The parkinsonian neurotoxin rotenone activates calpain and caspase-3 leading to motoneuron degeneration in spinal cord of Lewis rats

Abstract

Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinson's disease (PD). Rotenone is a neurotoxin that has been used to induce experimental Parkinsonism in rats. We used the rotenone model of experimental Parkinsonism to explore a novel aspect of extra-nigral degeneration, the neurodegeneration of spinal cord (SC), in PD. Rotenone administration to male Lewis rats caused significant neuronal cell death in cervical and lumbar SC as compared with control animals. Dying neurons were motoneurons as identified by double immunofluorescent labeling for terminal deoxynucleotidyl transferase, recombinant-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells and choline acetyltransferase (ChAT)-immunoreactivity. Neuronal death was accompanied by abundant astrogliosis and microgliosis as evidenced from glial fibrillary acidic protein (GFAP)-immunoreactivity and OX-42-immunoreactivity, respectively, implicating an inflammatory component during neurodegeneration in SC. However, the integrity of the white matter in SC was not affected by rotenone administration as evidenced from the non co-localization of any TUNEL(+) cells with GFAP-immunoreactivity and myelin basic protein (MBP)-immunoreactivity, the selective markers for astrocytes and oligodendrocytes, respectively. Increased activities of 76 kD active m-calpain and 17/19 kD active caspase-3 further demonstrated involvement of these enzymes in cell death in SC. The finding of ChAT(+) cell death also suggested degeneration of SC motoneurons in rotenone-induced experimental Parkinsonism. Thus, this is the first report of its kind in which the selective vulnerability of a putative parkinsonian target outside of nigrostriatal system has been tested using an environmental toxin to understand the pathophysiology of PD. Moreover, rotenone-induced degeneration of SC motoneuron in this model of experimental Parkinsonism progressed with upregulation of calpain and caspase-3.

Fig. 1

Effect of rotenone on body weight. Rats were injected with rotenone (2.5 mg/kg/day) subcutaneously (s.c.) once daily during the first 4 days, followed by single injection of the same dose on day 6, 9, 12, 15, 18, and 21 (total 25 mg/kg over 21 days). Daily records of body weight of rats injected with rotenone (closed circles) or vehicle (open circles) are represented in g ± SEM. *P<0.05 (n=9).

Fig. 2

Effect of rotenone on neuronal viability in cervical and lumbar SC. (A) Representative photomicrographs of double immunofluorescent staining for NeuN (green) and TUNEL (red) performed in cervical and lumbar SC coronal sections (5 μm). No TUNEL⁺ neurons were identified in the sections from control animals (see Merge); however, cell death as evaluated by TUNEL-positive IR in many dorsal and ventral neurons was found in sections from cervical and lumbar SC segments in rotenone-injected animals (yellow, merge). Images taken at 200X magnification (n≥4). (B) The lower panels show semi-quantitative analysis of TUNEL mean fluorescence intensities (MFI) per unit area of neurons. Data show significant differences in the arbitrary units between samples from control animals and after rotenone exposure. *P≤0.05 (n≥ 4).

Fig. 3

Effect of rotenone on motoneurons in cervical and lumbar SC. Representative photomicrographs of double immunofluorescent staining for TUNEL (red) and ChAT (green) a marker of motoneuron, performed in cervical and lumbar SC coronal slices (5 μm). In cervical and lumbar slices from control rat SC, ChAT-positive motoneurons did not show TUNEL-positive IR (A, B, C and G, H, I). TUNEL-positive IR in ventral motoneurons appeared after rotenone exposure, depicting motoneuronal cell death, evaluated by ChAT and TUNEL co-localization sites (D, E, F and J, K, L, respectively, arrows indicate co-staining). Images taken at 200x magnification (n≥4). Semi-quantitative analysis of TUNEL mean fluorescence intensities (MFI) per unit area of ventral motoneurons has been represented in the respective right panels. Data showed significant differences in the arbitrary units between samples from control animals and after rotenone exposure. *P≤0.05 (n≥4).

