Transthyretin neuroprotection in Alzheimer's disease is dependent on proteolysis

Abstract

The deposition of amyloid β peptide (Aβ) in the hippocampus is one of the major hallmarks of Alzheimer's disease, a neurodegenerative disorder characterized by memory loss and cognitive impairment. The modulation of Aβ levels in the brain results from an equilibrium between its production from the amyloid precursor protein and removal by amyloid clearance proteins, which might occur via enzymatic (Aβ-degrading enzymes) or nonenzymatic (binding/transport proteins) reactions. Transthyretin (TTR) is one of the major Aβ-binding proteins acting as a neuroprotector in AD. In addition, TTR cleaves Aβ peptide in vitro. In this work, we show that proteolytically active TTR, and not the inactive form of the protein, impacts on Aβ fibrillogenesis, degrades neuronal-secreted Aβ, and reduces Aβ-induced toxicity in hippocampal neurons. Our data demonstrate that TTR proteolytic activity is required for the neuroprotective effect of the protein constituting a putative novel therapeutic target for AD.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder clinically characterized by progressive memory deficits and cognitive decline (Blennow et al., 2006). The 2 major hallmarks of AD are the intraneuronal presence of neurofibrillary tangles constituted by tau and the extracellular accumulation of senile plaques consisting of amyloid-β peptide (Aβ) (Kosik et al., 1986). Aβ results from the cleavage of the amyloid precursor protein (APP) by β- and γ-secretases (De Strooper et al., 2010). Lowering Aβ levels is a major therapeutic goal in AD, which might be achieved by interfering with the production, aggregation, or degradation of the peptide. Proteases have an important role in controlling Aβ homeostasis, and several Aβ-degrading enzymes were described, among them neprilysin and insulin-degrading enzymes have a well-established role in the regulation of Aβ levels (Iwata et al., 2000, Kurochkin and Goto, 1994).

Transthyretin (TTR), a protein with metalloprotease activity (Liz et al., 2012), was shown to cleave Aβ in vitro (Costa et al., 2008a). TTR has been characterized as a neuroprotective protein in AD as: (1) TTR levels are decreased in the cerebrospinal fluid (CSF) of AD patients (Riisoen, 1988); (2) overexpressing human TTR wild type (WT) in an AD mouse model normalizes cognition and memory, and diminishes neuropathology and Aβ deposition (Buxbaum et al., 2008); and (3) in vitro, TTR reduces Aβ fibrillization (Costa et al., 2008b). TTR neuroprotection in AD has been mainly attributed to its ability to bind Aβ (Schwarzman et al., 1994), but the relevance of Aβ cleavage by TTR remains unknown.

We have characterized the TTR active site as a triad formed by the residues His88, His90, and Glu92 that bind to a catalytic zinc ion and by Glu72, which acts as the general base (Liz et al., 2012). The identification of TTR catalytic residues enables assessing TTR devoid of proteolytic activity by mutation of the above mentioned residues, being therefore a powerful tool to study the relevance of TTR proteolysis. As such, in this work, we aimed to understand the impact of TTR proteolytic activity in AD by comparing the effect of TTR WT and TTR proteolytically inactive on Aβ aggregation, degradation, and neurotoxicity.