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-synuclein activates microglia: a process leading to disease progression in Parkinson’s disease
,1
* Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina, USA;
Department of Neurology, First Clinical Hospital,
Department of Physiology, Dalian Medical University, Dalian, China; and
Department of Pathology, University of Washington, Seattle, Washington, USA
1Correspondence: Division of Neuropathology, Harborview Medical Center, University of Washington School of Medicine, 703C Research and Training Bldg., Campus Box 359635, 325 9th Ave., Seattle, WA 98104-2499, USA. E-mail: zhangj{at}u.washington.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-synuclein (a major component of Lewy bodies that can cause neurodegeneration when aggregated) in microglial activation was investigated. The results demonstrated that in a primary mesencephalic neuron-glia culture system, extracellular aggregated human -synuclein indeed activated microglia; microglial activation enhanced dopaminergic neurodegeneration induced by aggregated -synuclein. Furthermore, microglial enhancement of -synuclein-mediated neurotoxicity depended on phagocytosis of -synuclein and activation of NADPH oxidase with production of reactive oxygen species. These results suggest that nigral neuronal damage, regardless of etiology, may release aggregated -synuclein into substantia nigra, which activates microglia with production of proinflammatory mediators, thereby leading to persistent and progressive nigral neurodegeneration in PD. Finally, NADPH oxidase could be an ideal target for potential pharmaceutical intervention, given that it plays a critical role in -synuclein-mediated microglial activation and associated neurotoxicity.—Zhang, W., Wang, T., Pei, Z., Miller, D. S., Wu, X., Block, M. L., Wilson, B., Zhang, W., Zhou, Y., Hong, J. S., Zhang, J. Aggregated -synuclein activates microglia: a process leading to disease progression in Parkinson’s disease.
Key Words: microglia • phagocytosis • innate immunity • aging • oxidative stress • protein aggregation • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Once activated, microglia display conspicuous functional plasticity and ultimately transform into a macrophage-like phenotype that involves morphological changes, proliferation, increased expression of cell surface receptors and the production of neurotrophic factors and neurotoxic factors, e.g., reactive oxygen species (ROS) and proinflammatory mediators such as tumor necrosis factor (TNF-) (2
, 3)
. NADPH-oxidase has been determined to be one of the major sources for production of ROS or related reactive nitric species in activated microglia (4
5
6
7)
.
Even though there is copious evidence to suggest that inflammation significantly contributes to neurodegeneration not only in rodent models of PD (8
9
10
11)
but possibly in PD itself (1
, 12
13
14)
, the mechanisms underlying microglial activation in PD or even in parkinsonian animal models are largely unknown. Pathologically, PD is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) with accompanying neuromelanin (NM) pigment in neuropil and pigment-laden activated macrophages (15)
, suggesting that NM released from dying or dead dopaminergic neurons could be one of the factors activating microglia in the SNpc (16)
. Nonetheless, direct nigral injection of human NM in rats failed to produce such effects (17)
. On the other hand, several studies have demonstrated that extracellular Lewy bodies, the pathological hallmark of PD, and nigral aggregates immunoreactive to -synuclein are often surrounded by activated microglia or inflammatory mediators such as complements (1
, 14)
. This phenomenon mirrors what has been described in AD; amyloid plaques, the pathological hallmark of AD, are usually colocalized with clusters of activated microglia (18)
. Hence, an alternative explanation for microglial activation in PD could be that aggregated -synuclein released from dying or dead dopaminergic neurons in neuropil may set off a chronic inflammation that attempts to clear up released -synuclein as well as neuromelanin.
The hypothesis that free aggregated -synuclein in nigral neuropil may trigger microglial activation is particularly interesting. It is believed that missense mutations (A30P (19)
, A53T (20)
, and, more recently, E46K (21)
) or overexpression of the -synuclein gene (22)
and -synuclein damaged during the aging process or environmental exposure (reviewed in ref 23
) appear to contribute at least partially to familial and sporadic PD, respectively, via their resistance to the ubiquitin proteasome system (UPS) -mediated proteolysis, leading to formation of soluble and insoluble -synuclein aggregates and eventually cell death (24)
. Although detailed mechanisms resulting in -synuclein aggregation in familial or sporadic PD remain to be defined, understanding the role of aggregated -synuclein in microglial activation is of major clinical relevance not only because microglial activation can maintain or even aggravate the disease process, but also because treatment with anti-inflammatory drugs could easily be initiated in PD patients with minimal risk to impede the disease progression.
