Inhibition of calpains prevents neuronal and behavioral deficits in an MPTP mouse model of Parkinson's disease

Abstract

The molecular mechanisms mediating degeneration of midbrain dopamine neurons in Parkinson's disease (PD) are poorly understood. Here, we provide evidence to support a role for the involvement of the calcium-dependent proteases, calpains, in the loss of dopamine neurons in a mouse model of PD. We show that administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) evokes an increase in calpain-mediated proteolysis in nigral dopamine neurons in vivo. Inhibition of calpain proteolysis using either a calpain inhibitor (MDL-28170) or adenovirus-mediated overexpression of the endogenous calpain inhibitor protein, calpastatin, significantly attenuated MPTP-induced loss of nigral dopamine neurons. Commensurate with this neuroprotection, MPTP-induced locomotor deficits were abolished, and markers of striatal postsynaptic activity were normalized in calpain inhibitor-treated mice. However, behavioral improvements in MPTP-treated, calpain inhibited mice did not correlate with restored levels of striatal dopamine. These results suggest that protection against nigral neuron degeneration in PD may be sufficient to facilitate normalized locomotor activity without necessitating striatal reinnervation. Immunohistochemical analyses of postmortem midbrain tissues from human PD cases also displayed evidence of increased calpain-related proteolytic activity that was not evident in age-matched control subjects. Taken together, our findings provide a potentially novel correlation between calpain proteolytic activity in an MPTP model of PD and the etiology of neuronal loss in PD in humans.

Figure 1.

Schematic representation of time course and experimental manipulations for each set of experiments in this study. Each horizontal line represents the duration of each experiment. Numerals above the horizontal line indicate time point (in days) of experimental end-points, whereas the set of five vertical lines indicates the timing of MPTP dosing (25 mg/kg measured as free base, i.p., per day for 5 consecutive days). A, Mice used for experiments examining the time course of calpain activation after MPTP intoxication were taken either before MPTP (day 0) or 7 or 14 d after the first injection (+7, +14). B, Mice implanted with osmotic minipumps (Alza) 1 d (-1) before the initiation of the MPTP regime (0). In this set of experiments, the locomotor behavior of these mice was analyzed 1 d before (+13) the 2 week end point (+14), and additional groups were also analyzed for survival at 3 weeks (+21). C, Recombinant adenoviruses were administered 1 week (-7) before the start of MPTP dosing (0), and tissues were analyzed 2 weeks later (+14). See Materials and Methods for details.

Figure 2.

Increased calpain proteolysis in nigral dopamine neurons of MPTP-treated mice. Detection of calpain-cleaved α-spectrin was absent in the SNc of saline-treated mice (a). Administration of MPTP (25 mg/kg measured as free base, i.p.) daily for 5 consecutive days resulted in increased reactivity for calpain-cleaved α-spectrin 7 d (b) and 14 d (c) later. Intracerebroventricular coadministration of the calpain inhibitor MDL-28170 blocked MPTP-induced calpain-mediated cleavage of α-spectrin 14 d after MPTP (d). MPTP-treated mice that were coadministered the vehicle (e, f) also revealed a predominance of punctate and cytoplasmic patterns of immunostaining for calpain-cleaved α-spectrin, whereas nuclear immunostaining was weak and not pronounced (f, inset). Immunohistofluorescence confirmed the increased expression of calpain-cleaved α-spectrin (g, i) in tyrosine hydroxylase-immunoreactive cell bodies in the substantia nigra (h, j) of MPTP-treated mouse brain tissues 14 d (i, j) after treatment, but not in the SNc of saline-treated mice (g, h). Clusters of speckled immunoreactivity were nonreactive for glial acidic fibrillary protein (data not shown). Arrowheads indicate region of the SNc (a-d), and arrow in e indicates area magnified in cell inset (f). Scale bars: a-e, 100 μm; g-j, 15 μm; f, inset, 25 μm.

Figure 3.

Protection of nigral dopamine neurons by calpain inhibition. Representative coronal midbrain sections (at level - 3.52 mm caudal to bregma) from mice 2 weeks after treatment with saline (a), MPTP with vehicle (b), or MPTP with the calpain inhibitor MDL-28170 (c). d, Quantitative comparison of nigral dopamine (TH+) neuron survival in mice treated with saline (hatched bars), vehicle and MPTP (white bars), or MDL 28170 and MPTP (black bars). Bars represent mean number (±SEM) of neurons according to the rostrocaudal distribution measured (millimeters) from bregma (Franklin and Paxinos, 1997) from eight mice per group (ANOVA, p < 0.01; Newman-Keuls, p < 0.05).

Figure 4.

