Environmental Factors Exacerbate Parkinsonian Phenotypes in an Asian-Specific Knock-In LRRK2 Risk Variant in Mice

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder affecting nearly 10 million people worldwide, and for which no cure is currently known. Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene, age, as well as environmental factors such as neurotoxin exposure and stress, are known to increase the risk of developing the disease in humans. To investigate the role of a specific Asian variant of the LRRK2 gene to induce susceptibility to stress and trigger PD phenotypes with time, knock-in (KI) mice bearing the human LRRK2 R1628P risk variant have been generated and studied from 2 to 16 months of age in the presence (or absence) of stress insults, including neurotoxin injections and chronic mild stress applied at 3 months of age. Pathophysiological and behavioural phenotypes have been measured at different ages and primary neurons and fibroblast cells were cultured from the KI mouse line and treated with H₂O₂ to study susceptibility towards oxidative stress in vitro. KI mice displayed specific PD features and these phenotypes were aggravated by environmental stresses. In particular, KI mice developed locomotion impairment and increased constipation. In addition, dopamine-related proteins were dysregulated in KI mice brains: Dopamine transporter (DAT) was decreased in the midbrain and striatum and dopamine levels were increased. Primary fibroblast cells and cortical neurons from KI mice also displayed increased susceptibility to oxidative stress. Therefore, the LRRK2 R1628P KI mice are an excellent model to study the progressive development of PD.

Keywords: Asian specific variant; LRRK2; Parkinson’s disease; behaviour; environmental toxins; oxidative stress; physiological stress.

Figure 1

R1628P risk variant in mice differentially regulates key PD-related proteins. (A) Schematic representation of the WT and KI transgenic constructs. (B) PCR genotyping analysis of LRRK2 R1628P mouse model. Lane 1: WT/WT (WT), Lane 2: KI/KI (KI), Lane 3: WT/KI (Het), Lane 4: DNA ladder. (C) Western blot analysis of LRRK2, DAT (upper band), DD2R and α-synuclein in the midbrain and striatum of 8-month-old WT and KI mice. Protein levels normalized to aged-matched WT controls (n = 6/group). (D) Autoradiograph evaluation of DAT in the midbrain of KI mice and WT controls at age 3, 6 and 12 months old (n = 3/group). (E) HPLC analysis of midbrain DA levels in KI mice at 8 and 16 months old. Levels normalized to aged-matched WT controls (n = 3 to 4/group). Data are means ± SEM; One-way ANOVA followed by Bonferroni post hoc comparisons, * p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Combination of Paraquat/Maneb and unpredictable chronic mild stress accelerates behavioural deficits in LRRK2 R1628P KI mice (A) Experimental outline. PQMB: Paraquat/Maneb, UCMS: Unpredictable chronic mild stress, B: Behavior analysis, P: Protein analysis. Six PQMB or saline injections were given at 3 months old and the PQMB-treated group underwent additional six weeks of UCMS. (B) Open field test at 2, 6, 9, 12, and 16 months old; left: distance travelled, middle: total number of rearing, right: number of entries in the centre. Two-month-old mice outperform 6-, 9-, 12-, and 16-month-old mice in both WT and KI groups (n number: Control WT-9/Control KI-8/Stressed WT-8/Stressed KI-16) [Tests of between-subject effects: Distance genotype + age combination effect, F(4,18) = 2.714; p < 0.033/Distance age effect, F(4,18) = 13.264; p < 0.01/Rearing age effect, F(4,18) = 6.631; p < 0.01/Center entries age effect, F(4,18) = 10.663; p < 0.01]. (C) Cylinder test at 6, 9, 12, and 16 months old (n number: Control WT-8/Control KI-9/Stressed WT-6/Stressed KI-23) [Tests of between-subject effects: Rearing genotype + stress combination effect, F(1,16) = 5.132; p < 0.025]. (D) Analysis of stool water content and number of stool droppings at 6, 9, 12, and 16 months old (n number: Control WT-8/Control KI-8/Stressed WT-6/Stressed KI-15) [Tests of between-subject effects: Water content genotype effect, F(1,15) = 11.048; p < 0.001/Water content age effect, F(1,15) = 2.591; p < 0.056/Nb of droppings genotype effect, F(1,15) = 5.626; p < 0.019/Nb of droppings age effect, F(1,15) = 4.362; p < 0.006]. (E) Body weight data over time. (F) Number of TH+ cells in substantia nigra using DAB staining (n = 6/group), scale bar: 100 µm. Data are means ± SEM, Two-way ANOVA followed by Bonferroni post hoc comparisons, and repeated measures considered for (B–E); * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Oxidative stress modulates RAB10, AKT, and ERK activity in LRRK2 R1628P WT and KI primary fibroblast cells and primary cortical neurons. (A) Oxidative stress treatment. H₂O₂ was introduced to WT and KI primary fibroblast cells for 1 h (short-term) and 24 h (long-term). Subsequently, Western blot was performed using antibodies specific for LRRK2, p-RAB10, p-ERK, and p-AKT. Protein levels normalized to WT controls (mean ± SEM) (n = 3/group). (B) Western blot analysis of WT primary cortical neurons, KI primary cortical neurons, and KI primary cortical neurons that have been treated with H₂O₂ for 1 h (short-term) and 24 h (long-term). Antibodies specific for LRRK2, p-AKT, p-ERK, and p-RAB10 were used. Protein levels normalized to WT group (mean ± SEM) (n = 3/group). One-way ANOVA followed by Bonferroni post hoc comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

RAB10, AKT, and ERK activation is altered in primary midbrain neurons from LRRK2 R1628P KI mice. Western blot analysis of primary midbrain neurons from WT and KI mice. LRRK2, TH, p-RAB10, p-AKT, and p-ERK were analyzed. Protein levels normalized to WT controls, t-Test with Welch correction (mean ± SEM) (n = 3/group), * p < 0.05; ** p < 0.01.