Misfolded α-synuclein and Toll-like receptors: therapeutic targets for Parkinson's disease

Abstract

Parkinson's disease (PD) is typified by the loss of midbrain dopamine neurons, the presence of large proteinaceous α-synuclein-positive intracellular inclusions, oxidatively modified molecules and activated microglia. The etiology of sporadic PD is not fully understood but several lines of evidence suggest that genetic vulnerability and environmental toxicants converge to incite pathology--the multiple hit hypothesis. One gene linked to both familial and sporadic PD is SNCA, which encodes for the protein α-synuclein that has a propensity to misfold into toxic moieties. Here we show that α-synuclein directly activates microglia inciting the production of proinflammatory molecules and altering the expression of Toll-like receptors (TLRs). We discuss the role for α-synuclein-directed TLR expression changes in PD and the therapeutic potential of modifying this response.

Fig. 1

α-Synuclein activates primary microglia. Primary microglia derived from C57/BL6 mouse cortices were treated with buffer (A) or 50nM α-synuclein (B) for 24 hr as previously described [12]. Cells were subsequently fixed and subjected to immunocytochemistry using anti-Iba-1 primary antibody (1:750/Wako Chemicals) followed by incubation with goat anti-rabbit fluorescent secondary antibody. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei. The dashed arrow (A) points to a ramified microglia prototypic of a non-activated cell while the solid arrow (B) points to an amoeboid shaped cell, the typical morphology of classically activated microglia. All animals used (Figures 1, 2; Table 1) were maintained and treated in accordance with the regulatory standards of the Animal Welfare Act and approved for use by the Georgetown University Animal Care and Use Committee.

Fig. 2

α-Synuclein incites microglial activation via toll-like receptor activation. This schematic diagram depicts our current hypothesis that α-synuclein released from neurons and/or presynaptic terminals (e.g., activity dependent release or as a result of ongoing neurodegeneration) activates microglia [1]. We have previously demonstrated that α-synuclein-mediated activation of microglia results in increased expression of TLRs (2; lightning bolt; Table 1 and ref. [12]). Importantly TLRs can mediate downstream pathways that result in the translocation of NFκB to the nucleus, which in turn causes increased expression of proinflammatory molecules (3 & 4; Table 1) that are detrimental to dopamine neurons (5; black triangle “Dopamine Neuron Vulnerability”). Microglial activation is often accompanied by a change in morphology from highly branched cells (ramified) to rounded cells with little branching (amboid) as indicated in the lower yellow triangle. Our data indicates that 24-hours post-exposure α-synuclein-directed microglial activation results in increased expression of TNF-α, NOS, IL-1β, MMP-9, ROS and morphological changes consistent with enhanced classical inflammation (Table 1 and Figure 1). If this proinflammatory milieu continues progressive death of dopamine neurons releasing α-synuclein would ensue followed by continued activation of microglia. However, since TLRs can also promote cell growth and survival it will be important in future studies to establish the specific TLRs that are altered by cognate α-synuclein conformers and subsequently to determine the exact downstream TLR pathways activated (e.g., MyD88 and/or PI3K) so that targeted therapies can be developed.