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. 2013 Aug 9:549:57-62.
doi: 10.1016/j.neulet.2013.05.034. Epub 2013 May 28.

Diapocynin prevents early Parkinson's disease symptoms in the leucine-rich repeat kinase 2 (LRRK2R¹⁴⁴¹G) transgenic mouse

Brian P Dranka  1 , Alison Gifford, Anamitra Ghosh, Jacek Zielonka, Joy Joseph, Anumantha G Kanthasamy, Balaraman Kalyanaraman
Affiliations

Diapocynin prevents early Parkinson's disease symptoms in the leucine-rich repeat kinase 2 (LRRK2R¹⁴⁴¹G) transgenic mouse

Brian P Dranka et al. Neurosci Lett. .

Abstract

The most prominent mechanism proposed for death of dopaminergic neurons in Parkinson's disease (PD) is elevated generation of reactive oxygen/nitrogen species (ROS/RNS). Recent studies suggest that ROS produced during PD pathogenesis may contribute to cytotoxicity in cell culture models of PD. We hypothesized that inhibition of ROS production would prevent PD symptoms in the LRRK2(R1441G) transgenic (tg) mouse model of PD. These mice overexpress a mutant form of leucine-rich repeat kinase 2 (LRRK2) and are reported to develop PD-like symptoms at approximately 10 months of age. Despite similar expression of the transgene, our colony did not recapitulate the same type of motor dysfunction originally reported. However, tests of motor coordination (pole test, Rotor-Rod) revealed a significant defect in LRRK2(R1441G) mice by 16 months of age. LRRK2(R1441G) tg mice, or wild type littermates, were given diapocynin (200mg/kg, a proposed NADPH oxidase inhibitor) three times per week by oral gavage starting at 12 weeks of age. Decreased performance on the pole test and Rotor-Rod in the LRRK2(R1441G) mice was prevented with diapocynin treatment. No loss in open field movement or rearing was found. As expected, tyrosine hydroxylase staining was similar in both the substantia nigra and striatum in all treatment groups. Together these data demonstrate that diapocynin is a viable agent for protection of neurobehavioral function.

Keywords: LRRK2; NADPH; NADPH oxidase isoform 2; NOX2; NSAID; Neurobehavioral analysis; Neurodegeneration; PD; PMA; Parkinson's disease; RNS; ROS; TH; leucine-rich repeat kinase 2; nicotinamide adenine dinucleotide phosphate (reduced form); non-steroidal anti-inflammatory drug; phorbol-12-myristate-13-acetate; reactive nitrogen species; reactive oxygen species; tyrosine hydroxylase.

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Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. LRRK2R1441G tg mice genotyping
Wild type FVB and LRRK2R1441G tg genotype was confirmed using PCR (A) and Sanger sequencing (B). Transgene copy number was compared to animals with known genotype to confirm hemizygous expression of LRRK2R1441G (C). Samples from each mouse were run in triplicate. Data shown are means ± s.d. n≥2 mice per group as indicated.
Figure 2
Figure 2. Open field movement is not decreased in LRRK2R1441G tg mice
From open field photobeam tracking experiments, the total distance travelled was calculated (A). Location in the open field apparatus was tracked as percentage time occupying each coordinate for each animal. Averages were then plotted as a heat map (B). The average duration of each dwell (C) and total number of dwells ≥ 1 s (D) were calculated. Gait analysis was used to monitor stride count (E) and length (F) over a constant distance (G). Data shown are means ± s.d. Significance was assessed by two-tailed t-test, and p values are shown for each measurement. n≥4 mice per group.
Figure 3
Figure 3. Diapocynin corrects loss of motor coordination without affecting gross motor function
Diapocynin was synthesized through the reaction of two apocynin monomers in the presence of ferrous sulfate and sodium persulfate (A). Purity of the isolated compound was confirmed by HPLC (B) and MS analysis (C). An apocynin standard was run in parallel to confirm purity of the diapocynin solution. At 16 mo, brains were formaldehyde fixed, sliced, and slices containing the nigral region were stained for TH (D-G). Images shown are representative with 4x magnification in insets. Total movement (H) and rearing (I) were assessed over 20 min in an open field using a photobeam system. Latency to fall from a Rotor-Rod apparatus was also measured (J). Data are the means ± s.d. *, p≤0.05 as determined by one-way ANOVA with Bonferroni post-hoc. Encircled numbers represent the number of animals in each group.
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