Increased calpastatin in the SNc ipsilateral to intrastriatal adenovirus administration (a), when compared with the contralateral hemisphere (b), and colocalization within dopaminergic SNc neurons (c,d). Representative coronal midbrain section at the level of the MTN (-3.16 mm caudal to bregma) (Franklin and Paxinos, 1997) from a saline-treated mouse (f) and the ipsilateral hemisphere of MPTP-treated mice that received intrastriatal administration of recombinant adenoviruses containing either lacZ (g) or the calpain inhibitor protein calpastatin (h). Images are representative of dopamine neuron survival observed 2 weeks after treatment with MPTP (n = 6 per group). e, Quantification of dopamine neuron survival per section in the ipsilateral SNc of Ad.lacZ, Ad.CALP, and uninjected-MPTP-treated or unlesioned mice. *ANOVA, p < 0.0001; Newman-Keuls, p < 0.001; Ad.CALP versus Ad.lacZ or MPTP.

Figure 5.

The neuroprotective effects of calpain inhibition on MPTP-induced toxicity in mice is associated with normalized locomotor behaviors. Two weeks after saline or MPTP treatment, groups of mice were assessed for spontaneous motor activity in a novel environment (open field) for a 60 min period, as described in Materials and Methods (a). Horizontal locomotor activity was reported as distance traveled (mean ± SEM; *ANOVA, p < 0.001; Newman-Keuls, p < 0.01; either group vs VEH-MPTP). Additional groups of mice were administered amphetamine(2.0 mg/kg, i.p.), and hyperactivity was measured in their home cages using beam-break activity monitors (b). Total activity over 30 min is plotted as mean ± SEM. Data represent n = 6-9 per group per treatment for a and b.

Figure 6.

Calpain inhibition does not prevent striatal denervation. Representative immunohistochemical detection of tyrosine hydroxylase (TH) (a-c) and dopamine transporter (DAT) (d-f) in striatal sections from mice treated with saline (a, d), MPTP, and vehicle (b, e), or MDL-28170 and MPTP (c, f). g, Quantification of striatal TH fiber densities (ANOVA, p < 0.001; Newman-Keuls, **p < 0.001, and *p < 0.05, vs saline).

Figure 7.

HPLC analysis of striatal dopamine (a) and DOPAC concentrations (b) shows no preservation of striatal dopamine or metabolism after MPTP administration in either vehicle (VEH-MPTP) or calpain inhibitor-treated (MDL-MPTP) mice. Data represent mean ± SEM (n = 6-8 per group). Where no significant differences existed between VEH-MPTP and MPTP treatment groups, data were pooled as “MPTP.”

Figure 8.

MPTP-induced increased striatal FosB expression is attenuated by calpain administration. Representative coronal forebrain sections depict low basal expression of FosB in the striatum (a) and a dramatic elevation 2 weeks after chronic administration of MPTP (b). Striatal sections from mice administered MPTP and vehicle revealed a comparable upregulation of FosB (c), whereas tissues from mice administered MPTP and the calpain inhibitor MDL-28170 revealed an attenuation of postsynaptic FosB expression (d). e, Quantification of striatal FosB-positive nuclei show significant differences between treatment groups (*ANOVA, p < 0.0005; Newman-Keuls, p < 0.01 compared with saline control). FosB expression in MDL-MPTP-treated mice did not differ from saline-treated controls (p > 0.05). Data represent mean ± SEM (n = 5-6 per group).

Figure 9.

Radioimmunoassay of striatal neurotensin revealed increased expression after treatment with MPTP that was attenuated by calpain inhibitor treatment (*ANOVA; p < 0.05). Data represent mean ± SEM (n = 7-8 per group).

Figure 10.

Immunohistochemical evidence of enhanced calpain activity in nigral neurons in postmortem human tissues. A, Immunohistochemical detection of calpain-cleaved α-spectrin (38-4) viewed at low (20×) magnification revealed intense clusters of immunopositive staining in PD cases that were rarely observed in control cases. B, C, Confocal optical sectioning (0.6 μm) of calpain-cleaved α-spectrin immunoreactivity in pigmented dopamine neurons from a case without neurological condition (B) and from a case with Parkinson's disease (C) further revealed the cytoplasmic speckled appearance of 38-4-immunopositive staining that colocalized with neuromelanin (insets). D, Quantification of the proportion of pigmented neurons per section that displayed speckled calpain-cleavedα-spectrin immunoreactivity from PD and control tissues revealed a significant increase in calpain activity in subjects with PD (**52.83 ± 7.5 vs 9.17 ± 3.1%; Mann-Whitney rank-sum test; p = 0.0079). Positive immunostaining disappeared when the primary antibody was omitted (data not shown). Scale bars: A,25μm; B, C,50μm.