Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo

Abstract

Background: Abnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo.

Methods: Fluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively.

Results: THK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinson's disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe³⁺ form, as evinced by the STXM results.

Conclusion: We demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

Keywords: Parkinson’s disease; alpha-synuclein; fluorescence imaging; iron; magnetic resonance imaging; optoacoustic imaging; susceptibility weighted imaging.

Fig 1.. Characterization of THK-565 in recombinant αSyn fibrils and M83 mouse and post-mortem PD brain.

(a) Chemical structure of THK-565; (b) Transmission electron microscopy characterization of recombinant αSyn fibril; Scale bar = 200 nm; (c) Thioflavin T assay of recombinant αSyn (red) fibril and blank (gray); (d) Spectrofluorometric measurements of the binding of THK-565 to recombinant αSyn (red) fibril and blank (gray, dd. water); (e-h) Immunofluorescence staining using THK-565 (red), with anti-αSyn antibodies LB509, Syn303, anti-p-αSyn antibody pS129 (green) on cortex (Ctx) and striatum (Str) of M83 mouse brain; (i) Immunocytochemistry using Syn303 antibodies on M83 mouse Str; arrow indicates αSyn inclusions. (j) Lambda scan of THK-565-stained αSyn inclusions in the M83 mouse brain. (k, l) Immunofluorescence staining using THK-565 (red), with pS129 (green) on medulla oblongata of postmortem tissue from patient with PD; nuclei were counterstained using DAPI (gray). Scale bar = 10 μm (e-h, j, k) and 50 μm (i, l);

Fig 2.. In vivo concurrent epifluorescence and VmsoT using THK-565.

(a) Representative epifluorescence (FL) and vMSOT images at different time points from pre-injection of THK-565 until 320 min post-injection in the brain of one M83 mouse (horizontal view). (b) The time difference in the vMSOT signal during the injection of THK-565 was used to distinguish THK-565 from HbO₂/Hb and background. (c) Absorbance spectrum of THK-565 (retrieved from the in vivo vMSOT data) and HbO₂/Hb [41]. (d-f) Quantification of absolute fluorescence intensity at 580 nm and 635 nm (F. I), normalized differential fluorescence at 635 nm, normalized ΔvMSOT over the whole brain of M83 mice after THK-565 i.v. injection. (g-i) Stable normalized F.I., ΔvMSOT, and unmixed ΔvMSOT over 90 min in the brain of one M83 mouse without THK-565 injection.

Fig. 3. Increased THK-565 uptake in the brains of M83 mice compared to NTL mice.

(a) Representative epifluorescence images of NTL and M83 mouse brains at 40 min post THK-565 i.v. injection. (b) % increase in fluorescence intensity over 90 min in the brains of M83 and NTL mice after THK-565 i.v. injection. (c) Higher % increase of fluorescence intensity in the brain of M83 compared to NTL mice. (d) Representative THK-565 signal resolved by vMSOT at 40 min post THK-565 i.v. injection (coronal and horizontal view). (e) Mouse brain atlas overlaid on the vMSOT images of the mouse brain (coronal, sagittal, horizontal view). (f) Whole brain ΔvMSOT signal intensity over 90 min of M83 and NTL mice post THK-565 i.v. injection. (g) Regional analysis of the normalized ΔvMSOT signal at 20–40 min post THK-565 i.v. injection.

Fig. 4. Imaging evidence of intracranial iron deposition in the M83 mouse.

(a) Ex vivo SWI at 9.4 T and corresponding phase image showing hypointensities/negative phase shifts indicating paramagnetic iron deposition in the M83 mouse brain. (b) Scanning transmission X-ray microscopy showed Fe³⁺ deposits in the striatum of the adjacent brain slice of the Prussian blue-stained slice. (c) Prussian blue staining indicating the presence of iron deposition in the cortex and striatum of the M83 mouse brain.

Fig. 5. SWI and phase imaging reveal intracranial calcification in the M83 mouse.

(a) Ex vivo SWI MRI at 9.4 T and corresponding phase image showing hypointensities/positive phase shifts indicting diamagnetic calcification in the M83 mouse brain. (b) H&E staining indicating the presence of calcification and enlarged perivascular space.