 leads to phosphorylation of APP. c Phosphorylated APP is cleaved by the amyloidogenic pathway to release Aβ into the ER lumen, and Fe65 into the cytoplasm. Fe65 then translocates into the nucleus, forming a transcriptionally active complex with Tip60 that regulates expression of stress-related genes.

Fig. 2

Knockdown of Fe65 with siRNA in CAD cells decreases Aβ levels in the soma by approximately 50%. GFP marks cells transfected with Fe65 siRNA. The histogram on the right shows the levels of Aβ along the indicated line. Bar = 10 μm.

Fig. 3

Oligomeric species (anti-oligomer antibody, A11) accumulate in ER. CAD cells were transfected with HA-KLC-TPR. This construct expresses the 6 TPRs of KLC, which can simultaneously bind 2 proteins. Insets are magnifications of the cell in the lower, left corner of the images. The graph shows direct correlation between the levels of oligomers and expressed HA-KLC-TPR. Bars = 20 μm.

Fig. 4

Overexpressed KLC accumulates in the cell soma of transfected CAD cells, causing an approximately 70% increased translocation of Fe65 in the nucleus. Plane of focus is through the nucleus. Bar = 10 μm.