Abstract
LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.
Highlights
Parkinson’s disease (PD), the second-most common neurodegenerative disorder, is caused by degeneration of dopaminergic neurons in the substantia nigra (SN)
Consistent with the results, a qRT-PCR analysis showed that LPS (100 ng/mL)-induced expression of IL-1b, IL-6, and inducible nitric oxide synthase (iNOS) mRNA was dramatically reduced in Lrrk2-KD clones at 3 and 9 hours (Fig. 1E)
Using agonists for TLR2, TLR-7/8 (CL097, 500 ng/ml), and TLR-9 (ODN1668, 500 ng/ ml), we found that Lrrk2-KD could regulate inflammatory responses mediated by these TLRs since nitric oxide production induced by these TLR agonists were significantly reduced in Lrrk2 KD clones (Fig. 1F–H)
Summary
Parkinson’s disease (PD), the second-most common neurodegenerative disorder, is caused by degeneration of dopaminergic neurons in the substantia nigra (SN). 51 disease-associated mutations in LRRK2 have been identified in familial or sporadic cases. These mutations are scattered throughout the entire LRRK2 gene, and include R1441C/G/H in the ROC domain, G2019S in the kinase domain, and G2385R in the WD40 domain [1,2,3,4]. There is evidence showing that expression of pathological LRRK2 mutations is sufficient to cause neurotoxicity in vitro [5,6], transgenic LRRK2 mutant mice show little or no obvious degeneration of dopaminergic neurons [7,8,9,10]. It has been suggested that certain changes in the brain microenvironment may cooperate with genetic defects to promote the development of PD [11]
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