Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons

Abstract

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of αS toxic features.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase

“…Fibrils of α-synuclein form cytoplasmic Lewy Bodies, a hallmark of Parkinson’s Disease ( Gallegos et al, 2015 ). However, oligomers of α-synuclein can also be found in the ER ( Bellucci et al, 2011 ; Colla et al, 2012a , b , 2017 ) and the extracellular space where they promote a prion-like transmission to healthy neurons ( Steiner et al, 2011 ; Brundin et al, 2015 ; Gallegos et al, 2015 ). PDI may interact with α-synuclein at any of these sites: although it is primarily found in the ER, PDI also functions at other intracellular and extracellular locations ( Turano et al, 2002 ; Ali Khan and Mutus, 2014 ; Soares and Laurindo, 2017 ).…”

Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum

“…For immunoblotting, cells were lysed according to protocols previously described [ 25 , 26 , 29 , 30 ]. Briefly, cells were lysed in cold lysis buffer containing 1% v / v Triton-X 100 (Tx), with protease and phosphatase inhibitor cocktails (PhosSTOP™ and cOmplete™, Mini, EDTA-free kits, Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS).…”

In vitro models of synucleinopathies: informing on molecular mechanisms and protective strategies

“…The α‐Syn species can be extracted from diseases α‐Syn transgenic mice (Colla et al . ; Panattoni et al . ) or prepared from recombinant protein, and can consist of oligomeric (Danzer et al .…”