Analysis of the biological function of the Parkinson’s disease associated protein LRRK2 in knockdown models

Das Parkinson-Syndrom (PS) ist die zweithäufigste neurodegenerative Erkrankung. Aus diesem Grund ist die Erforschung ihrer Pathogenese von zentraler Bedeutung. In dieser Arbeit wurde die biologische Funktion der Leucine-rich repeat kinase 2 (LRRK2) untersucht. Dieses Protein, welches 2004 identifiziert wurde, ist nicht nur verantwortlich für 5-6% der autosomal dominanten, sondern auch für 1-2% der sporadischen Parkinson-Syndrome. Diese Arbeit beschreibt erstmals die gesamtgenomischen Auswirkungen des Verlustes von LRRK2 in humanen dopaminergen Zellen und gibt damit einen Einblick in LRRK2-abhängige Signalkaskaden. Dieser Verlust („Knockdown“) konnte auf RNA- und Proteinebene mit Hilfe von RNA-Interferenz erreicht werden. Die Analyse der Gene und Signalkaskaden, welche durch den Verlust von LRRK2 differentiell reguliert werden, führt in dieser Arbeit zu der Betrachtung von drei grundlegenden Fragestellungen. Apoptoseprozesse werden mit der Pathogenese des PS in Zusammenhang gebracht. Aus diesem Grund befasst sich die erste Fragestellung dieser Arbeit mit dem Einfluss von LRRK2 auf Apoptose-Signalkaskaden. Das gesamtgenomische Expressionsprofil des humanen Zellkulturmodells zeigt einen deutlichen Einfluss des LRRK2-Verlustes auf p53-abhängige Signalkaskaden. Dabei ist p53 ein entscheidender Regulator der Apoptoseprozesse. Auch die Expressionsanalyse von Gehirnen LRRK2-defizienter Tiere bestätigt dieses Ergebnis. Die anschließende funktionelle Analyse im humanen LRRK2-Knockdown-Modell verdeutlicht eine antiapoptotische Wirkung des LRRK2-Verlustes, welcher bei ER-Stress, oxidativem Stress und Kinase-Inhibition auftritt. Während der Pathogenese des PS kommt es zur Bildung von Lewy-Körperchen, welche als Hauptbestandteil das Protein Alpha-Synuklein beinhalten. Alpha-Synuklein und LRRK2 sind Proteine, welche mit der familiären Form des Parkinson-Syndroms assoziiert sind. Es gibt bisher noch keine Analysen, ob beide Proteine über ihre biologischen Funktionen einen gemeinsamen Pathogenesemechanismus ausüben. Aus diesem Grund wird in der vorliegenden Arbeit das Transkriptom von Alpha-Synuklein-reduzierten, humanen dopaminergen Zellen mit dem Transkriptom von LRRK2-reduzierten Zellen vergleichend analysiert. Deutlich kann herausgestellt werden, dass es Signalkaskaden gibt, welche durch den Verlust beider Proteine dereguliert vorliegen. Die Betrachtung der differentiell regulierten Transkripte verdeutlicht allerdings oft entgegengesetzte Regulationsrichtungen, so dass diese Analysen gegen einen direkten gemeinsamen Pathomechanismus sprechen. Die dritte Fragestellung dieser Arbeit befasst sich mit dem Einfluss von LRRK2 auf zytoskelettäre Prozesse. Durch die veröffentlichte Interaktion von LRRK2 mit Mikrotubuli und den Einfluss von LRRK2 auf die Neuritogenese, ist eine biologische Funktion von LRRK2 in Zytoskelett-Signalkaskaden zu vermuten. Die Ergebnisse dieser Arbeit zeigen, dass die mRNA-LRRK2-Expression in Zellkultur unter zytoskelettalem Stress ansteigt. Die Expressionsprofile des humanen wie auch des murinen LRRK2-Knockdown-Modells verdeutlichen, dass besonders Aktin-Zytoskelett-assoziierte Signalkaskaden durch den LRRK2-Verlust dereguliert vorliegen. Besondere Aufmerksamkeit erlangen im humanen Modell nach LRRK2-Verlust die differentiell regulierten Transkripte CDC42 und ARHGEF7. Beide Proteine spielen eine prominente Rolle in Aktin-Zytoskelett-Regulationskaskaden und werden deshalb gemeinsam mit Beta-Aktin auf eine Interaktion mit LRRK2 getestet. Alle drei Proteine zeigen in vitro eine Interaktion sowie in vivo eine Kolokalisation mit LRRK2. LRRK2 besitzt sowohl eine GTPase- als auch eine Kinase-Funktion, welche sich gegenseitig beeinflussen können. Die weitere Charakterisierung der Interaktion zwischen LRRK2 und ARHGEF7 verdeutlicht, dass die Interaktion in diesen zwei Hauptenzymdomänen von LRRK2 stattfindet. Bisher wurde noch kein Guanidin-Austauschfaktor (GEF) von LRRK2 identifiziert, welcher zur Aktivierung der GTPase-Funktion von LRRK2 nötig sein könnte. Die funktionellen in vitro Studien dieser Arbeit zeigen, dass ARHGEF7 das Potential hat, als GEF am LRRK2 wirken zu können, womit dies die Erstbeschreibung eines GEFs am LRRK2 darstellt. In weiteren Bindungsstudien zwischen der GTP-Hydrolyse-defizienten, pathogenen LRRK2 R1441C Mutation und ARHGEF7 kann verdeutlicht werden, dass ein reduziertes Bindungspotential zwischen ihnen vorliegt und dass die GTP-Bindung der R1441C Variante von LRRK2 durch ARHGEF7 ansteigt. Durch die identifizierte Bindung von LRRK2 an CDC42, Beta-Aktin und ARHGEF7 wird in dieser Arbeit eine mögliche Funktion von LRRK2 als Gerüstprotein bei dem Neuritenwachstum herausgestellt und diskutiert. Diese Arbeit verdeutlicht damit zum ersten Mal, dass gesamtgenomische Expressionsprofile die Basis für Interaktionsstudien sein können, wodurch