In this study, using well-established primary rat and mouse midbrain cultures (11
, 25)
, we demonstrated that aggregated -synuclein, the major component of Lewy bodies in patients with PD or dementia with Lewy bodies (DLB), indeed activated microglia and led to enhanced dopaminergic neurotoxicity induced by -synuclein. Microglial phagocytosis of -synuclein and activation of NADPH oxidase appeared to be pivotal in aggregated -synuclein-induced microglial activation and neurotoxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-synuclein (r-Peptide, Athens, GA, USA); cytosine ß-D-arabinofuranoside (Ara-c) and leu-leu methyl ester (LME) (Sigma-Aldrich, St. Louis, MO, USA); [3H] dopamine (DA) (28Ci/mmol) and [3H] gamma-aminobutyric acid (GABA) (81Ci/mmol) (Perkin-Elmer Life Science, Boston, MA, USA); polyclonal rabbit anti-human -synuclein (AB5038, Chemicon, Temecula, CA, USA); monoclonal antibody against the CR3 complement receptor or OX-42 (BD PharMingen, San Diego, CA, USA); polyclonal anti-tyrosine hydroxylase (TH) antibody (a gift from Dr. John Reinhard at GlaxoSmithKline, Research triangle Park, NC, USA); Neu-N (PharMingen); polyclonal antibody against glial fibril acidic protein (GFAP) (DAKO, Carpinteria, CA, USA); Vectastain ABC kit and biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA); fluorescence probe 2’, 7’-dichlorodi-hydrofluorescein (DCFH-DA) (Calbiochem, La Jolla, CA, USA); superoxide dismutase (SOD); and phagocytosis inhibitor cytochalasin D (CD) (Sigma-Aldrich).
Characterization of purified -synuclein
To determine the nature of the purchased -synuclein, its ability to aggregate after it was aged in vitro for up to 14 days was analyzed by size exclusion chromatography and transmission electron microscopy (TEM). Purified -synuclein (1 mg/mL) was incubated in PBS at 37°C with agitation for 0, 1, 3, 7, and 14 days. At the appropriate time, 50 µL aliquot of each sample was eluted with a superose 6 size exclusion column (Tricorn 10/300 mm; Amersham Biosciences, Piscataway, NJ, USA) in PBS at a flow rate of 0.3 mL/min and monitored by a FPLC system (BioLogic Duo-Flow; Bio-Rad Laboratories, Hercules, CA, USA) to determine the relative distribution of -synuclein aggregates in terms of molecular size. Aliquots of 5 µL sample from each fraction were applied to carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA), negatively stained with 1% (w/v) uranyl acetate, and visualized on a Philips 420 TEM operated at 80 kV. Finally, since potential contamination of endotoxin, a potent activator of microglia, may confound the results of -synuclein on microglial activation, the amount of endotoxin in the purified -synuclein was determined at r-Peptide per our request. The results demonstrated that the content of endotoxin was <1.3 U/mg of peptide, a concentration incapable of producing any significant changes in terms of neurotoxicity or induction of ROS production (26)
.
Rat primary mesencephalic cultures
Rat primary mesencephalic neuron-glia cultures were prepared from brains of embryonic day 14 Fisher 344 rats with methods described previously by us (27
, 28)
. Briefly, ventral mesencephalic tissues were isolated and dissociated with gentle mechanical trituration. Cells were seeded at 5 x 105/well to 24-well culture plates precoated with poly-D-lysine (20 µg/mL) and maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air in 0.5 mL/well maintenance medium. The medium consisted of minimum essential medium containing 10% heat-inactivated FBS, 10% heat-inactivated horse serum, 1 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, 50 U/mL penicillin, and 50 µg/mL streptomycin. Three days after the initial seeding, 0.5 mL of fresh maintenance medium was added to each well. Seven-day-old cultures were used for treatment. The composition of the cells at the time of treatment was 
48% astrocytes, 11% microglia, 40% neurons, 1% of which were TH immunoreactive (TH-ir). To obtain rat primary neuron-enriched cultures, 24 h after seeding the cells Ara-c was added to a final concentration of 7.5 µM to suppress glial proliferation (25)
. Three days later cultures were changed back to maintenance medium and used for treatment 7 days after initial seeding. This method typically can enrich neurons to >95% purity. Rat primary microglia- and astroglia-enriched cultures were prepared from the whole brains of 1-day-old Fisher 344 rat pups, following our described protocol (27
, 28)
. Briefly, brain tissues were triturated after removing the meninges and blood vessels. Cells (5x107) were seeded in a 150 cm2 cultures flask. After a confluent monolayer of glia cells had been obtained, microglia were shaken off and collected (a purity of >98%). The remaining astroglia were detached with trypsin-EDTA and seeded in the same culture medium as that used for microglia. After five consecutive passages, a purity of >98% of astroglia preparation can be achieved. Finally, rat microglia depleted cultures were obtained by suppressing microglial proliferation with LME at 1.5 mM 24 h after seeding the cells (25)
. Three days later, cultures were changed back to maintenance medium and were used for treatment 7 days after initial seeding. The composition of the cells at treatment was 54% astrocytes, 1% microglia, and 45% neurons.
