Abnormal localization of leucine-rich repeat kinase 2 to the endosomal-lysosomal compartment in lewy body disease

Abstract

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common causes of both familial and sporadic forms of Parkinson disease and are also associated with diverse pathological alterations. The mechanisms whereby LRRK2 mutations cause these pathological phenotypes are unknown. We used immunohistochemistry with 3 distinct anti-LRRK2 antibodies to characterize the expression of LRRK2 in the brains of 21 subjects with various neurodegenerative disorders and 7 controls. The immunoreactivity of LRRK2 was localized in a subset of brainstem-type Lewy bodies (LBs) but not in cortical-type LBs, tau-positive inclusions, or TAR-DNA-binding protein-43-positive inclusions. The immunoreactivity of LRRK2 frequently appeared as enlarged granules or vacuoles within neurons of affected brain regions, including the substantia nigra, amygdala, and entorhinal cortex in patients with Parkinson disease or dementia with LBs. The volumes of LRRK2-positive granular structures in neurons of the entorhinal cortex were significantly increased in dementia with LBs brains compared with age-matched control brains (p < 0.05). Double immunolabeling demonstrated that these LRRK2-positive granular structures frequently colocalized with the late-endosomal marker Rab7B and occasionally with the lysosomal marker, the lysosomal-associated membrane protein 2. These results suggest that LRRK2 normally localizes to the endosomal-lysosomal compartment within morphologically altered neurons in neurodegenerative diseases, particularly in the brains of patients with LB diseases.

Figure 1

Localization of leucine-rich repeat kinase 2 (LRRK2) in the brains of Parkinson disease (PD) and dementia with Lewy bodies (DLB) cases. (A) The location of peptides recognized by 3 LRRK2-specific polyclonal antibodies are shown aligned to the protein domain structure of LRRK2. (B-G) LRRK2 immunoreactivity in the core (arrowheads) and its outer rim (arrows) of brainstem-type Lewy bodies (LBs) in the substantia nigra pars compacta (B, E), locus coeruleus (C, D, G) or dorsal vagal nucleus (F) of PD (B, C, E, F) or DLB (D, G) brains. (H, I) Double immunolabeling for LRRK2 (brown) and phosphorylated α-synuclein (blue) shows colocalization of LRRK2 with α-synuclein-positive brainstem-type LBs (dark brown). (J) LRRK2 immunohistochemistry in the amygdala of a DLB brain; LRRK2-positive enlarged granules are observed within the cytoplasm of neurons (arrowheads). Scale bars = 20 μm.

Figure 2

Localization of leucine-rich repeat kinase 2 (LRRK2) in brains from Alzheimer disease (AD) cases. (A, B) LRRK2 immunoreactivity in the hippocampal CA region stained with antibody JH5514 (A) or NB300-267 (B). (C-E) Double immunofluorescent labeling for LRRK2 (C, D: JH5514, E: JH5517) and paired helical filament (PHF)-tau shows that LRRK2 either does not colocalize (arrowheads) or only partially colocalizes (arrows) with PHF-tau-positive neurofibrillary tangles. All scale bars = 20 μm.

Figure 3

Localization of leucine-rich repeat kinase 2 (LRRK2) in various neurodegenerative diseases. (A, B) Consecutive paired sections from the dentate gyrus (DG) of a case of Pick disease (PiD) stained with anti-paired helical filament (PHF)-tau (A) or anti-LRRK2, NB300-267 antibody (B). Arrowheads in (A) indicate tau-positive Pick bodies. (C, D) Consecutive sections from the entorhinal cortex (Ent) of a case of corticobasal degeneration (CBD) stained with anti-PHF-tau (C) or anti-LRRK2, NB300-267 antibody (D). Arrowheads in (C) highlight tau-positive pretangles. (E, F) Consecutive sections from the Ent of a case of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) stained with anti-TAR DNA binding protein-43 (TDP-43) (E) or anti-LRRK2, NB300-267 antibody (F). Arrowheads in (E) indicate TDP-43-positive dystrophic neurites. Panels A and B, C and D, and E and F are paired consecutive sections obtained from the same affected brain region of a single case for each disorder. All scale bars = 20 μm.

Figure 4

Leucine-rich repeat kinase 2 (LRRK2)-positive enlarged granules and vacuoles. (A, B) In normal aged controls, LRRK2 immunoreactivity has a small punctate pattern throughout the neuronal soma and processes in the entorhinal cortex (A) or neuromelanin-containing dopaminergic neurons (arrowheads) of the substantia nigra pars compacta. (C-E) In the entorhinal cortex of DLB cases, LRRK2 immunoreactivity in neurons is mostly in abnormal enlarged (C) or giant (D) granules or clustered vacuoles (E). (F, G) LRRK2-positive vacuoles or granules in neurons of the substantia nigra pars compacta (F) and entorhinal cortex (G) from PD cases. (H) LRRK2-positive enlarged granules in neurons of the entorhinal cortex from an AD case. Scale bar in (A) represents 20 μm in A-H. (I) Computerized image analysis for estimation of the volume of enlarged granules immunostained with anti-LRRK2 antibody (JH5514) or total cytoplasmic area outlined by brown (DAB) immunoreactivity, as described in Methods. (J) Percentage of total cell area occupied by LRRK2-positive granules in neurons of the entorhinal cortex of DLB brains (n = 6), AD brains (n = 6) and aged control brains (n = 7). Values are the mean percentage calculated from 25 neurons per brain.

Figure 5

Double colorimetric immunolabeling for leucine-rich repeat kinase 2 (LRRK2) and organelle markers in neurons of the entorhinal cortex from cases of Dementia with Lewy bodies (DLB). (A, B) Late-endosome (Rab7B); (C) lysosome (LAMP2); (D) trans-Golgi network (TGN38) (E) mitochondria (COX IV). Each color (brown or blue) corresponding to LRRK2 immunoreactivity or organelle maker immunoreactivity is indicated in each image. Insets in B–E are enlarged regions as indicated. Arrows in (A) highlight neurons with double-positive enlarged granules for LRRK2 and Rab7B (dark brown). LRRK2-positive granules mainly colocalize with Rab7B (A, B) and to a lesser extent with LAMP2 (C), but show minimal overlap with TGN38 (D) and COX IV (E). All scale bars = 10 μm.

Figure 6

Confocal double immunofluorescent images for leucine-rich repeat kinase 2 (LRRK2) and organelle markers in neurons of the entorhinal cortex from cases of Dementia with Lewy bodies (DLB). (A) late-endosome (Rab7B); (B) lysosome (LAMP2); (C) trans-Golgi network (TGN38); (D) mitochondria (COX IV). Insets in A–D represent enlarged regions as indicated. LRRK2-positive granules mainly colocalize with Rab7B (A) and to a lesser extent with LAMP2 (B), but show minimal overlap with TGN38 (C) and COX IV (D). Scale bar in (A) = 10 μm in A–D.