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,1
,1
,1,||,2
* Burke Medical Research Institute,
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, White Plains, New York;
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, South Korea;
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA; and
|| Kyung Hee University, College of Oriental Medicine, Seoul, South Korea
2Correspondence: Department of Neurology and Neuroscience, Weill Medical College of Cornell University, Rm. F610, 1300 York Ave., New York, NY 10021. E-mail: thjoh{at}med.cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: dopamine neuron protection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; DA neurons degenerate progressively in a self-perpetuating fashion (2)
. Although the molecular and cellular mechanisms underlying the degenerative process of DA neurons have not yet been defined, accumulating evidence suggests that microglia may play an important role (3
, 4)
. Activated microglia were found around surviving DA neurons in the brain of PD patients (2
, 4)
. In mesencephalic primary culture, activated microglia generated superoxide anion, O2– via NADPH oxidase (NADPHO) reaction (5
, 6)
and enhanced neurotoxin-elicited DA neuronal death. In addition, activated microglia could cause DA neuronal death (7
8
9)
; for example, lipopolysaccharide (LPS)-activated microglia generated superoxide and NO that caused DA cell death. These findings indicate that activated microglia may play an important role in the progressive degeneration of DA neurons in PD. We have been investigating novel signaling mechanisms that activate microglia in DA neuronal degeneration, including whether abrogation or inhibition of the signaling might prevent the progression of degeneration.
In our previous report (10)
, we demonstrated that the active form of matrix metalloproteinase-3 (actMMP-3) was released from apoptotic neuronal cells and activated microglia in vitro. Catalytically active recombinant MMP-3 (cMMP-3) also activated microglia, and both actMMP-3 and cMMP-3 led to production of microglial inflammatory cytokines such as TNF-
, which, in turn, exacerbated DA cell death (10)
. Because MMP-3 had only been known as a member of the MMP family that digests or modifies the extracellular matrix complex (EMC), the MMP-3-elicited activation of microglia was a newly discovered action of this enzyme. Moreover, whether MMP-3 participates in DA neuronal death in PD has never been addressed thus far.
In this manuscript, we present a novel mechanism by which actMMP-3 causes microglial activation, leading to DA neuronal degeneration, using primary cultures of mouse mesencephalon and enriched microglia, as well as the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected animal model of PD. This finding will contribute to the understanding of the significance of the presence of activated microglia in the substantia nigra (SN) and their role in DA neuronal degeneration in the brain of PD patients. Our present results may lead to a novel therapeutic approach for PD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
MMP-3 null mice (C57BL/6x129SvEv) were generously donated by Dr. John Mudgett (Merck) (11)
. Because the genetic background of these mice has been maintained in C57BL/6, the same strain from the Jackson ImmunoResearch Laboratory (Bar Harbor, ME, USA) was used as WT. WT C57BL/6J (gp91phox+/+) and NADPHO-null (gp91phox–/–) mice were also obtained from the Jackson ImmunoResearch Laboratory (5)
. All animals were maintained according to the Institutional Animal Care and Use Committee approved protocol.
Primary mesencephalic neuron-enriched culture
All experiments were conducted within National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee. Pregnant C57BL/6 mice were obtained from the Jackson ImmunoResearch Laboratory. The ventral mesencephalon was removed from 14 days gestation embryo and incubated with 0.01% trypsin in HBSS for 15 min at 37°C. After trituration, 3 x 105 cells were plated on each polystyrene cover slide that had been precoated with 100 µg/ml poly-L-lysine and 4 µg/ml laminin and placed in a 24-well culture plate. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 in neurobasal medium supplemented with B-27, 2 mM glutamine, 100 IU/l penicillin, and 10 µg/ml streptomycin. At day 5 or 6 in vitro, the neurons were fed with fresh medium and treated.
Primary mesencephalic neuron-glia cultures
Mouse ventral mesencephalic neuron-glia cultures of gp91phox+/+ and gp91phox–/– mice were prepared using a previously described protocol (9)
. Briefly, midbrain tissues were dissected from 12- to 13 day-old mouse embryos. Cells were dissociated via gentle mechanical trituration in minimum essential medium (MEM) and immediately seeded (5x105 cells/well) in poly D-lysine (20 µl/ml) precoated 24-well plates. Cells were seeded in maintenance medium and treated in treatment medium, as described previously (12)
. Three days after the seeding, the cells were replenished with 500 µl of fresh maintenance medium. Four days later, the cells were treated with either LPS (10 ng/ml) or cMMP-3 (250 ng/ml).