Primary mesencephalic neuron-glia cultures from wild-type and NADPH oxidase (PHOX) -deficient mutant mice
Primary mice mesencephalic neuron-glia cultures were prepared from the brains of embryonic day 12/13 PHOX+/+ and PHOX–/– mice (Jackson Laboratory, Bar Harbor, ME, USA) following our described protocol mentioned above. The composition of the cells at the time of treatment was 48% astrocytes, 11% microglia, and 40% neurons, 1% of which being TH-ir neurons.
Treatment with -synuclein and measurement of neurotoxicity
Purified -synuclein was aged in PBS at 37°C for 7 days, then incubated with various cultures for up to 10 days. Neurotoxicity was measured by determining uptake of [3H] DA and [3H] GABA (27)
as well as direct cell counting after immunostaining to visualize TH-ir or total neurons, respectively (see below). Cultures were incubated for 20 min at 37°C with 1 µM [3H] DA or GABA in Krebs-Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.3 mM EDTA, and 5.6 mM glucose; pH 7.4). After washing three times with ice-cold Krebs-Ringer buffer, cells were collected in 1N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA and GABA uptake observed in the presence of mazindol (10 µM) and NO711 (10 µM) with ß-alanine (1 mM), respectively, was subtracted.
Immunocytochemistry
TH-ir and total neurons were recognized with the anti-TH and anti-Neu N antibodies, respectively; microglia were detected with the OX-42 antibody, which recognized the CR3 receptor, as described (27
, 28)
. Briefly, formaldehyde (3.7%) -fixed cultures were treated with 1% hydrogen peroxide (20 min) followed by sequential incubation with blocking solution (20 min), primary antibody (overnight, 4°C), biotinylated secondary antibody (2 h), and ABC reagents (1 h). Color was developed with 3,3'-diaminobenzidine. For morphological analysis, images were recorded with an inverted microscope (Nikon, Tokyo, Japan) connected to a charge-coupled device camera (DAGE-MTI, Michigan City, IN, USA) operated with MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA). For visual enumeration of the immunostained dopaminergic and total neurons in the cell cultures, 16 representative areas/well of the 24-well plate were counted by three individuals in a blind fashion under the microscope at 100x magnification.
Measurement of oxidative stress
The production of superoxide anion (O2•–) in various cultures was determined by measuring the SOD-inhabitable reduction of tetrazolium salt WST-1 (25
, 29)
. Essentially, microglia-enriched cultures with density of 1 x 105/well were seeded and grown in a 96-well plate in Dulbecco’s minimum essential medium containing 10% FBS for 24 h and incubated with vehicle control or 25–250 nM -synuclein (aged in vitro for 7 days in PBS at 37°C with constant agitation) in HBSS, followed by 50 µL/well of 4 mM WST-1 in HBSS with or without SOD (250 U/well). Next, cultures were washed twice with Hanks’ balanced salt solution (HBSS) without phenol red. Absorbance at 450 nm was read for a period of 50 min at 37°C with a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The amount of O2•– production was determined as the increase of absorbance in 25 min and expressed as percentage of the control cultures.
Intracellular ROS (iROS) were determined by using a DCFH-DA assay as described previously with mild modification (30)
. DCFH-DA enters cells passively and is de-acetylated by esterases to nonfluorescent DCFH. DCFH reacts with ROS to DCF, the fluorescent product. DCFH-DA was dissolved in methanol at 10 mM and was diluted 500-fold in phenol red-free HBSS containing 2% FBS and 2% HS to yield a final concentration of 20 µM. 100 µL/well of DCFH-DA, diluted to a final concentration of 40 µM in phenol red-free HBSS containing 2% FBS and 2% HS was added to cultures and incubated for 30 min at 37°C. Then, 100 µL/well of 25–250 nM -synuclein was added to cultures and incubated for 2 h. Finally, fluorescence intensity was measured at 485 nm for excitation and 530 nm for emission using a SpectraMax Gemini XS fluorescence microplate reader (Molecular Devices). The value of the control cultures was viewed as background and the increase in value was viewed as the result of increased iROS.
