LRRK2 is a component of granular alpha-synuclein pathology in the brainstem of Parkinson's disease

Abstract

Classical Parkinson's disease (PD) is characterized by the appearance of Lewy bodies (LBs) in affected brain regions, showing mostly compact alpha-synuclein deposition, in contrast with punctate or granular deposition, hypothesized to represent early stages of aggregation. Leucine-rich repeat kinase 2 (LRRK2) is the commonest mutated gene in inherited and idiopathic PD. LRRK2 mutation carriers display a diverse neuropathology, including alpha-synuclein and tau inclusions, suggesting an upstream role for LRRK2 in protein aggregation. We studied LRRK2 expression throughout the normal human brain with three different antibodies. We also examined the pattern of LRRK2 expression in relation to alpha-synuclein aggregation and LB formation in the brainstem of sporadic LB disease. Physiological LRRK2 expression was not restricted to regions preferentially affected in PD and LRRK2 often localized to the nuclear envelope in addition to the known cytoplasmic expression. In PD, we were able to consistently detect LRRK2 in the halo of a minority (approximately 10%) of nigral LBs using three different antibodies. Only one antibody detected LRRK2 in the core of approximately 80% of classic LBs. In the lower brainstem, most notably in the dorsal motor nucleus of the vagus, we found previously unrecognized LRRK2 labelling of complex globular lesions, filled with LB-like matter showing a punctate or granular staining for alpha-synuclein. This was often accompanied by strong LRRK2 expression within dystrophic neurites. Our findings confirm widespread physiological LRRK2 expression in the human brain and suggest an association of LRRK2 with possible early-stage alpha-synuclein pathology in the brainstem of PD.

Fig. 1. LRRK2 detection by immunoblotting

(A) Detection of a LRRK2-GFP reporter gene overexpressed in HEK cells using the three anti-LRRK2 antibodies used in this study. Recognition of the appropriate band is confirmed with an anti-GFP antibody. (B) Detection of the predicted 286 KDa band on a brain homogenate from a temporal lobe biopsy (Bx) by the C- and N-terminal antibodies. (C) LRRK2 detection in human post-mortem samples. Both the C-terminal and the N-terminal antibodies detect the same band of about 286 KDa in human post-mortem brain high salt homogenate extracts from several controls and patients. Since post-mortem (PM) samples from control and diseased brain gave a different pattern to the Bx sample, possibly due to post-mortem degradation, we performed a degradation assay of 1 or 3 hours with portions of the Bx sample and reproduced the same pattern as with the PM samples.

Fig. 2. LRRK2 is extensively expressed in the cytoplasm of neurons of the normal CNS. LRRK2 localises to the nuclear envelope

All images from adult control tissue unless stated otherwise. (A) Substantia nigra. (B) Hippocampus. (C) Substantia nigra (infant control). (D) Dentate granule cells. (E) Striatum, showing large (arrow) and medium neurons (arrowhead). (F) Anterior horn of the spinal cord (infant control). (G) CA2 sector of the hippocampus. (H) CA4 sector of the hippocampus. (I) III nerve nucleus. (J) Red nucleus (PD case). (K) CA4 sector of the hippocampus. (L) CA4 sector of the hippocampus. Single slide from a z-stack three dimensional reconstruction. LRRK2 (red colour) in an apparent intranuclear rod is actually at the nuclear membrane. In G-L, arrows point at the nuclear membrane enhancement of staining. Arrowheads point at nuclear indentations and rods. The antibody used is shown in each image. Slides were counterstained with haematoxylin/DAPI (L). Scale bars: 100 μm in A and B, rest, 10 μm.

Fig. 3. Association of LRRK2 with brainstem lesions in PD/DLB

All images are from PD/DLB cases. (A-D) LB halo staining in the midbrain (A-C) or medulla (D). In D note the concomitant staining of the core; this was rare. (E-J) LB halo staining with immunofluorescence double labelling in the midbrain. In E, a faint inner ring was also present (arrowhead). In J, the green arrow points to the alpha-synuclein-positive ring. Red arrow points to the LRRK2-positive ring. (K and L) LB core staining in the midbrain. (M-O) LB core staining with immunofluorescence double labelling in the midbrain. (P-V) LRRK2-positive lesions in the lower brainstem associated with granular alpha-synuclein staining. Alpha-synuclein staining was stronger in the periphery. Note in R that the dense peripheral structures are associated with a halo-like compact alpha-synuclein staining. U and V show weak or absent staining, respectively, of the body of these LRRK2-positive lesions for Ub and p62, with a potential “aggregation centre” localized in the periphery. P-R and S-V are thin consecutive sections. (W-Y) LRRK2-positive neurites in the lower brainstem. X and Y are thin consecutive sections. No difference between PD and DLB was observed. The antibody used is shown in each image. Slides were counterstained with haematoxylin/TOPRO3. Scale bars: 10 μm.