Regular articleTransthyretin neuroprotection in Alzheimer's disease is dependent on proteolysis
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.
Section snippets
TTR production, purification, and labeling
Recombinant TTR WT and TTR H90A (proteolytic inactive mutant) (Liz et al., 2012) were produced in BL-21 pLys Escherichia coli cells transformed with pETF1 carrying TTR complementary DNA (Goldsteins et al., 1997). Proteins were isolated and purified as previously described (Liz et al., 2004). Briefly, after bacterial lysis, protein extracts were run on DEAE-cellulose (Whatman) ion-exchange chromatography, and TTR was subsequently isolated in native Prosieve agarose (Lonza) gels. For cellular
The proteolytically inactive TTR mutant H90A has no impact on Aβ fibrillization
Mutation of the catalytic residues of TTR creates the possibility to characterize the relevance of TTR proteolysis. We previously demonstrated that TTR active site is constituted by His88, His90, and Glu92, and mutation of any of these 3 residues leads to inactive TTR (Liz et al., 2012). In this work, we assessed the effect of proteolytically competent TTR WT and the proteolytically inactive mutant TTR H90A in Aβ fibrillization, degradation and toxicity. To confirm the proteolytic activity of
Discussion
TTR is mainly recognized as the transporter for thyroxin and retinol but is also a metalloprotease (Liz et al., 2012). In this respect, TTR was shown to cleave the Aβ peptide in vitro (Costa et al., 2008a), raising the unexplored hypothesis that TTR proteolysis could underlie TTR neuroprotective effect in AD. In this work, by using proteolytically competent versus proteolytically inactive TTR, we demonstrated that TTR cleavage of Aβ leads to inhibition and disruption of Aβ fibril formation.
Disclosure Statement
The authors declare no conflicts of interest.
Acknowledgements
This work was financed by Norte-01-0145-FEDER-000008-Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through European Regional Development Fund (FEDER); FEDER funds through the Operational Competitiveness Programme—COMPETE and by National Funds through FCT—Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-021392
References (17)
- et al.
Alzheimer's disease
Lancet
(2006) - et al.
Transthyretin binding to A-Beta peptide–impact on A-Beta fibrillogenesis and toxicity
FEBS Lett.
(2008) - et al.
Alzheimer's beta-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme
FEBS Lett.
(1994) - et al.
Transthyretin, a new cryptic protease
J. Biol. Chem.
(2004) - et al.
Recombinant soluble neprilysin reduces amyloid-beta accumulation and improves memory impairment in Alzheimer's disease mice
Brain Res.
(2013) - et al.
Transthyretin protects Alzheimer's mice from the behavioral and biochemical effects of Abeta toxicity
Proc. Natl. Acad. Sci. U. S. A.
(2008) - et al.
Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor
PLoS One
(2008) - et al.
The secretases: enzymes with therapeutic potential in Alzheimer disease
Nat. Rev. Neurol.
(2010)
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Authors contributed equally to this work.