Measurement of levels of nitric oxide (NO•), TNF-, and prostaglandin E2 (PGE2)
The production of NO• was determined by measuring the accumulated level of nitrite (an indicator of NO•) in the supernatant after 24, 48, and 72 h of -synuclein treatment using a colorimetric reaction with Griess reagent (31)
. Briefly, supernatants were collected and mixed with an equal volume of Griess reagent [0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% H3PO4]. The mixture was incubated in the dark for 10 min at room temperature and absorbance at 540 nm was measured with a SpectraMax Plus microplate spectrophotometer (Molecular Devices). The concentration of nitrite in the samples was determined from a sodium nitrite standard curve. Release of TNF- in the mesencephalic neuron-glia cultures was measured after 3, 6, and 24 h of -synuclein treatment with a rat TNF- enzyme-linked immunosorbent assay kit from R&D Systems (Minneapolis, MN, USA), as described (11
, 32)
. The production of PGE2 was evaluated after 24 h of -synuclein treatment with a PGE2 EIA kit-Monoclonal from Cayman (Ann Arbor, MI, USA) according to the manufacturer’s instructions.
Phagocytosis of -synuclein by microglia
Primary microglia-enriched cultures were exposed to different concentrations of fluorescently labeled -synuclein according to the manufacturer’s instructions at concentrations of 1 µg/mL, 10 µg/mL, and 100 µg/mL for 2 and 12 h at 37°C. The experiments were repeated in the presence of an inhibitor of microglial phagocytosis, cytochalasin D. Microglia with and without -synuclein treatment were examined under a confocal microscope (Zeiss LSM510 confocal laser scanning microscope, Carl Zeiss, Inc., Thornwood, NY, USA).
Statistical analysis
The data were expressed as the mean ± SE. Statistical significance was assessed with an analysis of 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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-synuclein produced relatively specific dopaminergic neurotoxicity-synuclein is largely caused by its soluble or insoluble aggregated oligomers and polymers; hence, we first determined the effect of aging of commercially purified human -synuclein for 0 to 14 days on dopaminergic cell toxicity. The results (not shown) demonstrated that human -synuclein aged for 7 days was most toxic. Remarkably, at day 7 of aging most -synuclein aggregates were those with a molecular mass between 44 and 158 kDa, consistent with formation of oligomers, which was further confirmed by electron microscopic examination (Fig. 1
). In contrast, by day 14 the majority of the protein aggregates became fibrillar polymers (not shown). These results agree entirely with observations by others: i.e., soluble oligomers are more toxic to neurons than fibrillary polymers (33
, 34)
. Consequently, all remaining experiments in this study were performed with -synuclein aged for 7 days. As illustrated in Fig. 2
, -synuclein aged for 7 days produced dose-dependent dopaminergic neurotoxicity when cells at a density of 5 x 105 with a culture medium of 1 mL were exposed to -synuclein at 25–250 nM for up to 10 days. The toxic effects of -synuclein on dopaminergic (TH-ir) neurons included suppression of DA uptake (Fig. 2A
), cell loss (Fig. 2B
), and apparent morphological alterations with loss of dendrites and dendrite beading in the remaining TH-ir neurons (Fig. 2C
). More important, however, -synuclein-mediated toxicity appeared to be specific to dopaminergic neurons, as GABA-containing neurons were largely preserved (assessed by GABA uptake; Fig. 2A
). Direct cell counting of total neurons after Neu-N staining revealed no significant difference between cells treated with or without -synuclein for up to 250 nM (Fig. 2B
). Enumeration of activated microglia and astroglia after immunohistochemical staining with OX-42 and GFAP, respectively, revealed that numbers of microglia and astroglial actually increased over controls after cultures were treated with up to 250 nM -synuclein for 10 days (data not shown), indicating that -synuclein had no overt toxicity on microglia or astroglia.