Microglia-enriched cultures
Microglia were prepared from whole brains of 1-day-old mouse pups, as described previously (12)
. Briefly, after removing meninges and blood vessels, the brain tissue was gently triturated and seeded (5x107) in 150 cm2 flasks on day 1. On day 8, the media were replaced. On day 15, when the cells had reached a confluent monolayer of glial cells, microglia were separated from astrocytes by shaking for 5 h at 150 rpm and replated at 1 x 105 in a 96-well plate precoated with poly D-lysine. The enriched microglia were >95% pure, as determined by immunostaining with antibodies against F4/80 and GFAP. The cultures were treated with either LPS (10 ng/ml) or cMMP-3 (250 or 125 ng/ml).
Measurement of MMP activity released from MPP+-treated mesencephalic cells
MMP activity in mouse mesencephalic cultures in the presence of MPP+, the oxidative product of MPTP (1
, 13
14
15
16
17)
, was measured using the MMP-3 assay kit. Briefly, the medium (100 µl) was collected from cell culture that had been treated with MPP+ for 6 h to 72 h and transferred to a 96-well plate, to which 100 µl of assay buffer (50 mM MES, 10 mM CaCl2, 0.05% Brij-35, pH 6.0) was added. After incubation for 30 min at room temperature, the reaction was started by the addition of 1 µl (4 µM final concentration) of the substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2). The samples were read continuously in Spectra Max Plus microtiter plate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) over 30 min at Ex/Em = 328/393.
Detection of MMP-3 released from MPP+-treated mesencephalic cells by immunoprecipitation and Western blot analysis
Culture medium (300 µl) was collected at 24 h and 48 h after the MPP+ treatment and incubated with 5 µl of anti-MMP-3 polyclonal antibody (pAb) (Santa Cruz) for 2 h, followed by 50 µl of protein G conjugated with agarose beads overnight at 4°C. The sample was then precipitated by centrifugation at 5000 g, washed with PBS three times, resuspended with loading buffer, and separated in 10–20% tricine gel (Invitrogen). Western blot analysis was carried out using anti-MMP-3 Ab (R&D Systems).
DA uptake assay
Cells were incubated in Krebs-Ringer buffer (130 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1 mM CaCl2, 3 mM KCl, 10 mM glucose, 10 mM HEPES) for 15 min at 37°C with 1 µM [3H]DA. Nonspecific uptake was blocked with 10 µM mazindole. The cells were then washed three times with ice-cold Krebs-Ringer buffer and lysed in 5 ml of 1 N NaOH. Radioactivity was measured by scintillation counting, and specific [3H]DA uptake was calculated by subtracting the counts in the presence of mazindole.
Measurement of superoxide release
Extracellular superoxide (O2–) production from microglia was determined, as reported previously by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1 (9)
. Briefly, microglia-enriched cultures were seeded (5x104 cells/well) in 96-well plates and incubated for 24 h. Immediately before treatment, cells were washed twice with HBSS. To each well, 100 µl of HBSS with or without SOD (600 U/ml), 50 µl of vehicle or LPS, and 50 µl of WST-1 (1 mM) in HBSS were added. The cultures were incubated for 30 min at 37°C and 5% CO2 and 95% air. The absorbance at 450 nm was read with a Spectra Max Plus spectrophotometer. The amount of SOD-inhibitable superoxide was calculated and expressed as a percentage of vehicle-treated control cultures.
