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. 2013 Oct:58:183-90.
doi: 10.1016/j.nbd.2013.05.017. Epub 2013 Jun 5.

Divergent α-synuclein solubility and aggregation properties in G2019S LRRK2 Parkinson's disease brains with Lewy Body pathology compared to idiopathic cases

Adamantios Mamais  1 , Meera Raja, Claudia Manzoni, Sybille Dihanich, Andrew Lees, Darren Moore, Patrick A Lewis, Rina Bandopadhyay
Affiliations

Divergent α-synuclein solubility and aggregation properties in G2019S LRRK2 Parkinson's disease brains with Lewy Body pathology compared to idiopathic cases

Adamantios Mamais et al. Neurobiol Dis. 2013 Oct.

Abstract

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). The most prevalent LRRK2 mutation is the G2019S coding change, located in the kinase domain of this complex multi-domain protein. The majority of G2019S autopsy cases feature typical Lewy Body pathology with a clinical phenotype almost indistinguishable from idiopathic PD (iPD). Here we have investigated the biochemical characteristics of α-synuclein in G2019S LRRK2 PD post-mortem material, in comparison to pathology-matched iPD. Immunohistochemistry with pS129 α-synuclein antibody showed that the medulla is heavily affected with pathology in G2019S PD whilst the basal ganglia (BG), limbic and frontal cortical regions demonstrated comparable pathology scores between G2019S PD and iPD. Significantly lower levels of the highly aggregated α-synuclein species in urea-SDS fractions were observed in G2019S cases compared to iPD in the BG and limbic cortex. Our data, albeit from a small number of cases, highlight a difference in the biochemical properties of aggregated α-synuclein in G2019S linked PD compared to iPD, despite a similar histopathological presentation. This divergence in solubility is most notable in the basal ganglia, a region that is affected preclinically and is damaged before overt dopaminergic cell death.

Keywords: Differential solubility; G2019S; Immunohistochemistry; LRRK2; α-Synuclein.

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Figures

Fig. S1
Fig. S1
Representative blots of cleared homogenates, SDS- and TBS-soluble fractions from G2019S, iPD and control brain tissue. Only the higher molecular weight (HMW) α-synuclein species are presented here and not the monomer. HMW α-synuclein species are abundant in basal ganglia homogenates of G2019S PD and iPD compared to controls (A). [Mann–Whitney U test: *p < 0.05].
Fig. S2
Fig. S2
The effect of PMD on the distribution of α-synuclein across fractions. (A) The G2019S and iPD cases have similar PMD values whilst the controls have a wider spread with no statistically significant divergence. (B, C, D) Linear regression analysis of the monomeric α-synuclein levels in the homogenates, TBS- and SDS-soluble fractions reveals no correlation with PMD. (E) A linear regression plot of the iPD values for the urea-fraction over PMD reveals no significant correlation to PMD.
Fig. 1
Fig. 1
Immunohistochemistry of pS129 α-synuclein inclusions in G2019S, iPD and control post-mortem brains. Formalin fixed sections were stained for pS129 α-synuclein. All of our G2019S cases are categorised as the limbic subtype of α-synuclein pathology, according to the McKeith criteria (McKeith et al., 2005). The regions examined were medulla, substantia nigra, basal ganglia (putamen), entorhinal cortex and frontal cortex. The DMN (dorsal motor nucleus of vagus) of the G2019S cases tended to have higher pS129 α-synuclein inclusions whilst comparable pS129 α-synuclein staining is observed in inclusion bodies in G2019S and idiopathic PD in the nigra, limbic cortex and frontal cortex. Scale bar correspond to10 μm in A, B, E, F, H, K, and L and 20 μm in C, D, G, I, J, M, N, and O.
Fig. 2
Fig. 2
Distribution of α-synuclein monomer across cleared homogenates, TBS and SDS soluble fractions of G2019S, iPD and control post-mortem brain. The levels of α-synuclein in cleared homogenates (A, B, C), TBS (D, E, F) and SDS (G, H, I) soluble fractions of three G2019S, iPD and control brain regions were examined. A trend of increased solubility of α-synuclein in G2019S compared to that in iPD is observed in basal ganglia (D, G), that is not evident in the less pathologically affected areas of the cortex (E, F, H, I). [Mann–Whitney U test: *p < 0.05].
Fig. 3
Fig. 3
Divergence of G2019S PD brains in levels of highly aggregated urea-soluble α-synuclein species. The urea-soluble fractions of basal ganglia (A) and limbic cortex (B) of G2019S, iPD and control brains were analysed for the presence of α-synuclein. Aggregated α-synuclein is observed in the basal ganglia and the limbic cortex of the iPD samples following pathology severity. Very little aggregated synuclein is detected in the urea-soluble samples of the G2019S cases compared to the iPD cases. A dot-blot of urea-fractions of the basal ganglia (C) reveals much lower levels of α-synuclein in the G2019S cases compared to that of the iPD samples, whilst no α-synuclein is detected in controls. [ANOVA with Bonferroni's correction: *p < 0.01].
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