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Vol. 69, No. 9 September 2010 pp. 959Y972

ORIGINAL ARTICLE

Leucine-Rich Repeat Kinase 2 Is Associated With the Endoplasmic Reticulum in Dopaminergic Neurons and Accumulates in the Core of Lewy Bodies in Parkinson Disease Je´re´mie Vitte, PhD, Sabine Traver, PhD, Andre´ Maue´s De Paula, MD, Suzanne Lesage, PhD, Giorgio Rovelli, PhD, Olga Corti, PhD, Charles Duyckaerts, MD, PhD, and Alexis Brice, MD

Abstract

Key Words: Endoplasmic reticulum, G2019S LRRK2 mutation, Lewy body, LRRK2, Parkinson disease. From the Laboratory of Molecular Basis, Physiopathology and Treatment of Neurodegenerative Diseases (JV, ST, SL, OC, CD, AB), Universite´ Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moelle e´pinie`re, UMR-S975, Paris, France; INSERM U975 (JV, ST, SL, OC, CD, AB), Paris, France; CNRS UMR 7225 (JV, ST, SL, OC, CD, AB), Paris, France; Service d’Anatomie Pathologique et de Neuropathologie (AMDP), CHU Timone, Marseille, France; Novartis Institute for BioMedical Research (GR), Basel, Switzerland; Alzheimer Disease Prion disease Team (CD), Universite´ Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moelle e´pinie`re, UMRS975, Paris, France; Laboratoire de Neuropathologie Raymond Escourolle (CD), Pierre and Marie Curie-Paris 6 University, Paris, France; and AP-HP, Assistance Publique Hoˆpitaux de Paris (AB), Groupe Hospitalier Pitie´-Salpeˆtrie`re, Paris, France. Send correspondence and reprint requests to: Alexis Brice, MD, INSERM UMR S975, Centre de Recherche Institut du Cerveau et de la Moelle, Groupe Hospitalier Pitie´ Salpeˆtrie`re, 47 Bd de l’Hoˆpital, 75 651 Paris Cedex 13, France; E-mail: [email protected] This research was supported by the Institut National de la Sante´ et de la Recherche Me´dicale and Agence Nationale de la Recherche (ANR-05NEUR-019-01/A05169DS). Online-only figures are available at http://www.jneuropath.com.

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Mutation of the leucine-rich repeat kinase 2 (LRRK2) gene is the most frequent genetic cause of Parkinson disease (PD). To understand the role of LRRK2 in the neuropathology of PD, we investigated the protein expression in a healthy brain and brains from patients with PD and its subcellular localization in dopaminergic neurons. LRRK2 was found to be widely expressed in healthy adult brain, including areas involved in PD. By double fluorescent staining, we found that endogenous LRRK2 is colocalized with the endoplasmic reticulum (ER) markers Neurotrace and KDEL in human dopaminergic neurons. Labeling of brain sections with anti-LRRK2 and antiY>-synuclein antibodies revealed localization of LRRK2 in the core of 24% of Lewy bodies (LBs) in the substantia nigra and 11% of LBs in the locus coeruleus in idiopathic PD patients. The percentage was increased to 50% in both areas in a patient with the G2019S LRRK2 mutation. The finding of ER localization suggests the possibility that LRRK2 is involved in the ER stress response and could account for the susceptibility to neuronal degeneration of LRRK2 mutation carriers. The localization of LRRK2 protein in the core of a subset of LBs demonstrates the contribution of LRRK2 to LB formation and disease pathogenesis.

INTRODUCTION Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease; it is the most common movement disorder affecting 0.6% to 2.6% of people older than 65 years (1). Major symptoms are progressive motor impairment with variably associated resting tremor, muscular rigidity, bradykinesia, and postural instability (2, 3). The neuropathological hallmark of PD is a progressive loss of dopaminergic neurons in the substantia nigra (SN) and the presence of Lewy bodies (LBs), often associated with Lewy neurites (LNs) in surviving neurons (4, 5). Although most PD cases are idiopathic, a small proportion of patients carry a single gene defect (monogenic forms). In the last decade, at least 13 loci and 9 genes have been discovered that are associated with both autosomal dominant or recessive forms of PD (6). Mutations in the leucine-rich repeat kinase 2 gene (LRRK2, PARK8 locus) were identified in families with autosomal-dominant, late-onset parkinsonism (7, 8), and in more than 3% of idiopathic PD cases (9). More than 50 different variants have been found (6, 10); only 11 have been proven to be pathogenic with age-dependent penetrance (10, 11). One particularly common mutation, G2019S, located in the LRRK2 exon 41, is of major importance because it accounts for 4% of patients with familial PD and for more than 1% of apparently idiopathic cases (11). The prevalence of this mutation depends on the origin of the population (12Y14) and its penetrance seems to be age-dependent and incomplete (11). It seems that the clinical phenotype in LRRK2 patients is indistinguishable from that of idiopathic PD and their responses to dopamine-replacement therapy are generally excellent (11). LRRK2-associated PD may have a more benign progression than idiopathic PD (11), although DOPA-induced dyskinesia might be more frequent (15). Most patients with the LRRK2 mutation, with or without a familial history, have typical LB neuropathology and SN degeneration as in idiopathic cases (16). In several cases, however, no inclusions have been detected (17Y21), or the disease may present with a tauopathy (22, 23) or as frontotemporal dementia with ubiquitin+ inclusions (24). Remarkably, pathological markers may vary among carriers of the same mutation, even in the same family (21, 22). LRRK2 is a large multidomain protein (286 kDa) belonging to the Roco protein family (25, 26). It possesses

