Header
Header
Article

Pathophysiological evaluation of the LRRK2 G2385R risk variant for Parkinson’s disease


Background information of patients and controls

The G2385R patient’s family was from the eastern area of Japan with no consanguinity reported. No other members in the G2385R family developed PD. We prepared samples from three patients with no PD as controls, four patients with sporadic PD, and two patients harboring LRRK2 R1441H or I2020T29,30,54. This study (M08-0477) was approved by the ethics committee of Juntendo University, Tokyo, Japan. Written consent was obtained from all the patients who were included in Supplementary Table 1.

Clinical and genetic analysis

PD diagnosis was made using the Movement Disorder Society clinical diagnostic criteria for PD55. Brain MRI, melanin imaging MRI, SPECT with intravenous injection of N-isopropyl-p-[123I] iodoamphetamine, DAT-SPECT, and myocardial scintigraphy with [123I] MIBG were performed as neuroimaging examinations. Genomic DNA was extracted from peripheral blood samples using a standard protocol. We screened genes related to familial PD or dementia using target sequencing by Ion Torrent system (Thermo Fisher Scientific, Waltham, MA, USA); the panel (IAD103177_182) was set up to screen SNCA, PARK2, UCHL1, PINK1, DJ-1, LRRK2, ATP13A2, GIGYF2, HTRA2, PLA2G6, FBXO7, VPS35, EIF4G1, DNAJC6, SYNJ1, DNAJC13, CHCHD2, GCH1, NR3A2, VPS13C, RAB7L1, BST1, c19orf12, RAB39B, MAPT, PSEN1, GRN, APP, and APOE56. The identified variants were confirmed by Sanger sequencing. The panel for sequencing was designed with Ion AmpliSeq Designer (Thermo Fisher Scientific, https://www.ampliseq.com). We also collected genomic DNA from the brain tissues of 10 individuals, which included control cases, using QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany). LRRK2 exon 48 was sequenced using the Sanger method, as reported previously2. These genetic markers were genotyped by Sanger method as reported previously57.

Neuropathological analysis

We obtained brain autopsies from the patients and carried out neuropathological examinations, comparing them to those of control cases. Brains were fixed with 15% neutral buffered formalin, and the selected tissues were embedded in paraffin. The paraffin embedded blocks were sliced at 6 μm thickness. Brain sections were stained with hematoxylin and eosin, Klüber-Barrera, methenamine-silver stain, Gallyas–Braak stain, and immunohistochemical staining for proteins related to neurodegenerative diseases. For immunohistochemistry, brain sections underwent antigen retrieval either by heat activation in a microwave oven or by reaction in formic acid, before being incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: anti-phospho-Ser129 αS (pSyn#64, monoclonal, 1:1,000 dilution, Wako, Osaka, Japan or EP1536Y, 1:400, Abcam, Cambridge, UK), anti-phospho-tau (AT8, monoclonal, 1:200, Thermo Fisher Scientific), anti-tau RD3 (8E6/C11, 1:100, Merck, Darmstadt, Germany), anti-tau RD4 (1E1/A6, 1:100, Merck)58, anti-4R tau (TIP-4RT-P01, 1:3,000, Cosmo Bio, Tokyo, Japan), anti-amyloid ß (1–42, polyclonal, 1:100, IBL, Gunma, Japan), anti-TH (TH-16, 1:2,000, Sigma-Aldrich, Merck), anti-phospho-TDP-43 (11–9, Ser 409/410, monoclonal, 1:3,000, Cosmo Bio), anti-GFAP (G-25-8-3, monoclonal, 1:500, IBL), and anti-ionized calcium-binding adapter molecule 1 (Iba1; polyclonal, 1:500, Wako). Immunosignals were visualized using the peroxidase-polymer-based method with a Histofine Simple Stain MAX-PO kit (Nichirei, Tokyo, Japan) and diaminobenzidine as the chromogen.

Biochemical analysis using brain autopsies

Biochemical fractionation using human brain tissues was performed as described previously59. Briefly, 20 mg of the putamen dissected from each frozen tissue sample was homogenized in 200 μL Buffer A (10 mM Tris–HCl, pH 7.4, 0.8 M NaCl, 1 mM EGTA, 10% sucrose) with a complete protease inhibitor cocktail (Merck) and spun at 100,000 g for 20 min at 4 °C. The supernatant was retained as a Tris buffer-soluble fraction. The pellet rinsed with 200 μL Buffer A was homogenized in 150 μL Buffer A containing 1% Triton X-100 and incubated for 30 min at 37 °C. After centrifugation at 100,000 g, the supernatant was retained as a Triton X-soluble fraction. The pellet was then rinsed with 150 μL Buffer A and further homogenized in 100 μL Buffer A containing 1% sarkosyl, incubated at 37 °C for 30 min and spun at 100,000 g for 20 min. The supernatant was retained as a sarkosyl-soluble fraction. The sarkosyl-insoluble pellet was resuspended in Laemmli sodium dodecyl sulfate (SDS) sample buffer and used as a sarkosyl-insoluble fraction. Tris buffer-soluble, Triton X-soluble, sarkosyl-soluble, and sarkosyl-insoluble fractions were subjected to SDS-polyacrylamide gel electrophoresis/western blotting. These sequential extraction procedures were performed repeatedly by using 20 mg of the frontal, temporal, occipital, entorhinal cortexes, and cerebellum dissected from each frozen tissue sample. All blots derive from the same experiment and were processed in parallel. The full-length uncropped images of western blot results were shown in Supplementary Fig. 2.



Source link

Back to top button