Overexpression of Parkinson’s Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and α-Synuclein Pathology

Generation of TET-inducible conditional LRRK2 transgenic mice. A, Schematic diagram of the TetP-LRRK2-TAP construct. B, Relative transgene copy number determined by semiquantitative PCR performed on genomic DNA expressed in arbitrary units as the ratio of the TetP-LRRK2-G2019S (GS) transgene to GAPDH for each founder mouse. C, Relative transgene copy number determined by semiquantitative PCR performed on genomic DNA expressed in arbitrary units as the ratio of the TetP-LRRK2-GS2019S/D1994A (GS/DA) transgene to GAPDH for each founder mouse. D, Schematic diagram of the generation of LRRK2-inducible transgenic mice using the “tet-off” system. E, Representative genotyping PCR for TetP-LRRK2 and CamKIIα-tTA using genomic DNA. GAPDH PCR was used as an internal control. F, Western blot analysis of LRRK2 expression from LRRK2 transgenic mouse brain. Each number represents a single LRRK2 transgenic founder line: 569, 648 and 597 of LRRK2-GS, 767, 763, and 768 LRRK2-GS/DA transgenic mice. G, Quantification of LRRK2 expression in mouse brains normalized to β-actin, n = 3. Differences between transgenic and control groups were assessed by unpaired, two-tailed Student’s t test. Quantified data are expressed as the mean ± SEM. *p < 0.05, ***p < 0.001. H, Representative Western blots of LRRK2 distribution in brain subregions from control and LRRK2-GS (line 569) and LRRK2-GS/DA (line 763) transgenic mice (OB; CTX; STR; HIP; VMB; BS; CER). I, Quantification of LRRK2 distribution in mouse brains normalized to β-actin, n = 3. J, Western blot analysis of total protein from mouse brains of control, LRRK2 GS, and LRRK2 GS/DA by anti-LRRK2 and LRRK2 phosphor S1292 antibodies. K, Quantification of phosphor Ser1292 LRRK2 levels in mouse brains normalized to total LRRK2 protein level, n = 3. Quantified data are expressed as the mean ± SEM: *p < 0.05, **p < 0.01, ***p < 0.001, Differences between transgenic and control groups were assessed by two-way ANOVA. There was a nonsignificant difference between LRRK2 GS and LRRK2 GS/DA groups.

Behavioral deficits of conditional LRRK2-G2019S transgenic mice. A, Open field analysis under normal conditions and following amphetamine challenge. Mice were placed in the center of the open field arena and allowed to explore the area for 25 min, following by d-amphetamine injection (7 mg/kg, s.c.) and another 25 min exploration. The total activities of mice were recorded every minute (control, n = 7; GS, n = 9; GS/DA, n = 8). B, Pole test to monitor the behavioral abnormalities of 22-month-old LRRK2 GS and GSDA transgenic and age-matched littermate controls (control, n = 8; GS, n = 8; GS/DA, n = 7). C, Assessment of latency to fall in an accelerated rotarod test (control, n = 8; GS, n = 9; GS/DA, n = 8). Data are the mean ± SEM. Statistical significance was determined by two-way ANOVA. *p < 0.05. n.s., Nonsignificant.

Characterization of the nigrostriatal pathway of LRRK2 conditional transgenic mice. A, Representative TH immunohistochemistry of the midbrain coronal sections of 22-month-old LRRK2 GS and GSDA transgenic and age-matched littermate controls. B, C, Stereological assessment of TH-positive (B) and Nissl-positive (C) neurons in the SNpc (control, n = 9; GS, n = 9; GS/DA, n = 9). Data are the mean number of cells per region ± SEM, n = 9 mice per group. Statistical significance was determined by two-tailed unpaired Student’s t test. D, Representative images of TH immunostaining of nerve terminals in the striatum of LRRK2 conditional transgenic mice at 22 months of age. E, Quantitation of TH immunostaining in the striatum using ImageJ software (NIH; control, n = 7; GS, n = 7; GS/DA, n = 7). Differences between groups were assessed by two-way ANOVA. Bars represent the mean ± SEM (n ≥ 5 animals/genotype). n.s., Nonsignificant.