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outer mitochondrial membrane (45), and microtubule network (46), thus suggesting a role in membrane trafficking. Similar localization patterns were described in cells that overexpress LRRK2 (28, 30, 43). Several studies have demonstrated the presence of LRRK2 immunostaining in LBs in human brains (41, 47Y50), although at least 1 group has disputed this observation (18, 51). Thus, the patterns of expression of LRRK2 and its colocalization with LBs reported in the literature are controversial, possibly because of the differences in the accessibility of the epitopes recognized by the antibodies (Abs) used, but also to the questionable specificity of some of them (37, 42, 52). Here, we analyzed the regional, cellular, and subcellular localization of LRRK2 in the human brain using a polyclonal Ab selected for its specificity. We compared LRRK2 immunostaining in healthy individuals and in 6 idiopathic PD patients and searched for its presence in LBs. We also characterized a new PD patient carrying the common G2019S LRRK2 gene mutation at the histopathological level and compared the localization of LRRK2 in this and in idiopathic PD cases.

MATERIALS AND METHODS Patients and Controls Idiopathic PD cases (characterized histopathologically by a neuronal loss in the SN and the presence of LBs by routine and >-synuclein staining) and age-matched controls

FIGURE 1. Characterization of leucine-rich repeat kinase 2 (LRRK2) polyclonal antibody (AT106). (AYD) The cytoplasmic localization of LRRK2 was revealed by immunofluorescence on LRRK2-transfected Cos-7 cells (A), and by immunohistochemistry on dopaminergic neurons of the substantia nigra (C). The specific signal (arrow) disappeared after preabsorption of the AT106 antibody with the purified kinase domain in both experiments (B, D). Neuromelanin (arrowheads) is responsible for the remaining brown staining (C, D). (E) Western blot experiments on lysates from transfected HEK293 cells shows almost complete disappearance of overexpressed LRRK2 after preabsorption of the antibody with the purified kinase domain. Bars = (A, B) 20 Km; (C, D) 150 Km.

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several protein interaction motifs (ankyrin repeats, leucinerich repeat regions, a WD40 domain [27]) and 2 catalytic domains that confer dual enzymatic activity and carry the more frequent disease-associated mutations (6); these are a Ras in complex proteins domain, with similarity to the Ras/ GTPase superfamily, and a serine/threonine kinase catalytic domain, highly similar to the catalytic domains of mitogenactivated protein kinase kinase kinases. LRRK2 kinase activity is variably affected by the identified gene mutations (28Y35); some mutations preserve it or reduce it slightly, whereas others (including G2019S) increase it significantly, suggesting that a dominant gain-of-function mechanism may underlie the disease (33). Although some alterations of the MAPK signaling pathway were reported in leukocytes isolated from LRRK2-associated PD patients (36), the cellular function of LRRK2 and the molecular mechanism linking LRRK2 mutations to neuronal toxicity remain unknown. Immunohistochemical studies have shown that the LRRK2 mRNA and protein are expressed in most regions of mouse and rat brains (37, 38) (including those areas affected in PD [39]) and in dopamine-innervated areas (40). LRRK2 protein is also widely expressed in the human brain (41, 42) and other organs (7, 17, 18). It is predominantly cytoplasmic, but cell fractionation experiments have demonstrated its partial association with membranous and vesicular structures, including lipid rafts (43), early endosomes, lysosomes, synaptic vesicles (44), plasma membrane, Golgi apparatus (43),

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TABLE 1. Relative Regional Expression of LRRK2 in the Healthy Human Adult Brain Brain Area

++ + ++ ++ ++ + + + + ++ ++ +++ ++ + + + ++ T +++ +++ +++ +++ +++ + ++ + +++ +++ + + + ++ +++ +++ + ++

TABLE 1. (Continued) Brain Area Nucleus vestibularis Cerebellum Granular layer (glomerulus) Purkinje cells Molecular layer

LRRK2 Neuronal Expression +++ + T 0

Semiquantitative analysis of neuronal LRRK2 expression in the healthy human brain using the AT106 anti-LRRK2 polyclonal antibody. Scale: 0 = absence, T = very weak, + = weak, ++ = moderate, or +++ = strong expression.

