Phosphorylation-state dependent intraneuronal sorting of Aβ differentially impairs autophagy and the endo-lysosomal system

ABSTRACT

Progressive accumulation of amyloid-β (Aβ) aggregates in extracellular plaques is a characteristic hallmark of Alzheimer disease (AD). Aβ is also found in intraneuronal deposits and associated with alterations of the endo-lysosomal system and impairment of macroautophagy/autophagy. Here, we assessed the effect of Aβ phosphorylation on neuronal autophagy and the endo-lysosomal pathway. Analysis of APP-PSEN1dE9 transgenic mice revealed a phosphorylation-state dependent intraneuronal accumulation of Aβ species in endo-lysosomal and autophagy-related compartments. Cell biological studies further demonstrate a differential uptake and sorting of phosphorylated Aβ variants in cultured neurons, and phosphorylation-state specific effects of Aβ variants on neuronal autophagy and lysosomal function. While Aβ phosphorylated at serine residue 8 accumulated in autophagosomes, Aβ phosphorylated at serine residue 26 showed efficient transport to lysosomes. The selective sorting of phosphorylated Aβ species caused differential impairment of vesicular transport and lysosomal function associated with neurotoxicity. Thus, the relative occurrence of phosphorylated Aβ species and their intraneuronal accumulation could contribute to AD pathogenesis, and to the commonly observed aberrations of the vesicular transport system already at the early stages of the disease.

Abbreviations

AD: Alzheimer disease; APP: amyloid beta precursor protein; ATG: autophagy related; Aβ: amyloid-β; CTSD: cathepsin D; DAPI: 4’,6-diamidino-2-phenylindole; EEA1: early endosome antigen 1; FA: formic acid; GFP: green fluorescent protein; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2: microtubule-associated protein 2; nmAβ: non-modified amyloid-β; npAβ: non-phosphorylated amyloid-β; pAβ: phosphorylated amyloid-β; p-Ser26Aβ: amyloid-β phosphorylated at serine residue 26; p-Ser8Aβ: amyloid-β phosphorylated at serine residue 8; RAB: RAB, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestome 1; YFP: yellow fluorescent protein.

KEYWORDS:

• Alzheimer’s disease
• autophagic flux
• neurodegeneration
• phosphorylated Aβ
• post-translationally modified Aβ
• vesicular trafficking

Acknowledgements

The authors would like to thank T. Bajaj and Dr. N. Gassen (University Clinic Bonn), and Dr. J. Höhfeld (University of Bonn) for sharing GFP-LC3-LC3Δ-RFP and mCherry-GFP-LC3B reporter cDNA constructs, scientific discussions, and result interpretation. The monoclonal antibodies H4B4 and ABL-93c developed by J.T. August was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. We also thank the Microscopy Core Facility, in particular Lydia Maus and Pia Stausberg, of the Medical Faculty at the University of Bonn for providing support and instrumentation funded by the Deutsche Forschungsgemeinschaft, Project Number: 388169927 (Confocal microscope) and 388171357 (Electron microscope).

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data of this study is available from the corresponding author (JW) upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2252300

Additional information

Funding

The work was supported by the Alzheimer Forschung Initiative e.V. [12854]; Alzheimer Forschung Initiative e.V. [17011]; Alzheimer Forschung Initiative e.V. [T1803]; Deutsche Forschungsgemeinschaft [WA1477/6-6]; Deutsche Forschungsgemeinschaft [WA1477/6-3].