MPTP injection and immunohistochemistry
WT and MMP-3 null mice received 4 intraperitoneal (i.p.) injections of PBS or MPTP (4x15 mg/kg at 2-h intervals) and were sacrificed after 2, 5, and 10 days (12 mice per time point). Immunohistochemistry was performed using the protocol described previously (18
, 19)
. Briefly, mice were deeply anesthetized with sodium pentobarbital (120 mg/kg) and transcardially perfused with saline containing 0.5% sodium nitrite and 10 U/ml heparin sulfate, followed by cold 4% formaldehyde generated from paraformaldehyde in 0.1M PBS (pH 7.2). Brains were postfixed in the same solution for 1 h and infiltrated with 30% sucrose overnight. Free-floating sections (40 µm) were obtained from the striata and SN using a freezing microtome. Sections were washed in 0.1 M PBS, incubated in 0.1 M PBS containing 1% BSA and 0.2% TritonX-100 for 30 min and subsequently incubated overnight with either TH (1:10,000) or CD11b (1:1,000) antibodies. The sections were then incubated with appropriate secondary IgG (1:200) for 1 h, followed by avidin/biotin/peroxidase staining for 1 h in a humidified chamber. PBS (0.1 M, pH 7.4) containing 0.5% BSA was used to wash sections on slides between all steps. The antigen-antibody complexes were visualized by incubation for 5 min in 0.05% [abb0]3,3'-diaminobenzidine and 0.003% H2O2.
Quantitative morphometrics and statistics
The volume and number of neurons in the SN pars compacta (SNpC) were generated as previously reported (20)
. Two investigators blinded to the experimental groups used unbiased morphometric techniques to make the measurements. Briefly, tissue sections were systematically projected on a video monitor. Internal tissue landmarks defined the perimeter of the SNpC that could be outlined by a cursor, and a software program (IBAS/20 Zeiss, Thornwood, NY, USA) produced the area of target on a particular section. The target volume was calculated based on the information on this target area, distance between samples (160 µm), and the total number of samples.
SNpC neuron number was determined by a modification of the optical dissector techniques previously reported (20)
. Briefly, a counting frame (100 µmx100 µm) was passed over the entire SNpC from five sections at 320-µm intervals. As in prior studies, neurons were identified as the largest cells in the field, and by the presence of an axon hillock and at least one distinct projection. Neurons were counted when they appeared within a portion of the counting frame (a square, 50 µm on a side) but were not in contact with the left or bottom border (oil immersion objective=1.25 N.A.). A motorized stage permitted systematic sampling of the entire SNpC and control over the Z-plane. Summing the number of neurons in all the counting frames and dividing by the volume of the total number of counting frames generated neuron densities. Total number of neurons was then the product of density and the volume of the SNpC. An ANOVA with group and side of the brain as factors was used to test for changes in the number of SNpC neurons and volume of the SNpC. Post hoc contrasts were determined by Fisher’s probable least-squares difference (PLSD). Analysis was performed using StatView 5.0. P values of 0.05 were considered significant.
In situ visualization of superoxide
In situ visualization of superoxide and its derivative oxidant products was performed by hydroethidine histochemistry, as described previously (8)
. Briefly, on days 2 and 5 post-MPTP administration, mice were injected i.p. with 200 µl of PBS containing 1 µg/µl hydroethidine and 1% dimethysulfoxide. Animals were sacrificed 15 min later, and the brain was removed and frozen on dry ice. Both SNpC and striatal sections (14 µm thick) mounted onto gelatin-coated glass slides were examined for hydroethidine oxidation product, ethidium accumulation, by fluorescence microscopy.
Statistical analysis
Statistical significance was assessed by ANOVA followed by Bonferroni’s t test using the StatView program (Abacus Concepts, Berkeley, CA, USA). A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 to 2%, as determined by immunostaining of the DA biosynthetic enzyme TH. MPP+-mediated DA cell death was examined by measuring DA uptake and TH-positive DA neuronal fiber degeneration. At 24 h after the addition of 5 µM of MPP+, a mixed population TH-positive DA cells were observed: relatively healthy cells with intact fibers and degenerating cells with little fibers remaining, indicating asynchronous cell death. Interestingly, MMP-3 staining was observed exclusively in the latter group of DA cells, suggesting that MMP-3 was induced in degenerating DA cells at an early stage (Fig. 1
A). In the absence of MPP+, MMP-3 was barely detectable (Fig. 1A
, top). By immunoprecipitation followed by Western blot analysis (Fig. 1B
), actMMP-3 (48 kDa) was detected in the culture medium in the untreated control at a minute level, which was increased by the MPP+ treatment in a dose-dependent manner (1 µM, 5 µM, and 10 µM), demonstrating that actMMP-3 was released from the stressed DA cells. The level of released actMMP-3 was higher at 24 h than at 48 h, suggesting that the release occurred at early stage of DA cell death. MMP activity in the medium also increased (Fig. 1C
) in accordance with the increase in actMMP-3 protein level. Although this measures the total MMP activity, involvement of other MMPs seemed minimal, because other MMPs such as MMP-9 were not detected in the culture medium (not shown). To confirm that MMP-3 induction and actMMP-3 release were related to DA cell degeneration, DA uptake was measured at 24 h and 48 h post-MPP+ treatment. MPP+ caused impairment of DA uptake in a dose-dependent manner (Fig. 1D
).