without neurological lesions (6 per group) were selected from La Salpeˆtrie`re Escourolle Neuropathology Laboratory. The idiopathic PD and control cases were genotyped and did not harbor the more frequent mutations found in LRRK2 gene. An additional screen was performed in the Brain Bank BGIE NeuroCEB[ run by a consortium of Patients Associations (including France Parkinson Association) and declared to the Ministry of Research and Universities, as requested by French Law. An explicit consent had been signed by the patient, or by the next of kin in the name of the patient, in accordance with the French Bioethical Laws. The project was approved by the ad hoc committee of the Brain Bank. At the time of death, the body was transported to the pathology department of a university hospital belonging to the NeuroCEB network where the brain was removed. One hemisphere, randomly left or right, was fixed in buffered 4% formaldehyde for the neuropathological diagnosis of PD. The other hemisphere was immediately sliced and kept in a deep freezer at j80-C. Samples from the frontal lobe, approximately 3 mm3 in volume, were taken for DNA extraction in the 60 cases for which there was a specific consent allowing postmortem genetic analysis. The screen led to the identification of a single case carrying the G2019S mutation in LRRK2. The patient was a 79-year-old woman who presented with a resting tremor, severe muscular rigidity, and bradykinesia since the age of 48 years. She had been treated with L-DOPA for 25 years, initially with a good therapeutic response. Progressive motor decline and cognitive deficit with hallucinations were noted in the last years of her life. Samples of the right hemisphere were embedded in paraffin. The brain of a patient aged 74 years who died of ischemic cardiopathy without neurological lesions was used for the study of LRRK2 expression as a control. The family had given informed consent for research.

++ ++

Immunofluorescence and Immunohistochemistry

+++ +++ + +++ +++ +++ +++

Cos-7 cells were transfected to produce full-length LRRK2 protein with Lipofectamine reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated with 4% bovine serum albumin, 4% normal goat serum in phosphate-buffered saline (PBS) and processed sequentially with primary and secondary Abs. For tissue staining, 5-Km-thick sections were prepared

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Isocortex Pyramidal cells Hippocampal region Dentate gyrus CA4 CA2-CA3 CA1 Subiculum Presubiculum Entorhinal cortex (second layer) Amygdala Corticomedial amygdala Basolateral amygdala Centralis Meynert basal nucleus Claustrum Striatum Caudate Putamen Pallidum External pallidum Internal pallidum Hypothalamus Anterior region Supraoptical nucleus Paraventricular nucleus Dorsomedial hypothalamic nucleus Corpus mammillare Lateral mammillary nucleus Posterior hypothalamus Thalamus Anterior and laterodorsal nuclei Mediodorsal nucleus Ventrolateral nuclei Thalamus nucleus paratenialis Subthalamic nucleus Midbrain Superior colliculus Periaqueductal gray Griseum centrale III nerve nucleus Supratrochlear nucleus Red nucleus Substantia nigra Pons Locus coeruleus Nuclei pontis Medulla XII nerve nucleus Dorsal nucleus of the vagus nerve Nucleus of the solitary tract Nucleus centralis reticularis Nucleus olivaris principalis Nucleus olivaris accessorius Nucleus funiculi lateralis

LRRK2 Neuronal Expression

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from paraffin blocks of formalin-fixed central nervous system tissue obtained at the time of autopsy. After removal of paraffin with xylene and rehydration in graded ethanol solutions, sections were either stained with hematoxylin and eosin or processed for microwave heatYinduced antigen retrieval (3 times, 5 minutes at 350 W) in 10 mmol/L citrate buffer. For immunoperoxidase staining, endogenous peroxidase activity was blocked with 1% H2O2 in 20% methanol solution for 10 minutes at room temperature (RT). Sections were permeabilized, and nonspecific epitopes were blocked with 0.2% Triton and 4% bovine serum albumin and 4% normal goat serum in PBS. LRKK2 Abs were incubated 36 hours at 4-C. After washing with PBS Tween 0.1%, sec-

tions were successively incubated with biotinylated secondary antirabbit IgG Ab (Vector Laboratories, Burlingame, CA) for 1 hour at RT and then with avidin and biotinylated horseradish peroxidase complex (ABC Standard Elite Kit; Vector Laboratories). The signal was revealed by incubating the sections in 0.25 mol/L Tris-HCl, pH 7.4, containing 0.5% of the peroxidase substrate, 3,3¶-diaminobenzidine tetrahydrochloride, and 0.015% H2O2 (BioGenex Laboratories, San Ramon, CA). The sections were dehydrated in an ascending series of ethanol concentrations, cleared in xylene, and mounted with Eukitt Mounting Medium (BioGenex). For double immunofluorescent staining, the sections were incubated for 36 hours at 4-C with anti-LRRK2 Ab.

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FIGURE 2. Expression of leucine-rich repeat kinase 2 (LRRK2) in the healthy human brain. (AYH) Immunohistochemistry with the AT106 antibody reveals different levels of LRRK2 immunoreactivity (IR) in the cytoplasm of neurons in the substantia nigra (A), locus coeruleus (B), Meynert basal nucleus (C), and III nerve nucleus (D). Fibers with necklace-shaped IR (arrow) were found in the caudate nucleus (E). Strong LRRK2-IR is seen in the walls (arrow) of veins and arteries (F). At a higher magnification, the LRRK2 cytoplasmic IR has a mottled-shaped appearance (arrow, G), similar to that of Nissl staining (arrow, H) in dopaminergic neurons with neuromelanin (arrowheads in G and H). LRRK2-IR is also seen in glial cells (arrow) of the amygdala (I). Bars = (AYD) 100 Km; (E) 15 Km; (F) 50 Km; (G, H) 10 Km; (I) 30 Km.

