Analysis of α-synuclein species enriched from cerebral cortex of humans with sporadic dementia with Lewy bodies.

Valentina N Lagomarsino,David Klenerman,Haiyang Jiang,Matteo Rovere,Ming Jin,Laura De Boni,Tracy L Young-Pearse,Dennis W Dickson,Xinyue Liu,Suman De,John B Sanderson,Ludovica Zaccagnini,Bradley T Hyman,Tim Bartels,Thomas C Pochapsky,Aurelia Hays Watson,Dennis J Selkoe

doi:10.1093/braincomms/fcaa010

Abstract

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other ‘synucleinopathies’. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity.

Highlights

Dementia with Lewy bodies (DLB) is an aggressive neurodegenerative disease associated with progressive cognitive dysfunction and hallucinations
To minimize the significant variation we observed in both the levels of ‘insoluble’ aSyn and the degree of Lewy cytopathology by aSyn immunohistochemistry (IHC) (Fig. 1), we homogenized between three and six different tissue pieces dissected from frozen frontal cortex of each DLB brain that showed Lewy pathology by aSyn IHC in sections prepared from a piece of the adjacent frozen cortex
Our decision to look at several small tissue pieces per brain, as we feared that unburdened areas of larger sections of tissue might dilute any pathology-specific effects and we recommend future studies to employ a similar approach

Summary

Introduction

Dementia with Lewy bodies (DLB) is an aggressive neurodegenerative disease associated with progressive cognitive dysfunction and hallucinations. The presence of intraneuronal aggregates of a-synuclein (aSyn), similar to those of Parkinson’s disease (PD), is diagnostic (McKeith et al, 1996; Spillantini et al, 1997; McKeith et al, 1999; McKeith et al, 2005; McKeith et al, 2017) In both PD and DLB, aSyn forms hallmark perikaryal and neuritic aggregates called Lewy bodies and Lewy neurites, in addition to more recently described small synaptic aggregates (Kramer and Schulz-Schaeffer, 2007; Roberts et al, 2015). A large body of work, predominantly assessing recombinant aSyn, indicates that soluble oligomers show neurotoxic properties, including a propensity to alter cellular membranes (Volles et al, 2001; Putcha et al, 2010; Fusco et al, 2016) Validation of these in vitro results in human tissue is incomplete, . Previous in vivo and in vitro studies suggest the existence of a soluble, diffusible aSyn species that might cause the spreading of Lewy pathology and neurotoxicity

Methods

Results

Conclusion