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Activation of microglia by cMMP-3, production of microglial NADPHO-derived superoxide, and superoxide-dependent DA neuronal death
To investigate the mechanisms underlying the actMMP-3-mediated microglial activation that lead to DA cell death, cMMP-3 was used to activate microglia in an enriched mouse microglial culture. In the presence of either 125 ng/ml or 250 ng/ml cMMP-3, superoxide level was increased in the medium (Fig. 2
A). The release began within 5 min and lasted for nearly 60 min, reaching maximum at 30 min (data not shown). The superoxide measured at 30 min was higher with a higher concentration of cMMP-3 (250 ng/ml) (Fig. 2A
). Furthermore, the addition of cMMP-3 (250 ng/ml) to neuron-glia mixed culture caused DA cell death, as evidenced by the 40% decrease in (3)
[H]DA uptake in 2 days (P<0.01) (Fig. 2B
). In comparison, DA uptake was not decreased in similarly treated cultures of NADPHO subunit gp91 null mice (gp91phox–/–), which have impaired superoxide generation (Fig. 2B
). Taken together, the data indicated that NADPHO-generated superoxide mediated the DA cell death following cMMP-3-induced microglial activation.
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Dramatic attenuation of MPTP-elicited SN DA neuronal degeneration in the absence of MMP-3
Intraperitoneal injection of MPTP elicited a 35% (P<0.003) loss of TH-positive SN DA neurons after 5 days, and a 70% loss (P<0.001) after 10 days (Figs. 3A, B
). In MMP-3 null mice, 90% and 82% of SN DA neurons were intact at 5 and 10 d post-MPTP, respectively (Figs. 3A, B
). The TH-positive DA neurons in both groups had the same morphology, but those in MMP-3 null mice were more closely packed in the SNpC compared to the WT. The striatal DA level is an important indicator for dopaminergic neuronal degeneration because the striatum is the first site of MPP+ uptake by DA transporter and hence the first site of degeneration. DA depletion in the striata of MMP-3 null mice was significantly lower (40%) than the WT (84%; P<0.05) at 7 days post-MPTP (Fig. 3
C). The amounts of striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in both WT and MMP-3 null mice were negligible, and the difference between the two was not significant. Striatal DA nerve endings, visualized by TH staining, were drastically decreased in the WT 4 days after the injection, but the loss was largely attenuated in MMP-3 null mice (Fig. 3D
).
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Absence of MPTP-elicited microglial activation in the SN and striatum of MMP-3 null mice
Activated microglia, stained with anti-CD11b Ab, were highly visible in the SNpC of the WT at 5 days post-MPTP, but minimal in MMP-3 null mice (Fig. 4
A). CD11b-immunopositive microglia were also detected in the striata of the WT, but not in MMP-3 null mice, 2 days post MPTP (Fig. 4B
). Microglial activation in striata was negligible 4 days post MPTP, even in WT, suggesting that the MMP-3-mediated microglial activation occurred in the striatum at an earlier time point than in the SN and subsided by 4 days.
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Absence of superoxide generation in the SN and striatum of MPTP-treated MMP-3 null mice
At 2 days post MPTP injection, superoxide was detected in both SNpC and striatum of the WT but was negligible in both regions of MMP-3 null mice (Fig. 5
). At this time, the DA neurons were morphologically intact in general, and microglial activation was visible in the SN and striatum of the WT but not the null mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that actMMP-3, unlike other members of the MMP family, is a signaling molecule for microglial activation, and postulated that the actMMP-3-elicited activation of microglia may play an important role in neuroinflammation and neurodegeneration (10)
. In fact, the present results demonstrate that actMMP-3 plays a pivotal role in DA neuronal degeneration. Abrogation of MMP-3 is highly effective in attenuating or preventing the MPTP-elicited degeneration of nigrostriatal DA neurons in the mouse brain. We have reported that the activity of MMP-9, which shares similar substrates with MMP-3, is up-regulated both in the striatum and SN after MPTP treatment, and that a common inhibitor of MMPs significantly reduces the MPTP-mediated DA neuronal degeneration (21)
. However, we have not detected any MMP-9 released from stressed DA cells such as apoptotic PC12 or SY5Y (10)
, or from mesencephalic cultures in the presence of MPP+ (data not shown).