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After washing in PBS, sections were incubated with Cy5conjugated antirabbit Ab (1:500, 111-175-144; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at RT. Then, the sections were processed sequentially with organelle primary Abs and Al488-conjugated antimouse Ab (1:200, A11029; Molecular Probes, Carlsbad, CA) or Neuro-

LRRK2 in ER and LB Core

trace (1:150, N21480; Molecular Probes) and finally mounted with fluorescent mounting medium (S2023; DakoCytomation, Glostrup, Denmark). Double immunofluorescent staining was analyzed with a confocal microscope (Leica SP2 AOBS, Wetzlar, Germany). Sections were observed with 63 objective, and

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FIGURE 3. Expression of leucine-rich repeat kinase 2 (LRRK2) protein in different cell types. LRRK2 immunoreactivity (IR) (arrow) in tyrosine hydroxylase (TH)+ dopaminergic neurons of the substantia nigra (A-Aµ). It is also seen in astrocytes (B-Bµ) as demonstrated by double fluorescent staining with anti-LRRK2 and anti-GFAP antibodies or in microglial cells with anti-CD68 antibody (C-Cµ) in human brain sections of amygdala. Bar = 10 Km. Ó 2010 American Association of Neuropathologists, Inc.

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LRRK2 in ER and LB Core

Z-stacks pictures of LBs were taken every 0.5 Km. Colocalization between LRRK2 and organelles was analyzed in dopaminergic neurons with ImageJ software using the Bcolocalization thresholds[ plug-in with an automatic thresholding. For each experiment, between 12 and 20 cells were selected as a region of interest, and the correlation intensity distribution between the 2 stainings was scored with the Pearson coefficient (PC).

plexes were revealed using chemiluminescent detection reagents (Pierce, Rockford, IL).

Competition Assays For competition assays, Abs were preincubated overnight at 4-C with a 500-fold excess of the purified kinase domain after production in bacteria (kind gift from Novartis). Abs were then used for Western blot, immunofluorescence, or immunohistochemistry.

Western Blot Analysis

Antibodies

Immunoblots were performed on total protein extract from HEK-293 cells transfected to produce human fulllength LRRK2 protein (a kind gift from Novartis, Basel, Switzerland). Proteins were resolved in a 3% to 8% gradient gel, transferred to nitrocellulose, and detected using antiLRRK2 Abs from the following sources with indicated dilutions: AT106, 1:1000 (Alexis Biochemicals, Farmingdale, NY); AP7099b, 1:1000 (Abgent, San Diego, CA); 2567S, 1:1000 (Cell Signaling Technology, Danvers, MA); and NB300-268, 1:1000 (Novus Biologicals, Littleton, CO). Antitubulin (T6557, 1:3000; Sigma-Aldrich, St Louis, MO) was used as a loading control. After washing in PBS and 0.05% Tween 20, membranes were incubated with antimouse or antirabbit IgG conjugated to horseradish peroxidase (1:20,000; Jackson ImmunoResearch Laboratories). The immune com-

The anti-LRRK2 Abs listed above were used for immunohistochemical and immunofluorescent experiments in the following concentrations: AT106, 1:2000; AP7099b, 1:200; 2567S, 1:200; and NB300-268, 1:200. Additional Abs used were as follows: tyrosine hydroxylase (TH, 1:500, 22941; Immunostar, Hudson, WI), glial fibrillary acidic protein (GFAP, 1:50, M0761; DakoCytomation), CD68 (1:100, M0814; DakoCytomation), KDEL (Lys-Asp-Glu-Leu, 1:150, SPA-827; Stressgen, Ann Arbor, MI), COX IV (1:100, A21347; Molecular Probes), LAMP2 (1:100, 25631; Abcam, Cambridge, MA), F-adaptin (1:200, 610385; BD Transduction Laboratories, Sparks, MD), >-synuclein (1:30, LB509; Zymed, Carlsbad, CA), tau (AT8; Innogenetics, Ghent, Belgium), and antiYamyloid AA (1:100, M0872; DakoCytomation).

FIGURE 4. Subcellular localization of leucine-rich repeat kinase 2 (LRRK2) in dopaminergic neurons. (AYE) Substantia nigra of control individuals were processed for double immunofluorescence with anti-LRRK2 antibody and different subcellular markers. LRRK2 showed colocalization (arrows) with Neurotrace, a fluorescent Nissl staining (A-AØ) and KDEL (B-BØ), markers localized in the endoplasmic reticulum. No or only faint colocalization was seen between LRRK2 (arrow) and COX IV, a mitochondrial marker (C-CØ), LAMP2, a lysosomal marker (D-DØ), and F-adaptin, a Golgi apparatus marker (E-EØ). Bar = 10 Km. Ó 2010 American Association of Neuropathologists, Inc.

