以MPTP傷害視網膜多巴胺細胞群探討影響巴金森氏症之因子

Abstr act

Parkinson's disease (PD) is a progressive neurological disorder in which the cardinal pathological feature is the loss of dopaminergic neurons in the substantia nigra pars compacta and their axons, which project to the striatum. The cause of PD remains unknown, but our understanding of mechanisms of dopaminergic neuronal death was advance by the discovery of 1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that selectively damages the nigrostriatal dopaminergic system and causes a parkinsonian syndrome in experimental animals. Systemically administered MPTP is not only selectively neurotoxic to substantia nigra neurons but also accumulates in the eye and induces damage in some retinal neurons. Acute histopathologic and chemical changes are also observed, including the degeneration of retinal cells as well as a downregulation of tyrosine hydroxylase, within a few hours of MPTP administration.

The retina is an outgrowth of forebrain and considered as part of the central nervous system. Our previous findings have shown that several substances, i.e., Bcl-2, are highly involved in neuroprotection in the degenerating retina under experimental conditions (Chen et al., 1994b, 1996, 1997, 1999). It is interesting to investigate whether appotosis-associated proteins, such as Bcl-2 family, regulate PD pathological lesions. In the present study, several important findings were described as follows:

1. This study indicates that intrastriatal injection of the neurotoxin has direct cytotoxic effect on neurons and glia, which are accompanied by thealteration of Bcl-2 and associated proteins in injured nigrostriatal pathway and retinal system.

2. A severe neuronal loss and a significant increase of Bcl-2 appears in the peritraumatic striatum and cortex, providing further evidence that Bcl-2 could be expressed in OX- 42-positive or activated microglia cells following MPP

insult.

3. In the injured SN, de novo synthesis of Bcl-2 is observed in the nigral neurons which are damaged indirectly and yet appear to survive.

4. Since Bcl-2 immunostaining within AD and PD brain tissues was shown to be increased with disease severity, their expressions may have a role in compensatory responses under PD/AD conditions, perhaps offering a neuroprotective mechanism to the remaining neurons.

5. The reduction of Bcl-2 family was different from the upregulation of Bcl-2 and Bax in the injured striatum and cortex, it may suggest that there are various neuronal and glial cytoarchitectonics between two nervous systems.

Background and Aims

The neurotoxic effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite ion MPP

on nigrostriatal dopaminergic neurons, as a model of Parkinson’s disease (PD), has been well documented (reviewed by Mari and Bódis–

Wollner, 1997). Following the neurotoxin injected into the striatum of experimental

animals, MPP

is taken up by dopaminergic nerve terminals and transported to the substantia nigra compacta, where it disrupts cellular respiration by inhibiting complex I of the electron transport chain. The neurotoxin is also accumulated in the eye and induces damage in some retinal neurons (Lydén et al., 1985; Wong et al., 1985). These cells are believed to be involved in the generation of the oscillatory potentials of the electroretinogram (Wachtmeister and Dowling, 1978). Further, several studies have also shown that MPTP or MPP

administration results in a marked depletion of tyrosine hydrolase immunoreactivity in the inner plexiform layer, as well as the activities of astrocytes and Müller glia (Guarnier et al., 1989; Adams et al., 1992). On the other hand, there is increasing evidence that Bcl-2 is capable of preventing cell death by blocking the release of cytochrome c and certain other mitochondrial reaction that precipitate apoptosis, whereas some studies have shown that Bcl-2 expression may also led to cellular redistribution of glutathione which could interfere with nuclear caspase activation and regulate apoptosis. The latter is of interest as we observed a downregulation of glutathione in response to kainate- or MPP

-induced apoptosis which can be prevented by melatonin (Chen and Chuang, 1999; Chuang et al., 1999). In the retinal system, our previous studies have indicated a significant increase of Bcl-2 expression in Müller glia and ganglion cells following optic nerve transection, ischemic injury or gliotoxin injection, suggesting that Bcl-2 plays an important role in regulating the survival of retinal populations (Chen et al., 1994, 1996, 1999). Besides, various homodimers and heterodimers formed by proteins in the bcl-2 family can either promote or inhibit cell death. For example, Bax homodimerizes and forms heterodimers with Bcl-2, countering the death repressor activity of Bcl-2. Bcl-X

respresses cell death, while its short form, Bcl-X

, favors cell death. Other pro-apoptotic members of this family include Bad, Bak, Bid and Hrk. Additional apoptotic inhibitors in this family include Bcl-w and A1 (Bfl-1). The propesisty of a cell to undergo apoptosis may be dependent on the ratio of these positive and negative regulators within the cell (Boise et al., 1993; Oltvai et al., 1993). In the present study, we investigate the effect of MPTP/MPP

intoxication on the survival/ cell death of retinal populations and the expression of apoptosis-regulators, Bcl-2 family. Three aims were performed, 1) To characterize the effect of MPTP/MPP

-induced neuronal degeneration in relation to glial action, 2) to determine the cellular source and pattern of expression of Bcl-2 and its associated proteins after neurotoxic treatment, and 3) to compare the expression of possible roles of these proteins in the retinal system and nigrostriatal pathway.

