Single low doses of MPTP decrease tyrosine hydroxylase expression in the absence of overt neuron loss

Abstract

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a prototypical neurotoxicant used in mice to mimic primary features of PD pathology including striatal dopamine depletion and dopamine neuron loss in the substantia nigra pars compacta (SNc). In the literature, there are several experimental paradigms involving multiple doses of MPTP that are used to elicit dopamine neuron loss. However, a recent study reported that a single low dose caused significant loss of dopamine neurons. Here, we determined the effect of a single intraperitoneal injection of one of three doses of MPTP (0.1, 2 and 20mg/kg) on dopamine neurons, labeled by tyrosine hydroxylase (TH⁺), and total neuron number (Nissl⁺) in the SNc using unbiased stereological counting. Data reveal a significant loss of neurons in the SNc (TH⁺ and Nissl⁺) only in the group treated with 20mg/kg MPTP. Groups treated with lower dose of MPTP (0.1 and 2mg/kg) only showed significant loss of TH⁺ neurons rather than TH⁺ and Nissl⁺ neurons. Striatal dopamine levels were decreased in the groups treated with 2 and 20mg/kg MPTP and striatal terminal markers including, TH and the dopamine transporter (DAT), were only decreased in the groups treated with 20mg/kg MPTP. These data demonstrate that lower doses of MPTP likely result in loss of TH expression rather than actual dopamine neuron loss in the SN. This finding reinforces the need to measure both total neuron number along with TH⁺ cells in determining dopamine neuron loss.

Keywords: Dopamine; Dopamine transporter; MPTP; Stereology; Tyrosine hydroxylase; Vesicular monoamine transporter.

Fig. 1. Effect of MPTP on dopamine neuron loss in the substantia nigra

Unbiased stereology counts of (A) TH⁺ neurons and (B) Nissl stained neurons of one brain hemisphere in mice treated with 0.1, 2 or 20 mg/kg.* indicates significantly different from control (p<0.05, n=5–6). (C) Representative TH immunostaining of saline and MPTP treatment groups, scale bar shows 250 μm.

Fig. 2. Effect of MPTP on dopaminergic neurochemistry in the striatum of C57BL/6J mice

(A) MPTP treatment reduced striatal DA levels in a dose response manner. (B) MPTP treatment had a significant effect on DOPAC in all the MPTP treatment groups. (C) MPTP treatment (20 mg/kg) significantly reduced the striatal level of HVA. * indicates significantly different from control (p<0.05, n=5–6).

Fig. 3. Effect of MPTP on DA turnover as determined by DOPAC/DA and HVA/DA ratios in the striatum of C57BL/6J mice

High dose of MPTP treatment significantly increased (A) DOPAC/DA ratio, but no significant effect on the (B) HVA/DA ratio was observed. * indicates significantly different from control (p<0.05, n=5–6).

Fig. 4. Effect of MPTP treatment on dopaminergic terminal markers

Upper panel: Quantification of the WB for DAT, TH and VMAT₂ in the striatum. Lower panel: Striatal levels of the dopaminergic terminal markers (DAT, TH, VMAT2) measured by western blot assay. Tubulin levels were used to ensure equal loading. * indicates significantly different from control (p<0.05, n=5–6).

Fig. 5

Quantification of the striatal TH (A) and DAT (B) immunofluorescent images. C) Representative Immunostaining of TH and DAT in the striatum. Scale bar represents 500 μm.

Fig. 5

Quantification of the striatal TH (A) and DAT (B) immunofluorescent images. C) Representative Immunostaining of TH and DAT in the striatum. Scale bar represents 500 μm.