Amyloid-\u03b2 interrupts canonical Sonic hedgehog signaling by distorting primary cilia structure

Aleister J. Saunders,Anna G. Vorobyeva

doi:10.1186/s13630-018-0059-y

Abstract

BackgroundPrimary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. These small organelles serve as sensory structures sampling the extracellular environment and reprogramming the transcriptional machinery in response to environmental change. Primary cilia are decorated with a variety of receptor proteins and are necessary for specific signaling cascades such as the Sonic hedgehog (Shh) pathway. Disrupting cilia structure or function results in a spectrum of diseases collectively referred to as ciliopathies. Common to human ciliopathies is cognitive impairment, a symptom also observed in Alzheimer’s disease (AD). One hallmark of AD is accumulation of senile plaques composed of neurotoxic Amyloid-β (Aβ) peptide. The Aβ peptide is generated by the proteolytic cleavage of the amyloid precursor protein (APP). We set out to determine if Aβ affects primary cilia structure and the Shh signaling cascade.MethodsWe utilized in vitro cell-based assays in combination with fluorescent confocal microscopy to address our study goals. Shh signaling and cilia structure was studied using two different cell lines, mouse NIH3T3 and human HeLa cells. To investigate how Aβ levels affect Shh signaling and cilia structure in these cells, we utilized naturally secreted Aβ as well as synthetic Aβ. Effects on Shh signaling were assessed by luciferase activity while cilia structure was analyzed by fluorescent microscopy.ResultsHere, we report that APP localizes to primary cilia and Aβ treatment results in distorted primary cilia structure. In addition, we demonstrate that Aβ treatment interrupts canonical Shh signal transduction.ConclusionsOverall, our study illustrates that Aβ can alter primary cilia structure suggesting that elevated Aβ levels, like those observed in AD patients, could have similar effects on neuronal primary cilia in the brain. Additionally, our study suggests that Aβ impairs the Shh signaling pathway. Together our findings shed light on two novel targets for future AD therapeutics.

Highlights

Primary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons
We evaluated whether increased Aβ levels disrupt primary cilia structure and the mammalian Sonic hedgehog signaling pathway
Aβ decreases primary cilia length and frequency in NIH3T3 cells To determine whether elevated Aβ levels affect primary cilia structure, we exposed cells to increasing concentrations of extracellular Aβ and examined cilia structure by immunofluorescence microscopy using an anti-acetylated tubulin antibody, an established primary cilia marker

Summary

Introduction

Primary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. APP function is not well understood, studies that shed light on the physiological role of Aβ in non-diseased brains show that Aβ plays a role in synaptic transmission and is involved in learning and memory formation [18,19,20,21,22]. For this reason, it is critical to maintain adequate soluble Aβ levels that support synaptic plasticity while not exceeding the pro-amyloidosis threshold.

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