Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction

Abstract

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein's ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson's disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker's yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson's disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant "KLKEGV". Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.

Keywords: Alpha-synuclein; Lipids; Parkinson’s disease; Vesicle trafficking.

Figure 1.. αS ‘3K’ (E35K+E46K+E61K) YFP+ inclusions overlap with LD-like structures in live cells.

A, 17D-TR/αS-3K::YFP neuroblastoma cells were induced to express mutant αS-3K::YFP for 48 h. Live cells underwent bright-field and fluorescence microscopy. αS-positive inclusions were detected in the green channel (left panel, the arrow points at an example inclusion). The cells contained round/oval structures that were visible in the brightfield channel (middle panel, the arrow points at an example structure). A merge image of YFP and brightfield channel demonstrates pronounced, but not complete, overlap between YFP+ inclusions and round/oval cellular structures (right panel, the arrow points at an example of pronounced overlap). B, αS-3K::YFP signal strongly colocalizes with a lipid dye (HCS LipidTOX^™ deep red neutral lipid dye) within round/oval cellular inclusions. Note that many but not all YFP+ inclusions overlap with lipid dye staining and vice versa. Incucyte-based imaging. Scalebars: (A) 20 μm, (B) 50 μm. C, Examples of αS-rich inclusions that overlap with LDs (i), αS-rich inclusions that do not overlap with LDs (ii), LDs that do not overlap with αS-rich inclusions despite αS-3K::YFP expression in the same cell (iii), and LDs in non-αS-expressing cells (iv).

Figure 2.. Endogenous vesicular markers stain αS-3K inclusions in αS-3K::YFP inducible cells.

Antibodies to Transferrin receptor (TfR), early endosomal Rab5, late endosomal Rab7, recycling endosomal Rab11, Golgi-localized Giantin, mitochondrial Tom20, and ER-localized Calnexin were utilized to understand the membrane contents of αS-positive inclusions. Vesicular markers TfR, Rab5, Rab7, and Rab11 colocalize with membrane-enriched inclusions, whereas Giantin, Tom20, and Calnexin largely exclude the inclusions. Scalebar: 20 μm.

Figure 3.. In inducible cells membrane-enriched αS variants ‘3K’ and ‘KLK’ cause inclusions that are rich in vesicles, tubular structures, and LDs by electron microscopy.

A, Electron micrograph of inducible αS-3K::YFP-expressing cell exhibits a membranous inclusion consisting mainly of tubular structures (T). Immunogold labeling with the C20 antibody for αS (black arrow) shows punctate αS 3K decorating the membrane-enriched inclusion. Scale bar: 100 nm. B, Electron micrograph of inducible αS-KLK::YFP-expressing cell also shows the inclusion being composed of vesicle accumulation (VA), tubular structures (T), and lipid droplets (LD). In addition, there is a mitochondrion (M) in the inclusion periphery. Scale bar: 500 nm.

Fig. 4.. In transfected cells membrane-enriched αS variant ‘KLK’ cause inclusions that are rich in vesicles, lipid droplets, and massive stacked tubular structures by electron microscopy.

A transfected αS-KLK::YFP cell demonstrates by electron micrograph inclusion components of vesicle accumulation (VA), lipid droplets (LD), and peripheral mitochondria (M). Rather than single tubular structures, an expanse of tubular membranes (TM) are observed in these inclusions. These features are shown with zoomed-in images for, mitochondria (M), lipid droplet (LD), vesicle accumulation (VA), and tubular membranes (TM). Immunogold detection suggests that αS is associated with the tubular membranes. Scalebars as indicated.

Fig. 5.. In transfected cells membrane-enriched αS variant ‘KLK’ causes tubular structures that may result from remodeling of vesicle membranes.

A, A transfected αS-KLK::YFP cell demonstrates by electron micrograph similar inclusion components of vesicle accumulation, LDs, and peripheral mitochondria. An expanse of tubular membranes (TM) is observed with variations in its appearance depending on the respective section (cross, oblique, longitudinal), as indicated. Scalebar: 500 nm. B, Zoom-in images of vesicle accumulation and of stacked tubular membranes (various sections as indicated, see Fig. 5A and main text for more details). Scalebars: 100 nm.