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FIGURE 5. Comparison of leucine-rich repeat kinase 2 (LRRK2) immunoreactivity (IR) in the substantia nigra (SN) and III nerve nucleus of controls and idiopathic PD patients. (AYE) The SN of PD patients contains fewer dopaminergic neurons (E) than the control (A). The subcellular LRRK2-IR (arrow) is disorganized and less intense in dopaminergic neurons in a PD patient (F) compared with the Nissl-like pattern (arrow) in dopaminergic neurons in an age-matched control (B). Neuromelanin in dopaminergic neurons (arrowhead, B and F); a Lewy body is shown without LRRK2-IR (asterisk, F). No degeneration was observed in the III nerve nucleus of a PD patient (G) compared with an age-matched control (C). The Nissl-like pattern (arrow) of LRRK2-IR is very similar in a control (D) and a PD patient (H). Bars = (A, C, E, G) 100 Km; (B, D, F, H) 10 Km.

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RESULTS Commercially Available Antibodies Recognize Overexpressed and Endogenous Human LRRK2

LRRK2 Is Widely Expressed in Different Brain Regions and Cell Types in the Healthy Human Brain AT106 was used to characterize the distribution of LRRK2 protein in the healthy aged human adult brain. Different brain regions were stained, and relative levels of LRRK2-IR were evaluated in each region (Table). LRRK2-IR was observed throughout the brain and was found to be intense in the neurons of several nuclei in the medulla, midbrain, thalamus, hypothalamus, and in the Meynert basal nucleus; staining was moderate in the SN and locus coeruleus (LC) and weak in the caudate nucleus and putamen (Figs. 2, AYD). Strong LRRK2-IR was also found in the walls of veins and arteries (Fig. 2F), as described by Zhu et al (52). This staining disappeared when the primary Ab was omitted (data not shown). Occasionally, there were a few thin fibers with regularly spaced dilations (reminiscent of a necklace) in the SN, LC, motor cortex, and Meynert basal nucleus of several control brains analyzed with both AT106 and 2567S Abs

(Fig. 2E). LRRK2-IR was also observed in glial cells in the amygdala, hippocampus, and isocortex (Fig. 2I). To validate the expression of LRRK2 in different cell types, we performed double fluorescent staining. LRRK2-IR was observed in TH+ dopaminergic neurons of the SN (Figs. 3, A-Aµ). It was also seen in astrocytes and macrophages, as illustrated by double staining for LRRK2 and GFAP (Figs. 3, B-Bµ) or CD68 (Figs. 3, C-Cµ) in the amygdala.

LRRK2 Is Localized in the Endoplasmic Reticulum of Normal Dopaminergic Neurons Immunohistochemical staining with AT106 and 2567S Abs revealed that LRRK2-IR was mainly localized in the cytoplasm. The IR was variously punctate depending on the cell type (Figs. 3, AYC). On a more detailed examination, the LRRK2 protein seemed to be heterogeneously distributed; mottled staining with a Nissl-like appearance was prominent in large cells, including dopaminergic neurons of the SN and neurons of the III nerve nucleus (Figs. 2G, H, 3A, 5, B, D, and H); staining was absent when primary Ab was omitted or when the Ab was preabsorbed with LRRK2-purified kinase domain (Fig. 1D and data not shown). Double immunofluorescent staining of SN sections from 3 different nonaffected brains with AT106 Ab and a fluorescent probe for Nissl bodies (which are composed of ribosomal RNA associated with the rough endoplasmic reticulum [ER]) demonstrated colocalization of the 2 markers by confocal microscopy (Figs. 4, A-AØ); PC scored at a mean T SEM of 0.70 T 0.01. The ER localization was further confirmed by double labeling of LRRK2 and KDEL, a specific epitope of ER proteins (Figs. 4, B-BØ), which also showed colocalization of these markers (PC = 0.66 T 0.02). In contrast, only a small proportion of the LRRK2-IR colocalized with mitochondria (Figs. 4, C-CØ; PC = 0.27 T 0.01), lysosomes (Figs. 4, D-DØ; PC = 0.11 T 0.01), and Golgi complex (Figs. 4E-EØ; PC = 0.17 T 0.01). In addition to the mottled staining, there was a fine punctate pattern of LRRK2-IR revealed by the AT106 Ab in the cytoplasm of dopaminergic cells or outside, which was absent when primary Ab was omitted. This staining did not colocalize with mitochondria, lysosome, or Golgi vesicles (Figs. 4, A-E) but might indicate the presence of LRRK2 in vesicles or neuritic processes. This punctateYLRRK2-IR did not colocalize with punctate >-synuclein-IR (data not shown), which has been postulated to aggregate and form LBs (53, 54).

LRRK2 Staining Is Specifically Altered in Dopaminergic Neurons of Idiopathic PD Patients and Localizes to the LB Core In the SN from clinically affected PD cases, surviving dopaminergic neurons were less intensely immunolabeled

FIGURE 6. Leucine-rich repeat kinase 2 (LRRK2) immunoreactivity (IR) in Lewy bodies (LBs), cytoplasmic, and neuritic accumulations. With the AT106 antibody in the substantia nigra (SN) of PD patients, some LBs (arrow) showed no LRRK2-IR (A), but specifically, there was some LRRK2-IR in the core (arrow) of some LBs (B). Unusual LRRK2 accumulations were observed in the cytoplasm (C) or neurites (D) of some neurons of the SN. (E-G) Double immunofluorescent staining with anti-LRRK2 and antiY>synuclein antibodies. Confocal analysis demonstrated the localization of LRRK2-IR (arrow) in the core of intraneuronal (F-Fµ) or ectopic (G-Gµ) inclusions, whereas some LBs did not show any LRRK2-IR (E-Eµ). Lewy bodies with weak (F-Fµ) or strong (G-Gµ) LRRK2-IR are shown. A section following the horizontal or vertical dashed line in the Z-stack acquired with the confocal microscope is displayed below and on the right side of each picture, respectively. Bars = 10 Km. Ó 2010 American Association of Neuropathologists, Inc.

