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Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies
Published on: June 2, 2021
Targeting specific kinase substrates rescues increased colitis severity induced by the Crohn's disease-linked
George R Heaton1, Xingjian Li1, Xianting Li1
1Center for Parkinson's Disease Neurobiology, Departments of Neurology and Neuroscience, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Abstract:
LRRK2 contains a kinase domain where the N2081D Crohn's disease (CD) risk and the G2019S Parkinson's disease (PD) pathogenic variants are located. It is not clear how the N2081D variant increases CD risk or how these adjacent mutations give rise to distinct disorders. To investigate the pathophysiology of the CD-linked LRRK2 N2081D variant, we generated a knock-in (KI) mouse model and compared its effects with those of the LRRK2-G2019S mutation. Lrrk2N2081D KI mice demonstrated heightened sensitivity to induced colitis, resulting in more severe intestinal damage than in Lrrk2G2019S KI and WT mice. Analysis of colon tissue revealed distinct mutation-dependent LRRK2 RAB substrate phosphorylation, with significantly elevated phosphorylated RAB10 levels in Lrrk2N2081D mice. In cells, we demonstrated that the N2081D mutation activates LRRK2 through a mechanism distinct from that of LRRK2-G2019S. We also found that proinflammatory stimulation enhances LRRK2 kinase activity, leading to mutation-dependent differences in RAB phosphorylation and inflammatory responses in dendritic cells (DCs). Finally, we show that knockout of Rab12, but not pharmacological LRRK2 kinase inhibition, significantly reduced colitis severity in Lrrk2N2081D mice. Our study characterizes the pathogenic mechanisms of LRRK2-linked CD, highlights structural and functional differences between disease-associated LRRK2 variants, and suggests RAB proteins as promising therapeutic targets for modulating LRRK2 activity in CD treatment.
Insights
The Crohn's disease (CD) linked LRRK2 N2081D variant increases susceptibility to colitis by altering RAB10 phosphorylation. This highlights distinct pathogenic mechanisms for LRRK2 variants and identifies RAB proteins as potential therapeutic targets for CD.
Area of Science:
- Genetics and Molecular Biology
- Immunology
- Gastroenterology
Background:
- The leucine-rich repeat kinase 2 (LRRK2) gene harbors variants associated with distinct diseases, including Crohn's disease (CD) and Parkinson's disease (PD).
- The precise mechanisms by which the N2081D CD-risk variant and the G2019S PD-pathogenic variant of LRRK2 lead to different pathologies remain unclear.
- Understanding these distinct mechanisms is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the pathophysiology of the LRRK2 N2081D variant associated with Crohn's disease.
- To compare the effects of the LRRK2 N2081D variant with the LRRK2 G2019S variant using knock-in mouse models.
- To identify potential therapeutic targets for LRRK2-associated CD.
Main Methods:
- Generation of LRRK2 N2081D knock-in (KI) mouse models.
- Induction of colitis to assess disease severity and intestinal damage.
- Analysis of LRRK2 substrate phosphorylation, particularly RAB proteins, in colon tissue and dendritic cells (DCs).
- Investigation of LRRK2 activation mechanisms and the role of Rab12.
Main Results:
- Lrrk2N2081D KI mice exhibited heightened sensitivity to colitis, with more severe intestinal damage compared to Lrrk2G2019S KI and wild-type (WT) mice.
- Distinct mutation-dependent LRRK2 RAB substrate phosphorylation was observed, with significantly elevated phosphorylated RAB10 levels in Lrrk2N2081D mice.
- The N2081D mutation activates LRRK2 via a distinct mechanism compared to G2019S, and knockout of Rab12 significantly reduced colitis severity in Lrrk2N2081D mice.
Conclusions:
- The study elucidates the pathogenic mechanisms of LRRK2-linked CD, demonstrating the N2081D variant's role in heightened colitis susceptibility.
- Significant structural and functional differences exist between disease-associated LRRK2 variants (N2081D and G2019S).
- RAB proteins, particularly RAB10, are implicated in LRRK2-mediated CD pathogenesis and represent promising therapeutic targets.
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