Another novel finding of our study is that, in contrast to the general belief that the proform of MMP-3 already exists inside the cell and is released into the extracellular space to be activated by plasmin, MMP-3 is demonstrated to be newly induced in neurotoxin-stressed DA neurons (Fig. 1)
and apparently activated inside the cell before release. Although the molecular mechanisms that govern induction and activation of MMP-3 in neurotoxin-stressed DA neurons are still unknown, we found a novel mechanism that underlies activation of MMP-3 in the brain. Whether the induction and activation of MMP-3 is a phenomenon specific to degenerating DA neurons or dying cells in general needs to be investigated further.
A role of actMMP-3 in microglial activation and DA cell death
The MMP-3-activated microglia produce NADPHO-derived superoxide that causes DA neuronal death in mesencephalic culture (Fig. 2B
). The similar culture of gp91phox–/– mice showed attenuation of the cell death (Fig. 2B
). The results are consistent with previously published reports that NADPHO-generated superoxide from activated microglia enhances or causes death of DA neurons in mesencephalic culture (5
, 6)
and in MPTP-treated mice PD model (8)
. These results strongly indicate that actMMP-3 released from stressed DA cells is a candidate molecule that activates microglia, leads to production of superoxide, and plays a pivotal role in DA neuronal death.
Microglia are known to play a neuroprotective role by releasing tropic factors and removing dead cells. However, these brain macrophages are also implicated as the pivotal immune cells mediating neuroinflammatory damage in animal models of PD (2
, 4
, 7
, 22)
. On the basis of the recent evidence about immune reactions of antigen-presenting phagocytes on apoptotic cells (23
, 24)
, it is possible that microglia are activated by apoptotic DA neurons in the PD brain. Because activated microglia generate superoxide anion, O2– (6)
and induce DA neuronal cell death (6
, 8)
, inhibition of microglial activation could protect DA neurons from degeneration (7
, 22)
. These results indicate that the function of activated microglia in neurodegeneration may not be limited to phagocytosis of dead neurons, but, in fact, microglia may actively contribute to neurodegenerative processes. Our previous results also suggest that actMMP-3-activated microglia participate in exacerbation of neuronal cell death (10)
. These results indicate that, regardless of the cause(s) of degeneration, actMMP-3 can be released from the degenerating neurons in PD and activates microglia, which then generates superoxide, leading to exacerbation of further DA neuronal death. Therefore, released actMMP-3 from stressed DA neuronal cells could be a key molecule in pathogenesis of PD.
A pivotal role of MMP-3 in MPTP-elicited degeneration of nigrostriatal DA neurons
The MPTP toxicity on SN DA neurons is thought to be initiated by inhibition of the mitochondrial complex I by MPP+ (13
, 14)
. However, MMP-3 activity has never been linked to this system. Our results show that the MPTP-elicited degeneration of DA neurons in the SN and striata was drastically attenuated in MMP-3 null mice (Fig. 3)
. One possible explanation for this would be that MPP+ inhibition of mitochondrial complex I might not be sufficient in itself to cause the full extent of SN DA neuronal death in vivo, and that the facilitation of the degeneration by the actMMP-3-activated microglia might be required. Reactive oxygen species (ROS) generated by microglial NADPHO play a crucial role in MPTP/MPP+-mediated DA neuronal degeneration in primary mesencephalic culture (6)
. In this article, we demonstrate that the addition of microglia to this culture causes exacerbation of the MPTP/MPP+-mediated DA degeneration. We also demonstrate that MPTP/MPP+ does not directly induce generation of superoxide, TNF- or NO in microglia. Microglial activation and superoxide generation are observed only in mixed neuron-microglia culture in the presence of MPTP/MPP+. This implies that microglial activation is not a direct effect of MPTP/MPP+ but is a reactive response to the neuronal damage. Furthermore, the MPTP-mediated SN DA neuronal degeneration is largely attenuated in gp91 KO mice (8)
, and microglia are activated early in degeneration of SN DA neurons elicited by MPTP (4)
or axonal injury (18
, 19)
. In the present study, activated microglia are highly visible both in the striatum and the SN at 2 and 5 days after MPTP injection in the WT mice but were minimal in MMP-3 null mice (Fig. 4)
, suggesting that MMP-3 plays a role in activating microglia. These results strongly suggest that microglia activated by actMMP-3 from degenerating SN DA neurons actively participate in exacerbating SN DA neuronal death.