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To select a specific anti-LRRK2 Ab, we performed Western blot analyses, immunofluorescent labeling of transfected cells and immunohistochemical labeling of human brain sections using 4 different commercially available polyclonal Abs. Cos-7 cells transfected to produce full-length human LRRK2 protein and subjected to immunofluorescent labeling with AT106, directed against the kinase domain of the LRRK2 protein, showed intense cytoplasmic staining that was not observed in nontransfected cells (Fig. 1A); this signal disappeared after preabsorption of the Ab with the purified kinase domain produced in bacteria (Fig. 1B). In contrast, no difference was observed with AP7099b and NB300-268 Abs between transfected or nontransfected cells (data not shown). Similarly, the LRRK2 immunoreactivity (IR) observed on paraffin-embedded brain sections incubated with AT106 (Fig. 1C) or 2567S (not shown) disappeared after preabsorption with the purified kinase domain (Fig. 1D) or when the primary Ab was omitted (data not shown). Because the quality of the immunostaining was optimal with AT106, we assessed its specificity further by competition assays with the kinase domain in Western blot experiments (Fig. 1E). The Ab recognized a 280-kDa protein in HEK-293 extracts from cells transfected to produce full-length LRRK2, whereas, as expected, the signal nearly completely disappeared after preabsorption. On the basis of these results we used the AT106 and occasionally the 2567S Abs in all subsequent experiments.

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Pathological Findings in a Patient With the G2019S Mutation In the brain of a carrier of the G2019S mutation of the LRRK2 gene, there was depigmentation of the SN and LC; neuronal loss was massive in these regions and moderate in the amygdala and Meynert basal nucleus (Fig. 7A). Immunohistochemical characterization revealed >-synuclein+ LBs and dystrophic neurites in the SN, LC and Meynert basal nucleus (Figs. 7, B-D). Lewy bodies were also found in pyramidal cells of the hippocampus, the cingulate gyrus, and the amygdala. Lewy neurites were present in the CA2-3 sector of the hippocampus. These alterations allowed the diagnosis of transitional form of LB disease (Braak stage 4 for LB disease) (55). Unusual >-synuclein staining was detected in the cytoplasm of astrocytes in the cingulate gyrus (Fig. 7E). The SN was devoid of neurofibrillary tangles and tau+ neurites (data

not shown). Neurofibrillary tangles and tau+ neurites were present in the entorhinal cortex, hippocampus, and amygdala (Braak neurofibrillary stage III) (56). AA+ deposits were present in the isocortex (Thal phase 1) (57). Some remaining large neurons in the SN had the typical Nissl-like staining pattern found in normal neurons (Fig. 6F). LRRK2 was also detected in the core of typical LBs in the SN, LC, and cortex, using the AT106 Ab (Fig. 6G). A total of 82 >-synuclein+ LBs identified in the SN, pons, and cortex (cingulate gyrus) were scored for LRRK2-IR after double staining in the SN: 22.0% T 3.0% of the LBs were intensely labeled, 28.5% T 1.5% were only weakly decorated, and 49.5% T 4.5% did not display LRRK2-IR (Figs. 7, H-Hµ). In the LC, 33.3% of the LBs were intensely labeled with AT106, 16.7% were weakly decorated, and 50% did not display LRRK2-IR. In the cortex of this patient, LRRK2-IR was found in the core of 5 (38.5%) out of 13 cortical LBs; no LRRK2-IR was found in LBs of a patient with dementia with LBs. As in idiopathic PD patients, approximately 20% of the >synuclein+ LNs had moderate LRRK2-IR in the G2019S patient (data not shown).

DISCUSSION To elucidate the role of LRRK2 in PD, we characterized the cellular and subcellular distributions of the LRRK2 protein in the healthy and pathological human brain. Controversial subcellular localizations have been previously reported, mostly in membranous and vesicular structures (43Y45), and it is still unclear whether the protein is a component of LBs (18, 37, 41, 47Y52, 58). Because disputed specificities of the LRRK2 Abs might explain inconsistencies among the studies (59), we first tested 4 commercial Abs for their ability to recognize the endogenous and overproduced proteins. We found that the AT106 Ab directed against the kinase domain of LRRK2 showed the most reliable signals in Western blot, immunofluorescence, and immunohistochemical experiments. Competition assays with the purified LRRK2 kinase domain demonstrated the specificity of the observed immunoreactivities. These observations are in agreement with previous studies reporting on the use of AT106 in Western blot experiments. Although Covy et al (51) were not able to detect LRRK2 in human and mouse protein extracts, Zhu et al (52) showed specific recognition of overproduced LRRK2 in HEK 293T cells; a specific signal was detected for both endogenous and overproduced human LRRK2 by Biskup et al (60). Here, we occasionally used the 2567S Ab to confirm the results obtained with AT106; it also proved to be specific in our experiments, although the intensity of the immunostaining was

