Caspase inhibitors attenuate 1-methyl-4-phenylpyridinium toxicity in primary cultures of mesencephalic dopaminergic neurons

Abstract

Parkinson's disease is characterized by a loss of dopaminergic nigrostriatal neurons. This neuronal loss is mimicked by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). MPP+ toxicity is mediated through inhibition of mitochondrial complex I, decreasing ATP production, and upregulation of oxygen radicals. There is evidence that the cell death induced by MPP+ is apoptotic and that inhibition of caspases may be neuroprotective. In primary cultures of rat mesencephalic dopaminergic neurons, MPP+ treatment decreased the number of surviving dopaminergic neurons in the cultures and the ability of the neurons to take up [3H]dopamine ([3H]DA). Caspase inhibition using the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) spared MPP+-treated dopaminergic neurons and increased somatic size. There was a partial restoration of neurite length in zVAD-fmk-treated cultures, but little restoration of [3H]DA uptake. Peptide inhibitors of caspases 2, 3, and 9, but not of caspase 1, caused significant neuroprotection. Two novel caspase inhibitors were tested for neuroprotection, a broad spectrum inhibitor and a selective caspase 3 inhibitor; both inhibitors increased survival to >90% of control. No neuroprotection was observed with an inactive control compound. MPP+ treatment caused chromatin condensation in dopaminergic neurons and increased expression of activated caspase 3. Inhibition of caspases with either zVAD-fmk or a selective caspase 3 inhibitor decreased the number of apoptotic profiles, but not expression of the active caspase. We conclude that MPP+ toxicity in primary dopaminergic neurons involves activation of a pathway terminating in caspase 3 activation, but that other mechanisms may underlie the neurite loss.

Fig. 1.

Effects of MPP⁺ on survival (A) and [³H]DA uptake (B) in primary cultures of mesencephalic dopaminergic neurons. MPP⁺ was added at concentrations ranging from 0.01 to 100 μm for 48 hr. Cultures were then either fixed and immunostained for TH, and the surviving TH-immunoreactive cells were counted, or [³H]DA uptake was assayed. Data shown in each case are the mean ± SEM of three independent experiments and are expressed as percentage of untreated control cultures (**p < 0.01; established by one-way ANOVA followed by Dunnett's test). Representative photomicrographs of control (C) or 10 μm MPP⁺-treated (D) TH-immunoreactive neurons are shown. Cultures were treated for 48 hr, then fixed and immunostained for TH.

Fig. 2.

zVAD-fmk attenuates 10 μmMPP⁺ toxicity and increases somatic size in mesencephalic dopaminergic neurons. For survival quantification (A), cultures were exposed to 10 μmMPP⁺ for 48 hr in the presence of various zVAD-fmk concentrations. Cultures were then fixed and immunostained for TH. Slides were blinded, and TH-immunoreactive cells were counted. Data points shown are from three independent experiments, each consisting of four independent wells, and are expressed as percentage of untreated control cultures (*p < 0.05, **p < 0.01; established by one-way ANOVA followed by Dunnett's test). Somatic size measurements (B) were made from each of four wells from one representative experiment. Random fields of view were visualized using MCID image analysis, and densitometry was used to establish the area occupied by the soma of TH-immunoreactive neurons. One hundred cells per well were measured for each data point. Photomicrographs of control (C), 10 μmMPP⁺-treated (D), and 10 μm MPP⁺- and 300 μmzVAD-fmk-treated (E) mesencephalic cultures are shown. Cultures were immunostained for TH, and representative photomicrographs were taken.

Fig. 3.

Effects of zVAD-fmk on [³H]DA uptake in primary mesencephalic dopaminergic neurons exposed to 1 or 10 μm MPP⁺ and effects of 300 μm zVAD-fmk treatment on neurite length of dopaminergic neurons. For [³H]DA uptake assays (Fig.3A), cultures were exposed to 1 or 10 μmMPP⁺ in the presence of various concentrations of zVAD-fmk for 48 hr, and then the ability of the cells to take up [³H]DA was assayed. Each data point is the mean ± SEM of three independent experiments, each consisting of four independent wells, and is expressed as percentage of untreated control cultures (*p < 0.05, **p < 0.01; established by one-way ANOVA followed by Dunnett's test). Neurite length measurements were made from TH-immunoreactive neurons in control cultures, cultures exposed to 10 μm MPP⁺ for 48 hr, and cultures treated for 48 hr with MPP⁺ and 300 μmzVAD-fmk. MCID image analysis was used to quantify the length of the longest neurite in each of 100 TH-immunoreactive neurons in four independent wells per treatment group. B shows the mean neurite length of TH-immunoreactive neurons. C shows the percentage of TH-immunoreactive cells in each treatment group with no, or only rudimentary, neurites; this was defined as a longest process of <10 μm in length.

