14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein

Abstract

α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies. Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.

Rab27b regulates the release, autophagic clearance, and toxicity of alpha-synuclein

“…We fractionated the CM into exosomal and non-exosomal fractions by sequential, high ultracentrifugation techniques (54) to test if Rab27b KD possibly preferentially inhibited release through non-exosomal pathways. The vast majority of released αsyn in our model is associated with the non-exosomal fraction from isyn cells (51). When Rab27b was knocked down in isyn cells, the amount of αsyn released in the non-exosomal fraction was decreased by 44% compared to nt-shRNA control ( Fig.…”

“…To biochemically characterize αsyn released into the CM, we used size exclusion chromatography (SEC). We and others have previously shown that much of the released αsyn is found in higher molecular weight fractions representing molecular sizes greater than 14kD, the expected monomeric αsyn size by SEC (5,51,55). We fractionated CM from induced nt-shRNA/isyn cells and induced isyn/Rab27b KD cells by SEC and found that the amount of released αsyn released into higher molecular weight fractions was significantly increased by Rab27b KD (Fig.…”

“…Our previous work in the paracrine αsyn model revealed that the type of αsyn species released was the critical factor that mediates toxicity, not the total amount of αsyn released (51). This finding suggests that oligomeric, toxic αsyn species are potentially available for release.…”

“…We have developed a paracrine αsyn model using a doxycycline (doxy)-inducible neuroblastoma cell line, termed isyn, in order to evaluate the toxicity associated with neuronally-released αsyn ( Fig. 1a) (51). We have previously shown that induction of αsyn expression with doxy in isyn cells leads to the detection of αsyn in the CM in a dose-dependent manner, and that fractionation of the CM into exosomal and non-exosomal fractions by sequential, high ultracentrifugation techniques shows that αsyn is released into both exosomal and non-exosomal fractions (51).…”

“…Additionally, we have previously established that released αsyn is toxic to separately cultured neurons: transfer of αsyn-enriched CM from induced isyn cells promotes cell death of separately cultured differentiated SH-SY5Y neuroblastoma cells or primary mouse neurons (51). Toxicity from αsyn-enriched CM depends on αsyn, as immunodepletion of αsyn from the CM eliminated toxicity in a dose-dependent manner (51). An initial PCR screen for detectable expression of potential exosome-related proteins in isyn cells pointed to Rab27b and Rab35 expression in isyn cells.…”