LRRK2: Biological Roles and Therapeutics Strategies

• Gene Information: Leucine-rich repeat kinase 2 (LRRK2) is a large multi-domain protein encoded by the LRRK2 gene. It possesses both GTPase and kinase activities and is widely expressed. The G2019S point mutation in LRRK2 is one of the most common pathogenic variants. This gain-of-function mutation markedly enhances LRRK2 kinase activity, and is a well-established major genetic risk factor for Parkinson’s disease.
• Protein Expression: LRRK2 is broadly distributed across multiple tissues. In the central nervous system, it is highly expressed in the midbrain, striatum, cerebral cortex, hippocampus, cerebellum and thalamus.
• Signaling Pathway: The hyperactive LRRK2 G2019S mutant induces multiple neuronal pathologies, which manifest as dysregulated Rab GTPase signaling, compromised lysosomal function and impaired protein quality control systems.
• Therapeutic Inhibition: Therapeutic targeting of the LRRK2 G2019S mutant holds great clinical potential. Common intervention approaches include suppressing mutant LRRK2 expression and blocking its aberrant kinase activity.

LRRK2

• Exons 1-53 of mouse Lrrk2 gene that encode the whole molecule (ATG to STOP codon), including the promoter, 5’UTR and 3’UTR were replaced with the corresponding human sequences.
• Human LRRK2 expression is driven by the human promoter, while endogenous mouse Lrrk2 transcription and translation are abolished.

• Human LRRK2 mRNA was specifically and correctly expressed in B-hLRRK2*G2019S mice.

Strain specific analysis of LRRK2 mRNA expression in wild-type C57BL/6JNifdc and B-hLRRK2*G2019S mice by RT-PCR and sequencing. Brian RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hLRRK2*G2019S mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human LRRK2 primers. Mouse Lrrk2 mRNA was only detectable in wild-type mice, human LRRK2 mRNA was exclusively detectable in homozygous B-hLRRK2*G2019S mice but not in wild-type mice. The point mutation of G2019S was confirmed via Sanger sequencing.

• Mouse Lrrk2 mRNA was only detectable in wild-type mice, human LRRK2 mRNA was exclusively detectable in homozygous B-hLRRK2*G2019S mice but not in wild-type mice.

Strain specific analysis of LRRK2 mRNA expression in wild-type C57BL/6JNifdc mice and B-hLRRK2*G2019S by RT-qPCR. Striatum, mid-brain, cortex and hippocampus RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hLRRK2*G2019S mice (H/H, n=3 per sex, 12-week-old), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human LRRK2 primers. Mouse Lrrk2 mRNA was only detectable in wild-type mice, human LRRK2 mRNA was exclusively detectable in homozygous B-hLRRK2*G2019S mice but not in wild-type mice. Mouse Gapdh served as the internal reference gene, and strain-specific LRRK2 expression in other tissues was normalized to mid-brain expression in male mice. Values are expressed as mean ± SEM.

• Mouse Lrrk2 mRNA was only detectable in wild-type mice, human LRRK2 mRNA was exclusively detectable in homozygous B-hLRRK2*G2019S mice but not in wild-type mice.

Strain specific analysis of LRRK2 mRNA expression in wild-type C57BL/6JNifdc mice and B-hLRRK2*G2019S by RT-qPCR. Lung, kidney and spleen RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hLRRK2*G2019S mice (H/H, n=3 per sex, 12-week-old), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human LRRK2 primers. Mouse Lrrk2 mRNA was only detectable in wild-type mice, human LRRK2 mRNA was exclusively detectable in homozygous B-hLRRK2*G2019S mice but not in wild-type mice. Mouse Gapdh served as the internal reference gene, and strain-specific LRRK2 expression in other tissues was normalized to mid-brain expression in male mice. Spleen samples were only collected from female mice, therefore, no spleen data are available for male mice. Values are expressed as mean ± SEM.

• The human LRRK2 were detected in both wild-type mice and homozygous B-hLRRK2*G2019S mice, as the antibody cross-recognized both human and mouse LRRK2.

Western blot analysis of LRRK2 protein expression in homozygous B-hLRRK2*G2019S mice. Various tissue were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hLRRK2*G2019S mice (H/H), and then analyzed by western blot with anti-LRRK2 antibody (Abcam, ab133474). 40 μg total proteins were loaded for western blotting analysis. LRRK2 was detected in both wild-type mice and homozygous B-hLRRK2*G2019S mice, as the antibody cross-recognized both human and mouse LRRK2.

• The human LRRK2 levels in the treatment group were reduced compared to the control group, demonstrating that B-hLRRK2*G2019S mice provide a powerful preclinical model for in vivo evaluation of human LRRK2-targeted nucleic acid drugs.

The inhibitory efficiency of the LRRK2-targeted small nucleic acid drug in homozygous B-hLRRK2*G2019S mice. B-hLRRK2*G2019S mice were randomly divided into 2 groups (n=4, 8-week-old, male). The human LRRK2-targeted nucleic acid drug (Test Article 1, TA1, produced in-house according to a patent) and artificial cerebrospinal fluid (αCSF) were administered to the mice individually. The mice were sacrificed on day 14, and the brains (cortex, hippocampus, mid-brain and striatum) were collected to detect the human LRRK2 expression by qRT-PCR. (A) The schematic diagram of experimental processing. (B) The expression of human LRRK2 mRNA in cortex, hippocampus, mid-brain and striatum. Gapdh served as an internal reference gene, and LRRK2 expression in other tissues was normalized to that in the mid-brain. The human LRRK2 mRNA in the treatment group was significantly reduced compared to the control group. Values are expressed as mean ± SEM. Significance was determined by 2-way ANOVA.  *P < 0.05, **P < 0.01, ***P < 0.001.