Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation

Abstract

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Figure 1

A) Molecular structure of heme B. B) Time course Thioflavin T (ThT) fluorescence, excited at 446 nm, in the absence and presence of heme with αSyn. 90 µM αSyn was incubated at 37 °C with 250 rpm shaking over a period of 115 hours in PBS pH 7.4. In the absence of 90 µM heme there is an increase in the fluorescence at 490 nm with increasing incubation time, corresponding to the formation of amyloid structures. In the presence of heme, there is an extension of the lag phase, as well as an overall decrease in ThT fluorescence intensity.

Figure 2

PAGE of αSyn. A, SDS-PAGE of αSyn at time 0; the protein runs as a single band at ~17kD. B, Native PAGE of αSyn at time 0 to 4 hours; no change is observed in the size or oligomer distribution of αSyn.

Figure 3

Native-PAGE of αSyn from 0 to136 hours of incubation in the absence and presence of heme. During the aggregation in the absence of heme the initial species of αSyn forms protofibrillar and fibrillar structures after 6 hours of aggregation as shown on the left of the figure. In contrast, in the presence of heme, a ladder of increasing sized oligomeric structures is resolved on the gel. The results presented are representative of three different experiments. On the left of the figure is the position of marker proteins, while the right shows MW calculated based on the relative migration compared to the marker.

Figure 4

Transmission electron micrograph of negatively stained αSyn at various stages of incubation. A) Small spherical oligomers of 10–15 nm are observed to be the primary species at this T=1 h, scale bar =100 nm, arrows indicate individual oligomers, inset scale bar =30 nm. B) 6 hours, C) 12 hours, D) 20 hours, E) 54 hours. Small spherical oligomers are observed during the initial stages of aggregation. Primarily fibrils exist after 30 hours. Large scale bar = 200 nm, inset scale bar = 30 nm.

Figure 5

Transmission electron micrograph of negatively stained αSyn in the presence of heme at A) 12 hours and B) 54 hours. Small spherical oligomers, which are ≈15 nm in width, are observed throughout the aggregation time-course, before the formation of fibrillar structures. Large scale bar = 200 nm, inset scale bar = 30 nm.

Figure 6

Atomic Force Microscopy of αSyn aggregates. A) Two hour incubation of αSyn showing prefibrillar oligomeric structures and annular protofibrils (arrow heads), B) Typical annular structures, note the scale bar is 59 nm. C) Histogram displaying the average height of 18 nm for annular structures formed at 2 hours of aggregation, (n=16). D) Two hour incubation of αSyn in the presence of heme, showing prefibrillar oligomeric structures and annular protofibrils (arrow heads), E) Typical annular structures, note the scale bar is 10 nm. F) Histogram of the heights of annular structures formed in the presence of heme throughout the aggregation time-course, showing an average height of 0.9 nm (n=87).

Figure 7

Circular Dichroism spectra showing the secondary structure changes of αSyn during the time-course of aggregation. A) Spectral changes at the following times: 0, 4, 8, 28, 52, 100, 136 hours, displaying a transition from unstructured to primarily β-sheet structure with a minimum at 218 nm. B) Spectral changes of αSyn in the presence of heme, at the identical time-points as in A, showing a secondary structure transition from unstructured to partially structured combination of α-helical and β-sheet structure at the end of the aggregation time-course.

Figure 8

Postulated model for amyloid fibril formation in the presence of heme. Heme (red squares) directly interacts with αSyn stabilizing oligomeric structures (red box) and inhibits the further aggregation into fibrillar structures. Low molecular weight oligomers are particularly stabilized by interaction with heme, reducing the chance of nuclei formation, and shifts the equilibrium away from fibril formation. The annular structures stabilized by heme are 10 nm in apparent diameter, whereas the annular structures typically formed are on the order of 60 nm.