MATERIALS AND METHODS I. Animals and chemicals treatment

Sprague-Dawley rats weighing 300-350 gm were used. Intravitreal injections were

made in the left eyeball at a volume of 3 µl of 100 nmol MPP

or saline, while

intrastriatal injections were made in the left striatum at a volume of 1 µl of 100 nmol MPP

or saline a blunt-tipped 30-gauge Hamilton syringe. The animals were allowed to survive for 3 hours, 1, 3, 7 days and 2 to 8 weeks before they were perfused with 4%

paraformaldehyde in phosphate buffer.

II. Immunocytochemistry and histological features

After animals were perfused, the eyeballs and brains were dissected and sectioned on cryostat. The retina and brain sections were reacted immunocytochemically for Bcl-2 (DAKO), Bax, Bcl-X, Bad, A1, Bcl-w and Bid (Santa Cruz) and tyrosine hydroxylase (TH, Sigma). Some of the adjacent sections were incubated with glutamine synthase (GS, Chemicon), GFAP (Sigma) and OX-42 (Serotec), makers for Müller glia, astrocytes and microglia/ macrophages, respectively. The sections were then processed for peroxidase reaction and developed according to the glucose oxidase-nickel-DAB method. Some of the sections were stained with cresyl violet to study the cytoarchitectonics and the pattern of cell death in the brain.

RESULTS

I. Structural analysis following MPP

injection

While only a small group of apoptotic cells along the tract of cannula was observed at the striatum and cortex 3 to 24 h postinjection, almost whole striatum and adjacent cortex showed a clear sign of severe degeneration 3 d to 2 mo after MPP

injury, as compared with the contralateral side. The morphological alteration and neuronal degeneration could be also detected in the SN ipsilaterally to MPP

-injected brain as revealed tyrosine hydrolase (TH) immunoreactivity, a marker for dopaminergic neurons. In the injured retina, the number of TH-positive neurons in the inner nuclear layer was also reduced.

II. Distribution of Bcl-2 and associated proteins in rat striatum, cortex and SN

In the contralateral uninjected side only faint Bcl-2 immunoreactivity was observed in

the striatal and cortical neurons, while a few of Bcl-2-positive cells appeared along the

tract of cannula 4 h postinjection. The cellular number and staining intensity were

increased significantly 3 to 7 d later. These ameboid-shaped small cells immunostained

for Bcl-2 were distributed in whole injured striatum 1 and 2 mo post-injection,

however, the number of positive cells was reduced. Except very faint expression in the

blood vessels, few Bcl-2 immunoreactive cells were observed in the SN of the intact

brain. To our surprise, Bcl-2-immunoreacted nigral cells appeared 3 to 7 d after MPP

intrastriatal injection. It is noteworthy that Bcl-2-immunoreactivity in the SN was

expressed in neurons showing medium-sized somata of varying morphology. The up-

regulation of Bcl-2 expression was eliminated 1 to 2 mo postinjury. While only faint

immunoreactivity for Bax was observed in the normal striatum and cortex, moderately

positive cells and fibers appeared in the lesioned cortex. Numerous small granules

could be detected in the cytoplasm of cortical pyramidal neurons 1mm away from the cannula.

III. Bcl-2 family in the normal and injured retinas

As previous studies, Bcl-2 was primarily distributed in the Müller glial processes in the normal retina. However, the staining intensity was decreased slightly 3h to 7 d postinjury. Bad, Bcl-x and Hrk immunoreactivities were mainly present in the retinal ganglion cells and nerve fiber layers of normal tissues, whereas the expression was downregulated by the MPP

intoxication. Bax was expressed in the nerve fiber layer in the normal and injured retinas. For A1 and Bcl-w, only faint immunoreactivity was found in both groups. Interestingly, Bid was expressed in the inner/outer segments of photoreceptors and outer limiting membrane, resembling the outer part of Müller glial processes. The pattern was not altered following MPP

intravitreal injection.

IV. Glial response in the MPP

-lesioned brain and retina

GFAP-immunoreactive cells (astrocytes) were evenly distributed in the nigrostriatal pathway of intact brain, however, a massive loss of GFAP-positive cells in the injected region was observed within a few days after lesion. On the other hand, OX-42-reactive cells were upregulated within 1 to 4 h after MPP

administration and expressed throughout the ipsilateral striatum within a few days. The increase of OX-42 expression reduced 1 mo postinjury as previous described for Bcl-2 and APP. In the SN a few hours to days subsequent to MPP

injection, the staining intensity increased and the ramified processes of OX-42-positive cells became much thicker, representing characteristic features of activated microglia. In the retinal system, the expression of OX-42 and GFAP was not changed postlesion. However, the staining intensity of GS, a marker for Müller cell body and processes, was decreased significantly several hours to days after intoxication.

CONCLUSIONS

- This study indicates that intrastriatal injection of the neurotoxin has direct cytotoxic effect on neurons and glia, which are accompanied by thealteration of Bcl-2 and associated proteins in injured nigrostriatal pathway and retinal system.

- A severe neuronal loss and a significant increase of Bcl-2 appears in the peritraumatic striatum and cortex, providing further evidence that Bcl-2 could be expressed in OX-42-positive or activated microglia cells following MPP

insult.

- In the injured SN, de novo synthesis of Bcl-2 is observed in the nigral neurons which are damaged indirectly and yet appear to survive.

- Since Bcl-2 immunostaining within AD and PD brain tissues was shown to be

increased with disease severity, their expressions may have a role in compensatory

responses under PD/AD conditions, perhaps offering a neuroprotective mechanism to

the remaining neurons.