Fig. 4.

Effects of peptide caspase inhibitors on survival of TH-immunoreactive neurons after 10 μmMPP⁺ treatment. Primary cultures of mesencephalic dopaminergic neurons were exposed to 10 μmMPP⁺ for 48 hr in the presence of zDEVD-fmk (A), zLEHD-fmk (B), zVDVAD-fmk (C), or zYVAD-cmk (D). Cultures were fixed and immunostained for TH. Slides were then blinded, and the number of surviving TH-immunoreactive neurons was counted. Each data point represents the mean ± SEM of three independent experiments, each consisting of four independent wells, and is expressed as percentage of untreated control cultures (**p < 0.01; established by one-way ANOVA followed by Dunnett's test).

Fig. 5.

Effects of novel caspase inhibitors on survival of dopaminergic neurons treated with MPP⁺. Two novel caspase inhibitors were tested for survival-promoting effects in primary cultures of dopaminergic neurons, M-920 and M-791, together with an inactive analog, M-725. Compounds were coadministered with 10 μm MPP⁺ for 48 hr, and then surviving TH-immunoreactive cells were quantified. Results shown are the mean ± SEM of four independent wells per treatment group (M-791 response, **p < 0.01; M-920 response,⁺⁺ p < 0.01; both established by one-way ANOVA followed by Dunnett's test).

Fig. 6.

Colocalization of apoptotic nuclei with TH immunoreactivity in primary mesencephalic cultures after MPP⁺ exposure. Cultures were stained with a primary antibody raised against TH and visualized using FITC. Nuclei were visualized by counterstaining using Hoechst 33342.A–C, Control cultures;D–F, cultures exposed to 10 μm MPP⁺ for 48 hr;G–I, cultures exposed to 10 μm MPP⁺ in the presence of 300 μm zVAD-fmk. Each of the photomicrographs within a condition is of the same field of view, stained with tyrosine hydroxylase (A, D, G) or Hoechst 33342 (B, E,H) or dual exposed to show colocalization (C, F, I). Apoptotic dopaminergic nuclei are shown by white arrows(and magnified in E, inset). Representative non-apoptotic dopaminergic nuclei are indicated byyellow arrows, and non-dopaminergic apoptotic nuclei byred arrows.

Fig. 7.

TH-immunoreactive neurons exposed to 10 μm MPP⁺ for 24 or 48 hr express activated caspase 3. Cultures were treated with MPP⁺for the required time, then fixed and double immunostained using a monoclonal tyrosine hydroxylase antibody and a polyclonal antibody raised against active caspase 3. A–C, Control cultures; D–F, cultures treated with 10 μm MPP⁺ for 24 hr;G–I, cultures exposed to MPP⁺ for 48 hr. A, D, and F show immunostaining using an antibody to TH;B, E, and H show the same field of view stained with the activated caspase 3 antibody. Colocalization of these antibodies is shown in C,F, and I.

Fig. 8.

Effects of MPP⁺ treatment in the presence and absence of caspase inhibition on the number of apoptotic profiles and active caspase 3 immunoreactive cells. Cells were double labeled for TH and active caspase 3, and the nuclei were counterstained with Hoechst 33342. Cells were visualized using a 20× objective, and the total number of nuclei, the number of apoptotic nuclei, the number of TH-immunoreactive cells, and the number of active caspase 3-immunoreactive cells were quantified, together with the number of cells coexpressing TH and apoptotic nuclei, and TH and active caspase 3. Ten fields of view were quantified in each of three wells per treatment group. Cultures that were quantified were untreated control cultures, cultures exposed to 10 μmMPP⁺ for 48 hr, or cultures exposed to 10 μm MPP⁺ for 48 hr in the presence of either 300 μm zVAD-fmk or 100 μm M-791.A shows the expression of apoptotic profiles and active caspase 3 as a percentage of the total cell population.B shows the cells coexpressing either activated caspase 3 or apoptotic profiles with TH expressed as a percentage of the total number of TH-immunoreactive cells.