FIGURE 7. Parkinson disease patient with the leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. (A) Hematoxylin and eosin staining of a section of the substantia nigra shows a small number of pigmented neurons (arrows). (B) Numerous immunoreactive inclusions such as Lewy bodies (LBs, arrows) and Lewy neurites (LNs, arrowhead) are >-synuclein; >-synuclein staining also revealed intraneuronal (arrow) and ectopic (arrowhead) LBs (C) and LNs (D). Unusual >-synuclein staining was seen in the cytoplasm of astrocytes in the cingulate gyrus (arrow, E). LRRK2 staining displayed a Nissl-like pattern of distribution in some remaining neurons of the midbrain (F) and moderate staining in the core (arrow) of some ectopic inclusions (G). Double immunofluorescent staining with anti-LRRK2 (H) and antiY>-synuclein (H¶) antibodies demonstrates localization of LRRK2 in the LB core (arrow, Hµ). A section following the horizontal or vertical dashed line in the Z-stack acquired with the confocal microscope is displayed below and on the right side of the picture, respectively. Bars = (A, B) 200 Km; (C, E-G and H-Hµ) 10 Km; (D) 20 Km. Ó 2010 American Association of Neuropathologists, Inc.

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than in the control cases; they had occasionally a few small clumps of Nissl substance or more diffuse staining (Figs. 5, A, B, E, and F). By contrast, most of the large neurons in the III nerve nucleus were intensely stained with Nissl-like pattern, regardless of the disease status of the individuals (Figs. 5, C, D, G, and H). The density of the necklace-shaped fibers (Fig. 2E) was variable among the cases but was not significantly different between controls and PD patients, and no colocalization of necklace-shaped LRRK2-IR with >-synuclein labeling was observed (data not shown). In contrast, the AT106 Ab revealed abnormal immunopositive accumulations in the cell body or neurites of neurons in the affected SN (Figs. 6C, D). Moreover, intraneuronal and ectopic Lewy inclusions with more or less intense LRRK2-IR were found in the PD cases (Figs. 6A, B); the LRRK2-IR most often identified the central core of the LB and was absent when the primary Ab was omitted (data not shown). Double immunofluorescent staining of midbrain and pons sections was performed with AT106 and antiY>-synuclein Abs. In the SN, more than a total of 90 >-synuclein+ LBs were analyzed by confocal microscopy: 7.8% T 2.0% had strong LRRK2-IR in the core, 15.7% T 5.8% were only moderately labeled in the core, and 76.4% T 6.2% were LRRK2-negative (Figs. 6, E-Gµ). In the LC, more than 80 >-synuclein+ LBs were analyzed: 1% T 0.6% had strong LRRK2-IR in the core, 10.6% T 2.6% were only moderately labeled in the core, and 88.4% T 2.7% were LRRK2negative. The neuritic pattern showed in Figure 6D was confirmed in the LRRK2- and >-synuclein double stainings. Moderate LRRK2-IR was found in 20% of >-synuclein+ LNs of idiopathic PD patients. No LRRK2-IR was found in thin >-synuclein+ neurites (data not shown).

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stabilizes ER transmembrane kinases implicated in the ER stress pathway (63). The identification of other physiological protein targets for LRRK2 kinase activity and LRRK2interacting partners will be central to unraveling the physiological role of LRRK2, and the mechanisms underlying its potentially dynamic association with the ER. Of note, the in silico analysis of LRRK2 did not reveal the presence of transmembrane domains or organelle targeting sequence (28), suggesting that other proteins may be necessary to anchor LRRK2 to ER membranes. The identification of LRRK2-IR in the core of a substantial proportion of LBs in the brains of PD patients is the second major finding of the present study: 24% of the LBs were decorated with the AT106 Ab in the SN of cases with idiopathic PD and 50% in a carrier of the G2019S mutation with pathological features characteristic of PD. Moreover, in the LC, the percentage of LRRK2+ LBs was also higher in the patient with the G2019S LRRK2 mutation than in the idiopathic PD cases. LRRK2 was also localized in a substantial proportion of the LNs (20%) in both idiopathic PD cases and the LRRK2 patient. With a single exception (41), previous studies have either failed to detect LRRK2-IR in LBs (64Y66) or reported its presence in the halo of a small population of these inclusions, ranging from 10% to 15% (37, 41, 42, 48, 49, 52). To explain the absence of staining in LBs with Abs directed against internal domains of LRRK2, it has been previously hypothesized that folded domains/regions of LRRK2 are masked in LBs, such that only sequences outside the folded domains are exposed to Abs (49, 52). Although our observations show that the internal kinase domain of LRRK2 can be detected by AT106, Abs directed against external LRRK2 segments might more efficiently recognize the protein in LBs. Accordingly, using an Ab against the C-terminus of the protein, Alegre-Abarrategui et al (41) recently found LRRK2-IR in the core of 77% of SN LBs. The presence of LRRK2 in the central core of the LBs in idiopathic PD and LRRK2associated patients shown by our study and by AlegreAbarrategui et al indicates that LRRK2 is not simply passively entrapped at the periphery of the inclusions but could contribute to their buildup. The protein may have the propensity to accumulate within the >-synuclein ring, or it could be associated secondarily with the misfolded proteins in both LBs and LNs. The presence of LRRK2 in LNs suggests that the formation of LNs and LBs is closely related. LRRK2 may coprecipitate with >-synuclein in the cell body and then be targeted to the neuronal processes; alternatively, it could initially accumulate with >-synuclein in the cell processes before being concentrated in the cell body, thus evolving from LNs to LBs, as suggested by Kanazawa et al (67). Interestingly, a significantly higher proportion of LBs had intense LRRK2-IR in 2 areas (SN and LC) of the brain of the PD patient carrying LRRK2 mutation than in that of idiopathic PD cases (27% compared with 8%, p G 0.05). The degree of this strong colocalization of LRRK2 with the core of LBs, ranging from 19% to 33% of the inclusions depending on the brain area analyzed, was significantly higher than that found in the brains of idiopathic PD patients, in which only 0% to 15% of the LBs presented intense LRRK2-IR. Although this finding needs to be confirmed in additional Ó 2010 American Association of Neuropathologists, Inc.