Our attempts to identify MMP-3, as well as actMMP-3 in degenerating DA neurons in the SNpC, pars reticulata and striata have not been successful. One can think of several possible reasons for this. First, the commercially available antibodies to MMP-3 may not be suitable for immunohistochemical staining of MMP-3 in the brain. While they could stain MMP-3 induced in stressed DA neurons in primary culture (Fig. 1A
), they failed to stain MMP-3 in vivo. Second, the expression level of MMP-3 in stressed DA neurons of MPTP-injected mouse might be too low to be detected. It is possible that a minute but steady amount of MMP-3 is continuously induced, activated, and released. In this scenario, it may be the chronic release of actMMP-3 and activation of microglia that are critical in PD. Third, DA neuronal cell death in response to acute MPTP injection (4x15 mg/kg at 2-h intervals) may take place early in time, and the MMP-3 induction and release might be over by the time of analysis in the present study.
However, there is a line of evidence to suggest that actMMP-3 is released by degenerating DA neurons: 1) MMP-3 is detectable in the medium of mixed neuron-glia culture in the presence of MPP+ by immunoprecipitation. 2) MMP-3 is induced only in MPP+-treated TH-positive DA neurons in mesencephalic culture, suggesting that the MMP-3 in the medium must be released from these cells only; 3) actMMP-3 has been demonstrated to be released from apoptotic neuronal cells and to activate microglia (10)
; 4) the possibility that MMP-3 is released from activated microglia can be eliminated because MPP+ does not activate microglial; 5) superoxide is released from enriched microglia cultures in the presence of catalytically active recombinant MMP-3 (cMMP-3) (Fig. 2B
). Because proMMP-3 does not activate microglia (10)
, it is likely that it is actMMP-3 that activates microglia to generate superoxide; 6) Generation of superoxide was also observed in vivo, in the SN and striatum of MPTP-treated WT mice but negligible in MMP-3 null mice (Fig. 5)
, suggesting that the superoxide generation is actMMP-3 dependent.
A possible mechanism for progressive degeneration of DA neurons in PD by MMP-3-activated microglia
Outside the brain, macrophages and dendritic cells are known to direct the death of unwanted or neighboring cells by causing apoptosis and phagocytosis (25
, 26)
. The presence of activated microglia in the SN in PD has been reported (2
, 3)
, and microglial NADPHO-derived superoxide has been linked to apoptosis of developing Purkinje cells (24)
. The present results demonstrate that neurotoxin-mediated DA cell damage activates superoxide production that facilitates DA cell death. Rotenone, a common herbicide, also causes DA neuronal degeneration (27)
, induces generation of NADPHO-derived superoxide in primary mixed neuron-microglia culture, and activated microglia markedly increase rotenone-induced DA cell death (5)
. In contrast, LPS by itself does not cause DA cell death but activated microglia do, through NADPHO-derived superoxide (9)
. If microglia are activated in DA neuronal degeneration and produce superoxide, they may cause apoptosis of neighboring DA neurons. This supports the notion that, once the degeneration process of SN DA neurons in PD is initiated by environmental toxins or mutated gene products in familial PD, the actMMP-3-activated microglia not only enhance death of the damaged DA neurons but also cause apoptosis of neighboring DA neurons. This cycle may create progressive, self-perpetuating degeneration of SN DA neurons in PD. This idea is in agreement with the findings of progressive degeneration of SN DA neurons in humans who had self-administered MPTP 10 to 12 yr prior to death (2)
, and in monkeys 5 to 14 yr before death (4)
. In this scenario, the active form of MMP-3 plays a pivotal role in DA neuronal degeneration in PD. Abrogation of MMP-3 or inhibition of MMP-3 activity in early neuronal degeneration may therefore be an effective method to prevent progressive degeneration of DA neurons. Our present results may lead to a novel therapeutic approach for PD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 22, 2006. Accepted for publication August 21, 2006.
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