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

Abstract

Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic 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. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the 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.

FIGURE 1

Characterization of THK‐565 in recombinant alpha‐synuclein (αSyn) fibrils, M83 mouse brains, and post‐mortem Parkinson's disease (PD) and non‐demented control brains. (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); (E–H) Immunofluorescence staining using THK‐565 (red), with anti‐αSyn antibodies LB509, Syn303, and anti‐p‐αSyn antibody pS129 (green) on the cortex (Ctx) and striatum (Str) of the M83 mouse brain. (I, J) Immunocytochemistry using Syn303 antibodies on the M83 mouse Str; the arrow indicates αSyn inclusions. (K) Lambda scan of THK‐565‐stained αSyn inclusions in the M83 mouse brain. (L) Staining using THK‐565 (red) on the striatum of nontransgenic littermate mouse brain. (M–O) Immunofluorescence staining using THK‐565 (red), with pS129 (green) on the medulla oblongata of post‐mortem tissue from a patient with PD (M, N); and non‐demented control (O). Nuclei were counterstained using diamidino‐2‐phenylindole (DAPI) (gray). Scale bar = 10 μm (E–J, L, M); 50 μm (N, O).

FIGURE 2

In vivo concurrent epifluorescence (epiFL) and volumetric multispectral optoacoustic tomography (vMSOT) using THK‐565. (A–D) Representative normalized epiFL and vMSOT images at different time points from preinjection of THK‐565 until 320 min post‐injection in the brain of one M83 mouse (A, B), and until 90 min post‐injection in the brain of one nontransgenic littermate (NTL) mouse (C, D) (horizontal view). (E) The time difference in the normalized vMSOT signal during the injection of THK‐565 was used to distinguish THK‐565 from oxy‐/deoxyhemoglobin (HbO₂/Hb) and background. (F) Absorbance spectrum of THK‐565 (retrieved from the in vivo vMSOT data) and HbO₂/Hb [46]. (G, H) Quantification of absolute fluorescence intensity (FI) at 580 and 635 nm excitation, normalized differential FI at 635 nm, (I, J) Quantification of unmixed THK‐565 optoacoustic (OA) intensity, and normalized ΔvMSOT intensity over the whole brain of M83 mice after THK‐565 intravenous injection. (K–M) Stable normalized FI, ΔvMSOT, and unmixed ΔvMSOT over 90 min in the brain of one M83 mouse without THK‐565 injection. ΔvMSOT = ΔOA.

FIGURE 3

Increased THK‐565 uptake in the brains of M83 mice compared to nontransgenic littermate (NTL) mice. (A) Representative epifluorescence images of NTL and M83 mouse brains at 40 min post‐THK‐565 intravenous injection. (B) Percent increase in fluorescence intensity over 90 min in the brains of M83 and NTL mice after THK‐565 intravenous injection. (C) Percent increase in fluorescence intensity in the brains of M83 compared to those of NTL mice. (D) Representative THK‐565 signal resolved by volumetric multispectral optoacoustic tomography (vMSOT) at 40 min post‐THK‐565 intravenous injection (coronal and horizontal view). (E) Mouse brain atlas overlaid on the vMSOT images of the mouse brain (coronal, sagittal, and horizontal view). (F) Whole‐brain normalized ΔvMSOT signal intensity over 90 min in M83 and NTL mice after THK‐565 intravenous injection. (G) Regional analysis of the normalized ΔvMSOT signal at 20–40 min post‐HK‐565 intravenous injection. ΔvMSOT = ΔOA. FI, fluorescence intensity.

FIGURE 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 (indicated by arrow). (B) Scanning transmission x‐ray microscopy showed iron‐rich 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. (D) Optical density of Fe L_2,3‐edge spectrum of the deposit indicates that the iron species is Fe³⁺.

FIGURE 5

Susceptibility weighted imaging (SWI) and phase imaging reveal intracranial calcification in the M83 mouse. (A) Ex vivo SWI magnetic resonance imaging at 9.4 T and corresponding phase image showing hypointensities/positive phase shifts indicting diamagnetic calcification in the M83 mouse brain, including striatum, thalamus, hippocampus, and midbrain (indicated by arrow) (B) Hematoxylin & eosin staining indicate the presence of calcification in the cerebellum of the mouse brain.