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generally less than that observed with AT106. Neither of the Abs used in 2 previous studies of the LRRK2 distribution in different regions of the human (41) or mouse (37) brain yielded specific signals in Western blots or immunochemical labeling in our hands. Our analysis of the physiological expression of LRRK2 confirmed the presence of the protein in most human brain regions. As in other immunohistochemical studies on human tissues (41, 42, 45, 48), we found LRRK2-IR at moderate levels in the SN and, as observed by Alegre-Abarrategui et al (41), LRRK2-IR was also present in moderate levels in the LC, another region subject to neurodegeneration in PD. In contrast to several previous studies (42, 45), however, we observed only weak LRRK2-IR in dopaminoceptive regions, such as the caudate and putamen. In general, we observed widespread neuronal staining with moderate expression in areas affected by neurodegeneration in PD (SN and LC), but stronger immunoreactivities in regions where LBs are usually found (i.e., nucleus basalis of Meynert, dorsal motor nucleus of the vagus nerve). We occasionally observed necklaceshaped neurites similar to the TH+ neurites with a necklace shape recently detected in a mouse model with overproduction of a pathogenic variant of human LRRK2 (61); however, these neurites do not seem to be associated with disease in the human brain because they were found in both PD patients and healthy controls. Moreover, the LRRK2 protein was found in astrocytes and microglia as well as neurons. This observation is consistent with the previously reported ubiquitous expression of LRRK2 in the brain and other organs (47); it suggests a physiological role of LRRK2 in different cell types and tissues. Although the widespread expression of LRRK2 contrasts with the localized primary pathology of PD, it might underlie the pleomorphic pathologic findings observed in the brains of LRRK2-associated PD patients (16). Our study reveals for the first time that LRRK2 is mainly associated with the ER in neurons in the human brain. Colabeling of sections from the SN for LRRK2 and 2 ERspecific markers, namely, Neurotrace and the KDEL epitope, demonstrated correspondence of the mottled staining observed in many large neurons of the human brain with the ER. Interestingly, the Nissl-like pattern of LRRK2-IR was disturbed in the dopaminergic neurons of PD cases but not in neurons of the III nerve nucleus, suggesting specific disorganization of the normal LRRK2 distribution in dying cells. Partial colocalization of LRRK2 with the ER had been previously suggested by immunocytochemical analysis of HEK293 cells overproducing a LRRK2-GFP fusion protein (30). Moreover, LRRK2-IR was reported to be of Nissl-like appearance in motor neurons of the healthy human spinal cord, although the association of the proteins with the ER was not demonstrated by double immunostaining in that study (41). The colocalization of LRRK2 with the ER is consistent with the previously reported association of the protein with membranous and vesicular structures in subcellular fractionation studies (30, 43, 45). It suggests that LRRK2, like other members of the Roco protein family (62), could play a role as a protein sensor in the ER stress signaling pathway; this might be in concert with its partner HSP90, which

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cases, it may indicate a direct pathogenic mechanism linking LRRK2 mutations to LB formation.

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ACKNOWLEDGMENTS The authors thank Christelle Condroyer and Philippe Martin-Hardy for DNA extraction and genotyping samples of the brain bank, Ve´ronique Sazdovitch for the management and advice concerning human tissue samples, Matthew Farrer (Mayo Clinic, Jacksonville, FL) for kindly providing the LRRK2 complementary DNA, and Novartis (Basel, Switzerland) for providing the purified LRRK2 kinase domain. We appreciated technical assistance from ClaudeMarie Bachelet and Aure´lien Dauphin at the Plate-forme Imagerie Cellulaire and from Muriel Condolf and the technical staff of Escourolle Laboratory. The frozen samples of the PD cases have been obtained through the national Brain Bank Neuro-CEB, funded by the Patients’ Associations France Alzheimer, France Parkinson, ARSEP, and Comprendre les Syndromes Ce´re´belleux.

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