Fig. 4

Effect of rotenone on GFAP and OX-42 immunoreactivity in cervical and lumbar SC. In control rat SC sections, there were a few GFAP and OX-42 positive cells. Intense astrogliosis, detected by increased GFAP-IR (A) and microgliosis, detected by OX-42-IR (C) seen in both cervical and lumbar SC slices from rotenone-injected animals. Data analyzed with NIH image 1.63 showed significant differences in the number of pixels in sections from control and rotenone-injected animals (B and D). *P≤0.05 (n≥ 4).

Fig. 5

Effect of rotenone on astrocytes in cervical and lumbar SC. Representative photomicrographs of double immunofluorescent staining for TUNEL (red) and GFAP (green) a marker for astrocytes, performed in cervical and lumbar SC coronal slices (5 μm). In cervical and lumbar slices from SC, GFAP-positive astrocytes did not show TUNEL-positive IR in control or rotenone administered rats. GFAP-immunostaining was enhanced in experimental rat SC. Images taken at 200x magnification (n≥4).

Fig. 6

Effect of rotenone on oligodendrocytes in cervical and lumbar SC. Representative photomicrographs of double immunofluorescent staining for TUNEL (red) and MBP (green) a marker of oligodendrocytes, performed in cervical and lumbar SC coronal slices (5 μm). MBP-positive oligodendrocytes did not show any TUNEL-positive IR in cervical and lumbar slices from control or the experimental rat SC. Images taken at 200x magnification (n≥4).

Fig. 7

Effect of rotenone on calpain content in cervical and lumbar SC. Determination of calpain content by Western blotting (A). Representative blot pictures (upper panels) showing bands of 80 kD (inactive) and 76 kD (active) calpain, as well as 42 kD β-actin (loading control), detected in cervical and lumbar SC tissues from control and rotenone-injected animals respectively. Densitometric analysis showed significant percent changes in 76 kD active calpain bands in both cervical and lumbar SC segments in rotenone-injected rats. *P≤0.05 (n≥4). Representative photomicrographs (B) of double immunofluorescent staining for 76 kD m-calpain (red) and NeuN (green), performed in cervical and lumbar SC coronal slices (5 μm). No active calpain-IR was found in the sections from control animals. Significant content of active calpain was observed in many ventral motoneurons in cervical and lumbar SC segments of rotenone-injected animals, evaluated by co-localization of 76 kD calpain-IR and NeuN-IR (Merge indicate co-staining in rotenone panel). Images are taken at 200x magnification (n≥4).

Fig. 8

Effect of rotenone on activation of caspase-3 in cervical and lumbar SC. Representative photomicrographs of double immunofluorescent staining for active caspase-3 (red) and NeuN (green), performed in cervical and lumbar SC coronal slices (5 μm). No active caspase-3-IR was found in the sections from control animals. Significant amount of active caspase-3 was observed in many ventral motoneurons in cervical and lumbar SC segments of rotenone-injected animals, evaluated by increased co-localization of active caspase-3-IR and NeuN-IR (Merge indicate co-staining in rotenone panel). Images are taken at 200x magnification (n≥4).

Fig. 9

Effect of rotenone on calpain and caspase-3 activities in cervical and lumbar SC. Representative blot pictures, showing bands of calpain-specific 145 kD SBDP and caspase-3-specific 120 kD SBDP, as well as 42 kD β-actin (loading control) in cervical and lumbar SC tissues from control and rotenone-injected animals respectively. Densitometrc analysis shows significant percent changes in calpain and caspase-3 SBDPs in both cervical and lumbar SC segments in rotenone-injected rats. *P<0.05 (n≥4).

Fig. 10. Effect of rotenone on TH-IR

Representative photomicrographs of immunofluorescent staining for TH-IR in coronal brain sections (10 μm) passing through SN and striatal region from control rats (A and C) and rotenone-injected rats (B and D). In control animals, numerous TH-positive cells and intense TH-IR are seen in SNpc (A) and striatum (C), respectively. Rotenone administration caused substantial decrease in TH-IR in both SN and striatum. Images were taken at 200X magnification. (n≥4).