The role of kalirin-7 in the pathogenesis of Parkinson disease and Huntington disease

Abstract

Huntington disease (HD) is caused by an expanded CAG repeat in the HD gene. Although the length of the CAG repeat strongly correlates with the age-at-onset (AAO), AAO in HD individuals may differ dramatically in spite of similar expanded CAG repeat lengths. Additional genetic or environmental factors are thought to influence the disease onset. To identify such a genetic modifier, we analyzed single nucleotide polymorphisms (SNPs) in the kalirin (KALRN) gene. Eleven SNPs in the kalirin gene were selected and SNP rs10934657 was examined in an association study in 680 independent European HD patients. However, the results did not reveal an association between the analyzed kalirin polymorphisms and the AAO in HD. In the second part of this study, we investigated the effect of kalirin-7 on synphilin-1 aggregate formation. Coexpression of kalirin-7 and synphilin-1 caused a dramatic relocation of synphilin-1 cytoplasmic small inclusions to a single prominent, perinuclear inclusion. These perinuclear inclusions were located in aggresomes according to the colocalization with microtubule organization center markers and their formation was microtubule-dependent. Furthermore, kalirin-7 increased the susceptibility of synphilin-1 inclusions to be degraded as shown by live imaging and quantification of aggregates. The GEF activity of kalirin-7 is the most prominent function of the protein, however, kalirin-7-mediated synphilin-1 aggresome response was not dependent on its GEF activity since various dominant negative small GTPases could not inhibit the formation of aggresomes. Interestingly, the aggresome response was blocked by HDAC6 catalytic mutants and the HDAC inhibitor trichostatin A (TSA), but not by sodium butyrate (NaBu). Moreover, kalirin-7 decreased the level of acetylated alpha-tubulin in response to TSA, which suggests an effect of kalirin-7 on HDAC6-mediated protein transportation and aggresome formation. In summary, we demonstrated that kalirin-7 leads to the recruitment of synphilin-1 into aggresomes in a HDAC6-dependent manner and also links kalirin-7 to microtubule dynamics. Our results implicate an important role of a GEF protein in the pathogenesis of PD and provide a link to the huntingtin pathway by binding the huntingtin associated protein 1 (HAP1) interactor. Kalirin-7 has been proposed as an important regulator for synaptic structure and function which is associated with cognitive processes in kalirin-7 knockout mice. In the third part of this study, we tried to generate and characterize kalirin-7 overexpressing mice to further crossbreed them with PD and HD animal models and evaluate the impact of kalirin-7 in these two diseases in vivo. Unfortunately, we were unsuccessful in characterizing and establishing the kalirin-7 animal model due to the lack of proper antibodies, including the one we generated, which was supposed to be human kalirin-7 specific. To further generate a kalirin-7 animal model, a stronger promoter and a small tag might have to be included.

Bei der Suche nach Proteinen, die im Pathogenesepathway des Morbus Parkinson (MP) involviert sind, haben wir kalirin-7, ein neuronaler GDP/GTP Austauschfaktor (auch genannt HAPIP), als interagierendes Protein von Synphilin 1 identifiziert. kalirin-7 selbst wurde bereits als interagierendes Protein des huntingtin-assoziierten Proteins HAP1 beschrieben. Damit konnten wir einen Stoffwechselweg des MP direkt mit einem Stoffwechselweg der Chorea Huntington (CH) koppeln. Daher kommt kalirin-7 eine zentrale Bedeutung für die Untersuchung der kausalen Ursachen neurodegenerativer Prozesse zu, welche in diesem Projekt mit Zellkultur- und Tiermodellen des MP und der CH untersucht werden soll. Während die Funktion von Synphilin-1 weitestgehend unklar ist, gibt es umfassendere Analysen für den Proteinkomplex des Huntingtin-Proteins, welches mit NMDAR2, PSD95 und IP3R interagiert. In ersten Analysen konnten wir nunmehr auch Synphilin-1 diesem Komplex zuordnen. Hier soll eine detaillierte funktionelle Charakterisierung von kalirin-7 bei der Aggregatformierung, beim Axonwachstum und der Dendritenausbildung sowie beim neuronalen Zelltod bzw. Überleben erfolgen. Darüber hinaus generieren wir kalirin-7 überexprimierende transgene Mäuse, um durch Kreuzung mit Tiermodellen des MP und der CH, die Relevanz von kalirin-7 als therapeutischen Angriffspunkt zu untersuchen.