Homozygous mutation of the LRRK2 ROC domain as a novel genetic model of parkinsonism

Abstract

Background: Parkinson's disease (PD) is one of the most important neurodegenerative disorders in elderly people. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in a large proportion of the patients with sporadic and familial PD. Mutations can occur at different locations in the LRRK2. Patients with LRRK2 ROC-COR mutations face an increased risk of typical motor symptoms of PD, along with cognitive decline. An animal model with a monogenic LRRK2 gene mutation is a suitable model for exploring the pathophysiology of PD and identifying potential drug therapies. However, the effect of homozygous (HOM) LRRK2 in PD pathophysiology is unclear.

Methods: We established human LRRK2 (hLRRK2) R1441G HOM transgenic (Tg) mice to explore the phenotype and pathological features that are associated with hLRRK2 R1441G Tg mouse models and discuss the potential clinical relevance. The open field test (OFT) was performed to examine motor and nonmotor behaviors. A CatWalk analysis system was used to study gait function. [¹⁸F]FDOPA PET was used to investigate functional changes in the nigrostriatal pathway in vivo. Transmission electron microscopy was used to examine the morphological changes in mitochondria and lysosomes in the substantia nigra.

Results: The R1441G HOM Tg mice demonstrated gait disturbance and exhibited less anxiety-related behavior and exploratory behavior than mice with hLRRK2 at 12 months old. Additionally, [¹⁸F]FDOPA PET showed a reduction in FDOPA uptake in the striatum of the HOM Tg mice. Notably, there was significant lysosome and autophagosome accumulation in the cytoplasm of dopaminergic neurons in R1441G hemizygous (HEM) and HOM mice. Moreover, it was observed using transmission electron microscopy (TEM) that the mitochondria of R1441G Tg mice were smaller than those of hLRRK2 mice.

Conclusion: This animal provides a novel HOM hLRRK2 R1441G Tg mouse model that reproduces some phenotype of Parkinsonism in terms of both motor and behavioral dysfunction. There is an increased level of mitochondrial fission and no change in the fusion process in the group of HOM hLRRK2 R1441G Tg mouse. This mutant animal model of PD might be used to study the mechanisms of mitochondrial dysfunction and explore potential new drug targets.

Keywords: Anxiety; Fission; GTPase activity; Gait; Homozygous; LRRK2; PET; Parkinsonism; R1441G.

Fig. 1

Detection of hLRRK2 and hLRRK2 R1441G expression and GTPase activity in Tg mice. A Real-time PCR assays of hLRRK2 and hLRRK2 R1441G mRNA expression in the brains of Tg mice. Data represent relative mRNA levels (mean ± SEM, n = 8) normalized to mouse apoB and are expressed in arbitrary units. B LRRK2 protein expression in the brains of non-Tg and Tg mice (n = 3). C hLRRK2 R1441G HEM and HOM mice lost less weight than non-Tg mice. Data are presented as the mean ± SEM (n = 6). D GTPase activity was measured using an enzyme-linked inorganic phosphate assay (ELIPA; n = 3). E Disruption of LRRK2 Ser935 phosphorylation in the brains of hLRRK2, R1441G HOM mice. Total lysates from the brains of 12-month-old hLRRK2 R1441G HOM mice were analyzed by western blotting for phosphorylation of LRRK2 at Ser935; actin was used as a loading control. *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Fig. 2

Locomotor activity in the open-field test was significantly reduced in hLRRK22 R1441G HOM mice. Data are presented as the mean ± SD (n = 5). *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Fig. 3

Several parameters of measured by the CatWalk system were affected in various ways in four mice. The swing velocity, cadence, and stride length were decreased in hLRRK2 R1441G HOM mice. In contrast, the stance duration and the pressure on the hind-paws BOS increased in hLRRK2 R1441G HOM mice. Data are presented as the mean ± SD (n = 5). *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Fig. 4

Comparison of [18F]FDOPA images from four groups of mice, showing significantly decreased uptake of the ligand among Tg mice: A coregistered coronal [¹⁸F]FDOPA images of the non-Tg mouse striatum; B coregistered coronal [¹⁸F]FDOPA images of the hLRRK2 mouse striatum; C coregistered coronal [¹⁸F]FDOPA images of the hLRRK2 R1441G HEM mouse striatum; D coregistered coronal [¹⁸F]FDOPA images of the hLRRK2 R1441G HOM mouse striatum; E average [¹⁸F]FDOPA uptake in the region of interest (striatum) in various groups. The uptake values are A, B, C, and D for Group 1,2,3, and 4, respectively Data are presented as the mean ± SD (n = 5–6). *Significant effect, p < 0.05; **Significant effect, p < 0.01; ***Significant effect, p < 0.005; ****Significant effect, p < 0.001

Fig. 5

Effect of SN in hLRRK2, R1441G HEM and HOM mice SNc region. A Ultrastructural analysis of SN in hLRRK2, R1441G HEM and HOM mice SNc region. Non-Tg and hLRRK2 represent a healthy mitochondrion; HEM and HOM represent a swollen mitochondrion. Selected regions in different magnification images (I, 10,000×; II, 20,000×). Asterisks indicate shrinkage and matrix condensation. HEM, R1441G HEM; HOM, R1441G HOM. B Western blot analysis of mitochondrial fission proteins in the mitochondrial fraction of the whole brain. Equal loading of the gel is demonstrated with Ponceau S staining for mitochondrial protein fragmentation performed on the blot before immunostaining. B Representative western blot for Drp1 and Fis1 expression. Densitometry of the Drp1 (C) and Fis1 (D) blots is shown as the fold increase (HEM or HOM hLRRK2). The figure shows a representative (of two) experiment. Values are means ± SD. *Significant effect, p < 0.05; **significant effect, p < 0.01; ***significant effect, p < 0.005; ****significant effect, p < 0.001

Fig. 6

LRRK2 R1441G HOM mice accumulated more autophagosomes in the SNc. A Transmission electron microscopic images of autophagosomes from the SN of 12-month-old hLRRK2 and R1441G transgenic mice. Selected regions in images at different magnifications (I, 10,000×; II, 20,000×). Arrows indicate autolysosomes, and asterisks indicate autophagosomes. Markers of autophagy (LC3 and p62) in the SNc of hLRRK2 and R1441G transgenic mice were determined by western blotting. p62 undergoes degradation at the early phase of autophagy. p62 in mitochondria serves as an adapter for autophagosome recognition. The data appear to downregulate the autophagy process, as observed by the increasing LC3-II conversion and the accumulation of p62, a marker of autophagic degradation