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Comparative Study
. 2009 Dec 16;29(50):15846-50.
doi: 10.1523/JNEUROSCI.4357-09.2009.

Unexpected lack of hypersensitivity in LRRK2 knock-out mice to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

Affiliations
Comparative Study

Unexpected lack of hypersensitivity in LRRK2 knock-out mice to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

Eva Andres-Mateos et al. J Neurosci. .

Abstract

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known cause of Parkinson's disease (PD). Whether loss of LRRK2 function accounts for neurodegeneration of dopamine neurons in PD is not known, nor is it known whether LRRK2 kinase activity modulates the susceptibility of dopamine (DA) neurons to the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To better understand the role of LRRK2 in DA neuronal survival and its role in the susceptibility of DA neurons to MPTP, we generated LRRK2 knock-out (KO) mice lacking the kinase domain of LRRK2. Here, we show that LRRK2 KO mice are viable and have no major abnormalities and live to adulthood. The dopaminergic system is normal in LRRK2 KO mice as assessed via HPLC for DA and its metabolites and via stereologic assessment of DA neuron number in young and aged mice. Importantly, there is no significant difference in the susceptibility of LRRK2 KO and wild-type mice to MPTP. These results suggest that LRRK2 plays little if any role in the development and survival of DA neurons under physiologic conditions. Thus, PD due to LRRK2 mutations are likely not due to a loss of function. Moreover, LRRK2 is not required for the susceptibility of DA neurons to MPTP.

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Figures

Figure 1.
Figure 1.
Targeted disruption of LRRK2 in LRRK2 KO mice. A, Schematic representation of the targeting strategy. B, Southern blot analysis of genomic DNA from WT (+/+), heterozygous (+/−) and homozygous LRRK2 KO (−/−) mice. C, PCR analysis of genomic DNA from heterozygous (+/−), WT (+/+), and homozygous LRRK2 KO (−/−) mice. D, A cDNA fragment of LRRK2 mRNA open reading frame was used as the probe for Northern blot analysis of total RNA from WT (+/+), homozygous LRRK2 KO (−/−), and heterozygous (+/−) mice. E, Immunoblot analysis of whole-brain lysate from WT, heterozygous (Het), and homozygous LRRK2 KO mice with affinity-purified rabbit polyclonal antibodies and monoclonal antibody 6.1 generated to LRRK2 protein using different synthetic peptides against human LRRK2. The blot was reprobed with anti-actin antibody as loading control.
Figure 2.
Figure 2.
Characterization of the nigrostriatal dopaminergic system in LRRK2 KO mice. A–D, Immunohistochemical staining for TH and Nissl was performed on coronal brain sections from LRRK2 WT (A, C) and KO (B, D) mice. Striatum (A, B) and ventral midbrain (C, D); scale bars, 500 μm. E, Stereological analysis of the number of TH-positive and Nissl-positive neurons in substantia nigra of 2–3 month and 18–22 month mice (n = 5). F, G, Striatal concentrations of dopamine and its metabolites were determined by HPLC with electrochemical detection in young (F) and aged (G) LRRK2 WT and KO mice (n = 6). The data are the mean ± SEM.
Figure 3.
Figure 3.
Evaluation of the effect of MPTP in LRRK2 KO mice. Stereological counting of dopaminergic neurons in the substantia nigra of mice treated with MPTP. TH immuno-staining in the substantia nigra of WT (A, B) and LRRK2 KO (C, D) mice treated (B, D) or nontreated (A, C) with MPTP; scale bars, 500 μm. E, Number of TH- and Nissl-positive neurons in the substantia nigra of WT and LRRK2 KO mice treated or nontreated (saline) with MPTP, determined by stereological counting. The data are the mean ± SEM (n = 6). F, Levels of DA and its metabolites (DOPAC and HVA) in the striatum of WT and LRRK2 KO mice treated or nontreated (saline) with MPTP. The data are the mean ± SEM (n = 7). *p ≤ 0.01.

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