No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts

<div><p>Mutations within the <i>LRRK2</i> gene have been identified in Parkinson’s disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.</p></div

Analysis of neurite outgrowth and branching complexity of primary hippocampal neurons.

<p><b>A-B:</b> Neurite parameters of neuronal cultures from LRRK2, GS-LRRK2 (line 2) and their respective non-tg littermate, which were treated with vehicle-control or LRRK2-IN-1 (0.1 M) for seven days (DIV7). Comparison of parameters describing neurite branching (A) included number of branches, number of neurite trees and number of segments. Comparison of neurite length parameters (B) included total neurite length, average neurite length and maximal neurite length. Data represent mean ± SEM and were analyzed with two-way ANOVA. No significant difference was detected. Number of neurons analyzed for cultures obtained from LRRK2 transgenic mice: non-tg = 1339, non-tg + LRRK2-IN-1 = 1609; LRRK2 = 1697, LRRK2 + LRRK2-IN-1 = 1542, n = 4 independent experiments; Number of neurons analyzed for cultures obtained from GS-LRRK2 transgenic mice: non-tg = 1268; non-tg + LRRK2-IN-1 = 1522; GS-LRRK2 = 1526; GS-LRRK2 + LRRK2-IN-1 = 1844, n = 4 independent experiments; <b>C-H:</b> Representative pictures of ß-Tubulin III stained neurons on DIV7 derived from wild type, GS- LRRK2 (line 2), their non-transgenic littermates. Pictures were obtained with the BD Pathway 855 high content Bioimager. <b>C1-H2:</b> Total neurite length (C1-H1) and number of branches (C2-H2) segmentation corresponding to ß-tubulin III staining images (C-H) obtained from Attovision Software.</p

LRRK2 mRNA and protein expression in brain regions in the three transgenic mouse lines.

<p><b>A:</b> RT-PCR semi-quantification of LRRK2 expression in whole brains at different embryonic and postnatal stages indicates robust transgene expression at postnatal day 2 (P2) in all three lines. Data represents means ± SEM; n = 3–5 animals per group. <b>B:</b> In-situ hybridization of coronal brain sections at the level of posterior hippocampus and midbrain with two human specific LRRK2 probes showed comparable transgene expression levels in hippocampus and cortex of 11-month-old LRRK2 and GS-LRRK2 lines 1 and 2. <b>C:</b> Western blot analysis of LRRK2 protein showed robust expression levels of LRRK2 in hippocampus (HC) and cortex (CTX) of 10-month-old animals with the human-specific LRRK2 antibody MJFF5; n = 3 animals per genotype.</p

No neuronal loss or neurodegeneration was detected in SNpc in transgenic mice.

<p><b>A:</b> Representative coronal section of midbrain sections from 12- to 13-month-old non-tg, LRRK2 and GS-LRRK2 (line 2) transgenic mice immunostained for TH. (Scale bars: 100μm). <b>B:</b> Cell counts of TH+ and Nissl+ neurons in SNpc from non-tg, LRRK2 and GS-LRRK2 transgenic mice. Data represent mean ± SEM; transgenic mice n = 2, non-tg mice n = 3. <i>n</i>.<i>s</i> = not significant, (one-way ANOVA, Tukey’s <i>post hoc</i> analysis).</p

Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.

<p>Neurite outgrowth parameters analyzed with the BD AttoVision^TM V1.6 Software from BD Bioscience.</p

Generation of human wild type and G2019S mutant LRRK2 transgenic mice.

<p><b>A:</b> Human full length wild type or G2019S mutant LRRK2 cDNA were cloned into the murine Thy-1.2 promoter element driving neuronal-specific transgene expression. <b>B:</b> Western blot analysis of LRRK2 protein expression in brain lysates from LRRK2, GS-LRRK2 line 1, GS-LRRK2 line 2 and non-tg littermates using MID antibody which recognizes human and murine LRRK2 protein. <b>C:</b> Densitometry quantification revealed approximately twice the amount of total LRRK2 protein in all transgenic lines compared to endogenous Lrrk2 levels in non-tg controls. Data represent means ± SEM; n = 2 for each transgenic line.</p

Treatment with the LRRK2-IN-1 kinase inhibitor does not alter adhesion properties in LRRK2 primary human skin fibroblasts.

