Expression of the LRRK2 gene in the midbrain dopaminergic neurons of the substantia nigra

Abstract

A hallmark of Parkinson's disease (PD) is the progressive loss of the A9 midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta. Recently, multiple causative mutations have been identified in the leucine-rich repeat kinase 2 (LRRK2) gene for both familial and sporadic PD cases. Therefore, to investigate functional roles of LRRK2 in normal and/or diseased brain, it is critical to define LRRK2 expression in mDA neurons. To address whether LRRK2 mRNA and protein are expressed in mDA neurons, we purified DA neurons from the tyrosine hydroxylase (TH)-GFP transgenic mouse using FACS-sorting and analyzed the expression of LRRK2 and other mDA markers. We observed that all mDA markers tested in this study (TH, Pitx3, DAT, Nurr1 and Lmx1a) are robustly expressed only in GFP(+) cells, but not in GFP(-) cells. Notably, LRRK2 was expressed in both GFP(+) and GFP(-) cells. Consistent with this, our immunohistochemical analyses showed that LRRK2 is expressed in TH-positive mDA neurons as well as in surrounding TH-negative cells in the rat brain. Importantly, in the midbrain region, LRRK2 protein was preferentially expressed in A9 DA neurons of the substantia nigra, compared to A10 DA neurons of the ventral tegmental area. However, LRRK2 was also highly expressed in the cortical and hippocampal regions. Taken together, our results suggest that LRRK2 may have direct functional role(s) in the neurophysiology of A9 DA neurons and that dysfunction of these neurons by mutant LRRK2 may directly cause their selective degeneration.

Fig. 1

LRRK2 mRNAs are expressed in dopaminergic and non-dopaminergic neurons. (A) GFP⁺ cells (DA) and GFP⁻ cells (non-DA) were sorted by FACS followed by RNAs extractions. RT-PCR analyses were performed using specific primers as described in Materials and Methods. The β-actin gene was used as a positive control.

Fig. 2

Real-time PCR reactions were performed using SYBR green I. The standard curve was generated using plasmid DNA containing the GAPDH cDNA. The amount of the TH mRNA in GFP⁺ cells was set as 100 to calculate the relative amounts of each mRNA. Each reaction has been performed in triplicate using an identical amount of cDNAs as the template. The expression of TH and LRRK2 was measured as described in Materials and Methods. GAPDH expression levels were used to normalize mRNA levels of each gene. Asterisk indicates statistically significant difference from GFP⁻ cells (*P< 0.01).

Fig. 3

LRRK2 is continuously expressed in SN regions. Rat brain sections were incubated with LRRK2 antibody (red) and tyrosine hydroxylase (green) showing colocalization (yellow) in dopaminergic neurons in the substantia nigra. In (A), (B) and C, the expression of LRRK2 proteins in regions A9 and A10 is shown. High magnification of (B) is shown in (D), (E) and (F). Scale bar = 50μm. By using confocal microscopy, LRRK2 immunoreactivities are detected in both TH-positive and TH-negative neurons (G). Arrows indicate co-localization of LRRK2 and TH. Arrow heads indicate that some TH-negative cells also express LRRK2 proteins. Scale bar = 10μm.

Fig. 4

LRRK2 is highly expressed in the cortical and the hippocampal regions. Rat brain sections were incubated with anti-LRRK2 antibody. Shown in the left column are representative fluorescence photographs of LRRK2 immunohistochemistry from rat brain at the hippocampus (A) and cortex (B) levels. In the right column, high magnification images corresponding to boxed areas from the overview image are shown for rat brain, hippocampal region (A) or cortical region (B). Scale bar=40μm.