Expression Analysis of Lrrk1, Lrrk2 and Lrrk2 Splice Variants in Mice

Abstract

Missense mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are linked to autosomal dominant forms of Parkinson’s disease (PD). In order to get insights into the physiological role of Lrrk2, we examined the distribution of Lrrk2 mRNA and different splice variants in the developing murine embryo and the adult brain of Mus musculus. To analyse if the Lrrk2-paralog, Lrrk1, may have redundant functions in PD-development, we also compared Lrrk1 and Lrrk2 expression in the same tissues. Using radioactive in situ hybridization, we found ubiquitous expression of both genes at low level from embryonic stage E9.5 onward, which progressively increased up until birth. The developing central nervous system (CNS) displayed no prominent Lrrk2 mRNA signals at these time-points. However, in the entire postnatal brain Lrrk2 became detectable, showing strongest level in the striatum and the cortex of adult mice; Lrrk1 was only detectable in the mitral cell layer of the olfactory bulb. Thus, due to the non-overlapping expression patterns, a redundant function of Lrrk2 and Lrrk1 in the pathogenesis of PD seems to be unlikely. Quantification of Lrrk2 mRNA and protein level in several brain regions by real-time PCR and Western blot verified the striatum and cortex as hotspots of postnatal Lrrk2 expression. Strong expression of Lrrk2 is mainly found in neurons, specifically in the dopamine receptor 1 (DRD1a) and 2 (DRD2)-positive subpopulations of the striatal medium spiny neurons. Finally, we identified 2 new splice-variants of Lrrk2 in RNA-samples from various adult brain regions and organs: a variant with a skipped exon 5 and a truncated variant terminating in an alternative exon 42a. In order to identify the origin of these two splice variants, we also analysed primary neural cultures independently and found cell-specific expression patterns for these variants in microglia and astrocytes.

Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice

“…LRRK2 protein is well documented to be found at highest levels in the striatum in mammals (Galter et al, 2006; Melrose et al, 2006; Taymans et al, 2006; Higashi et al, 2007a; Higashi et al, 2007b; Melrose et al, 2007; Westerlund et al, 2008a; Westerlund et al, 2008b; Mandemakers et al, 2012; Davies et al, 2013a; Giesert et al, 2013; West et al, 2014). Therefore we reasoned that the G2019S mutation might impact on the functional integrity of the nigro-striatal dopaminergic pathway.…”

Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon

“…Radioactive in situ hybridization was performed on 8-mm paraffin sections as previously described (Giesert et al 2013). Immunohistochemistry on paraffin sections was performed according to standard procedures.…”

Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1αmRNA in Mouse Skeletal Muscle

“…RT-PCR reactions were carried out in a 20  μ L volume containing 1XFast Start Universal SYBR Green Master (ROX) (Roche, Germany), 50 ng cDNA, 0.3  μ M forward and reverse primers (each). qRT-PCR reaction conditions for long products (~800 bp) were 95°C for 10 min and then 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min [21, 22]. Target gene expression in each sample was normalized to the endogenous control gene cyclophilin.…”