<p><b>A-B:</b> Western blot analysis of LRRK2 expression in primary skin fibroblasts from healthy subjects (LRRK2) and PD-patients with mutations in the kinase (A) and ROC domain (B) of LRRK2 after treatment with vehicle or 0.1μM LRRK2 IN-1. The MJFF#2 antibody we used recognizes both human and mouse LRRK2. Brain lysates from a Lrrk2 knock-down mouse (KD) and cortex lysate from a non-tg mouse served as negative and positive controls, respectively. One representative fibroblast line per mutation group is shown and 30μg protein was loaded on a 7% acrylamide SDS-gel. <b>C-D:</b> Percentage of adhered fibroblasts with LRRK2 mutations in the kinase (C) and ROC (D) domain at different time points. No significant differences in adhesion capacity were observed between lines. <b>E-F</b>: Percentage of adhered fibroblasts with LRRK2 mutations in the kinase (E) and ROC (F) domain after treatment with vehicle control (0), 0.1μM or 1μM LRRK2-IN-1 for 30 and 120 minutes. No differences were observed in fibroblasts with LRRK2 mutations in the kinase (E) and ROC domain (F). Data represent mean ± SEM; n = 4 independent experiments (C, E) and n = 3 independent experiments (D, F). Healthy-Subjects (LRRK2) = 4 lines; G2019S LRRK2 patients (GS) = 3 lines; N1437S LRRK2 patient (NS) = 2 lines; R1441C LRRK2 patients (RC) = 1 line. (Two-way ANOVA with Repeated Measures).</p

Microgliosis in end-stage haSN(A53T) transgenic mouse brain is unaltered by high LRRK2 levels.

<p>DAB-immunohistochemistry for Iba1 shows activated microglia on a representative sagittal brain section of a haSN(A53T) mouse (A and 20×higher magnification from brainstem in B) and a haSN(A53T)/hLRRK2(G2019S) double transgenic mouse (C and 20×higher magnification from brainstem in D). (E) Quantification of the brainstem results. Values represent % of the area in the brainstem that is covered by Iba1-positive microglia. p-value (p = 0.179) was determined by two-tailed, unequal variances Student’s t-test. Dots represent quantifications of single individuals. Control images obtained from a separate experiment but from littermate hLRRK2(G2019S) single transgenic (F and 20×higher magnification from brainstem in G) and from non-transgenic wildtype littermate control (Ntg) (H and 20×higher magnification from brainstem in I) mouse.</p

High LRRK2 transgene levels do not exacerbate α-synuclein-driven phenotypes.

<p>(A) Schematic representation of the four different transgenic lines used to generate double transgenics. (B) 3-Step accelerated rotarod performance of females and males comparing single and double transgenics. The different genotypes and the number of mice per genotype are indicated. p-values were determined by repeated measures ANOVA (group effects for the respective panels: 1: F(1,22) = 0.483, p = 0.494; 2: F(1,26) = 0.000, p = 0.983; 3: F(1,11) = 0.738, p = 0.409; 4: F(1,22) = 2.048, p = 0.166; 5: F(1,16) = 1.255, p = 0.279; 6: F(1,27) = 5.171, p = 0.031). (C) Kaplan-Meier curves showing the time-of-sacrifice when mice had to be killed because of too severe motor deficits (1 = 100% and 0 = 0% of mice alive). The different genotypes, gender, number of mice per genotype and the p-values (nonparametric Kaplan-Meier) are indicated.</p

aSN and phospho-S129-aSN protein levels in spinal cord and forebrain of end-stage disease single and double transgenic mice.

<p>Tris-soluble and -insoluble fractions of spinal cord and forebrain lysates were immunoblotted and stained with antibodies detecting total α-synuclein (aSN) or specifically phosphorylated S129-aSN (paSN). β-actin (βAc) levels were measured as loading control and for normalization. For reference, LRRK2 levels detected via immunoblot are shown comparing single and double-transgenics. Different α-synuclein protein species/forms are marked as follows: mo, monomer; ol, oligomer; tr, truncated. For reference, in the upper panels the performance and specificity of the antibodies are illustrated in the two right lanes comparing WT and KO (aSN knock-out) brain samples and were added to indicate unspecific cross-reactive proteins (taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036581#pone.0036581.s007" target="_blank">Figure S7</a>). Graphs represent quantifications of monomeric aSN and paSN/aSN, all normalized to βAc. Circles represent individual mice, the means are indicated as horizontal bars and % are normalized to the levels in haSN(A53T) single transgenics. p-values were determined by two-tailed, unequal variances Student’s t-test. Genotypes: aSN = haSN(A53T), aSN/LRRK2 = haSN(A53T)/hLRRK2(G2019S), Ntg = non-transgenic wildtype littermate control and KO = aSN knock-out mice.</p