The Effects Of Phenylethanoid Glycosides On The MPTP Mice

Parkinson's disease (PD) is a progressive neurodegenerative disease, pathologically characterized by selective loss of dopaminergic neurons in substantia nigra and neuronal cytoplasmic inclusions called Lewy bodies. The underlying mechanism of PD remains unknown. Interestingly, Dauer and coworkers found thatα-synuclein (α-syn) null mice displayed striking resistance to 1-methyl-4—phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced degeneration of dopaminergic neurons, and this resistance appeared to result from an inability of the toxin to inhibit complex I. Furthermore, the mice with Overexpression ofα-syn treated with MPTP have significantly greater mitochondrial abnormalities than those seen in either of the saline-treated controls or the MPTP-treated wild-type mice. Thus, the two questions arise if there is a physical link betweenα-syn and mitochondria, and if the link is involved in the deleterious cascade of events induced by MPTP. We and others have found that phenylethanoid glycosides from cistanchis salsa could inhibit the neuronal apoptosis induced by 1-methyl-4-phenylpyridinium, we want to know ifα-syn is the target of phenylethanoid glycosides.To examine those questions, firstly, we investigated the subcellular localization ofα-syn and potential co-localization ofα-syn and mitochrondia. Here, we show that a portion ofα-syn is present in the membrane of mitochondria in normal dopaminergic (DA) neurons. Then, we observed the response ofα-syn binding to mitochondria to MPTP in vivo, and found that aggregation of mitochondria induced by MPTP, which was co-localized withα-syn. As a subsequent result, massive reduction of mitochondria was found in some nigral dopaminergic neurons severely damaged by MPTP. Finally, we investigate the effects of phenylethanoid glycosides on the expression ofα-synResultsα-Syn is present in the membrane of mitochondria in the neurons of sustantia nigra of miceWe performed purification of mitochondria and western blot analysis, and found that, in purified mitochondrial pellet of ventral midbrain, the expression ofα-syn was detected. The expression ofα-syn in mitochondria of cerebral cortex were strikingly similar to those observed in ventral midbrain. This suggests that expression ofα-syn in mitochondria is a common characteristic ofα-syn-positive cells, not only in ventral midbrain, but also in other areas of brain.The double-immunofluorescence technique was used to investigate co-localization ofα-syn and COX IV in nigral neurons of mice. In the merged picture, the immunofluorescence ofα-syn is partially overlapped with that of COX IV, the yellow area is also distributed diffusely in the neuronal somata in the substantia nigra. The same phenomena also were found in theα-syn positive neurons of hippocampus, this suggests that co-localization ofα-syn and mitochondrial structure is not specific to nigral neurons.With the pre-embedding double labeled immunoelectron microscopic techniques, we found that, in the sections of substantia nigra, a portion of immunogold silver grains was detected in the membrane but not matrix of mitochondriaAbnormal aggregation of mitochondria co-localized withα-syn in substantia nigra neurons after chronic MPTP treatmentAfter the initial MPTP injection, the immunofluorescence ofα-syn increased dramatically and was distributed over the whole neuronal somata including the nuclear area in the SNc at 12 hours. At 24 hours after the initial MPTP injection, massive aggregated mitochondria were found around the nuclear area in neurons over the SNc, the location ofα-syn protein was coincided with that of the COX IV protein. From 2 -7 days after the initial MPTP injection, a few but not most of neurons in the SNc were also found with the increasedα-syn immunoreactivity, however, the aggregated mitochondria still were observed in many neurous in the SNc, which co-localized withα-syn protein. Furthermore, immunogold electron microscopy was performed to investigate the changes ofα-syn in mitochondria of dopaminergic neurons in the SNc induced by MPTP. Unexpectedly, we did not found massive immunogold silver grains (theα-syn immunoreactivity) importing into the mitochondrial matrix in any time points after MPTP administration, only in a few of mitochondria the immunogold silver grains were found into the matrix.α-Syn immunoreactivity was still detected mainly in the membrane of mitochondria within dopaminergic neurons in MPTP-intoxicated mice.Reduction of mitochondria in neurons in substantia nigra after MPTP intoxicationImmunogold electron microscopy revealed that a reduction of mitochondria occured in many dopaminergic neurons after 3 days (at 24 hours after second MPTP injection) MPTP treatment, and the surviving mitochondria still gathered and aggregated in the the dopaminergic neurons. We also observed that many mitochondria gathered in the proximal neuronal processes in some dopaminergic neurons, even if the reduction of mitochondria was present in the cytoplasmic area. Superimposed on the reduction of mitochondria, large, swollen and/or vacuolated mitochondria in the dopaminergic neurons were also observed.The effects of phenylethanoid glycosides on the MPTP mice. We found that, phenylethanoid glycosides could reduce the average time of pole test of MPTP mice, and increase the expression of TH in striatum after MPTP injection. Only high dose (400mg/kg/d)of phenylethanoid glycosides could inhibit the reduce of TH positive neurons within SNc at 7d and 14d after the initial MPTP injection significantly (P <0.05). Then, we performed immunohistochemistry to investigate the effects of phenylethanoid glycosides on expression ofα-synuclein within SNc at 4d after the initial MPTP injection, and found that, after MPTP administration, the number of neurons with upregulated expression ofα-synuclein increased within SNc(P<0.01). The high dose (400mg/kg/d)of phenylethanoid glycosides could reduce the the number of neurons with upregulated expression ofα-synuclein within SNc(P<0.01).Conclusion:α-Syn is present in the membrane of mitochondria in the neurons of sustantia nigra of mice, and binding to the membrane of mitochondria is the physiological nature ofα-syn and might also contribute to the pathological role of this protein in the mitochondrial dysfunction induced by MPTP. Phenylethanoid glycosides could improve the MPTP models in vivo, and the changes of the expression ofα-synuclein may play a role in the process.