thesengetrieben funktionelle Analysen möglich werden, welche die biologische Funktion von Proteinen aufzuklären helfen.Parkinson’s disease is the second most common neurodegenerative disease. Therefore further knowledge of the pathogenesis of this disease is necessary. In this study the biological function of the Leucine-rich repeat kinase 2 (LRRK2), which was identified in 2004, is analysed. Mutations in LRRK2 contribute to 5-6% of all cases of autosomal-dominant as well as to 1-2% of cases of sporadic Parkinson’s disease. This study elucidates for the first time the whole genome consequences caused by RNA-interference mediated knockdown of LRRK2 on protein and RNA level in human dopaminergic cells. This enables insights in LRRK2 dependent signaling cascades. Analyses of differentially regulated transcripts and signaling cascades, influenced by the loss of LRRK2, highlight 3 cardinal questions, which will be analysed in this study. Apoptosis has been proposed to contribute to neuronal loss in Parkinson’s disease. Therefore the impact of the knockdown of LRRK2 on apoptosis signaling is examined. Whole genome expression analysis of the LRRK2 knockdown in the human cell culture model reveals a deregulation of p53 depending signaling cascades. The tumor protein p53 is an important regulator of programmed cell death. This result could be confirmed by means of whole genome expression analysis of brains of LRRK2 knockdown mice. Further analysis in the human LRRK2 knockdown model indicates an anti apoptotic effect of the loss of LRRK2 under condition of ER-stress, oxidative stress and kinase inhibition. Pathological the Parkinson’s disease is characterised by the presence of Lewy bodies in surviving neurons, which contains alpha-synuclein (SNCA). SNCA as well as LRRK2 is a protein associated with autosomal-dominant Parkinson’s disease. Until now there is no investigation, if the biological function of both proteins contributes to the same pathogenesis inducing pathway. These study compares the differentially regulated transcripts of whole genome expression analysis of both SNCA knockdown and LRRK2 knockdown in human dopaminergic cells. The data highlight that there are common signaling cascades, which contain differentially regulated genes in both experiments. Nevertheless the direction of regulation is conflictive. Therefore a common mechanism of SNCA and LRRK2, which probably lead to neurodegeneration in Parkinson’s disease, could not be detected. The third topic, analysed in this study, is the impact of LRRK2 on cytoskeleton signaling cascades. It is conceivable that LRRK2 have a direct or indirect role in cytoskeleton maintenance because of its published interaction with microtubule and its influence on neuritogenesis. This study shows that the mRNA expression level of LRRK2 is induced under condition of cytoskeleton stress. The microarray analysis in LRRK2 siRNA treated human dopaminergic cells as well as whole genome expression analysis of murine LRRK2 knockdown brains reveal numerous differentially regulated transcripts involved in actin cytoskeleton signal transduction pathways. Detailed analyses point at special impact of the differentially up regulated transcripts CDC42 and ARHGEF7. Because of their major role in actin cytoskeleton signaling cascades, they are analysed for interaction with LRRK2 additionally to Beta-Actin. All three tested proteins highlight an in vitro interaction and an in vivo colocalisation with LRRK2. The multidomain protein LRRK2 harbours a kinase and GTPase domain and published results show, that the kinase activity is regulated via the intrinsic GTPase activity. The interaction of ARHGEF7 with LRRK2 occurs in these major protein domains. Although suggesting that LRRK2 requires co-factors like guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) for its GTPase activity, none of them are known until now. The in vitro data of this study reveal the potential of ARHGEF7 to be the first identified GEF for LRRK2. Further analysis between probably pathogenic LRRK2 variants and ARHGEF7 emphasize a reduced binding between the GTP hydrolysis deficient LRRK2 R1441C variant and ARHGEF7, additionally to an induced GTP binding capacity of the R1441C variant during the presence of ARHGEF7. By means of the new identified LRRK2 interacting proteins, the impact of LRRK2 as scaffold during neurite outgrowth is discussed. This work illustrates for the first time, that whole genome expression analyses could be the basis for interaction partner studies and lead to functional scientific hypotheses, which are essential for elucidation of the biological function of proteins

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

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

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

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

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