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Microglia enhanced -synuclein-mediated dopaminergic neurotoxicity
To evaluate the influence of various kinds of glia on -synuclein-induced dopaminergic neurotoxicity, three types of experiments were performed: 1) neuron-enriched cultures treated with 250 nM -synuclein in the presence of different percentages of astroglia; 2) neuron-enriched cultures treated with 250 nM -synuclein in the presence of different percentages of microglia; and 3) neuron-glia cocultures treated with 250 nM -synuclein when microglial differentiation and proliferation were inhibited. The data shown in Fig. 3
indicate that a protective effort was observed when an increasing percentage of astroglia was added to neuron-enriched cultures treated with -synuclein for 10 days (Fig. 3A
). In contrast, -synuclein-mediated dopaminergic neurotoxicity was progressively enhanced as an increasing percentage of microglia was added to neuron-enriched cultures treated with -synuclein for 10 days (Fig. 3B
). The possibility that microglia hasten -synuclein-induced dopaminergic neurotoxicity was further confirmed in an experiment showing that depletion of microglia in neuron-glia mixed cultures by LME, which decreased microglial component to <1% of total cells in the mixed cultures, profoundly prevented -synuclein-mediated dopaminergic neurotoxicity (Fig. 3C
).
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-Synuclein-induced microglial activation
We and others have shown that lipopolysaccharide (LPS), a classic and potent activator of microglia, and rotenone, a parkinsonian toxicant-causing nigral degeneration as well as formation of Lewy body-like cytoplasmic inclusions in the remaining nigral neurons, induce a distinct temporal pattern in microglial production of proinflammatory mediators, with elevation of extracellular O2•– being the earliest event followed by iROS, TNF-, NO•, and PGE2 (11
, 26
, 35)
. To determine whether or not -synuclein activated microglia, we measured levels of extracellular O2•–, iROS, nitrite (metabolite of NO•), TNF-, and PGE2 at the time when these mediators are typically peaked in microglia treated with LPS or rotenone (i.e., extracellular O2•– 25 min post--synuclein treatment; iROS production 2 h post--synuclein treatment; TNF- production 6 h post--synuclein treatment; and nitrite as well as PGE2 production 24 h post--synuclein treatment). As can be seen in Fig. 4
A, B, -synuclein clearly enhanced microglial production of extracellular O2•–, iROS, and PGE2. However, the levels of nitrite and TNF- with -synuclein treatment were not significantly different from those treated with vehicle controls (data not shown); basal levels of nitrite and TNF- were 0.524 ± 0.232 µM and 15.3 ± 3.8 ng/mL, respectively.
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Since the temporal pattern of increase in reactive species induced by -synuclein may be different from that induced by LPS or rotenone, levels of nitrite were measured 48 h and 72 h post--synuclein treatment whereas levels of TNF- were measured 3 and 6 h post--synuclein treatment to make sure that we did not miss the increasing phase of nitrite or TNF- by measuring only one time point, 24 h post--synuclein treatment. The results (not shown) demonstrated that neither nitrite nor TNF- increased at any time point measured after -synuclein treatment.
To compare the morphology of microglia treated with and without -synuclein, microglia were visualized directly with OX-42 immunohistochemical staining at 24 h post--synuclein treatment; the results are shown in Fig. 4C
, demonstrating that microglia treated with -synuclein had a significant enlargement of cell size from predominantly resting round and small cells to activated rod- and/or amoeboid-shaped cells with plenty of cytoplasm, characteristic of activated microglia (representative cells treated with vehicles are presented in the inset).
Microglial phagocytosis of -synuclein was required for -synuclein-mediated microglial activation
As demonstrated above, temporal production of proinflammatory mediators after -synuclein challenge was different from that provoked by LPS or rotenone. We investigated the mechanisms involved in -synuclein-mediated microglial activation. Specifically, we asked whether direct phagocytosis of -synuclein by microglia rather than signaling via toll-like receptor (typically involved in LPS-mediated microglial activation) was important in -synuclein-mediated microglial activation. Rat primary microglia cultures were incubated with -synuclein (labeled fluorescently with Aexa Fluor 488) 1, 10, or 100 µg/mL for 2 or 12 h before visualization with confocal microscopy. In this experiment, higher concentrations of -synuclein were required due to the limitation of sensitivity of detecting fluorescence intensity by confocal microscopy. The results demonstrated that internalization of -synuclein by microglia was dose and time dependent; representative images are shown in Fig. 5
A when microglia were treated with -synuclein at 10 µg/mL for 12 h (cells treated with vehicle controls are presented in the inset of Fig. 5A
).
|
More important, phagocytosis of -synuclein by microglia was impeded by cytochalasin D (CD), an inhibitor of phagocytosis, in a dose-dependant manner (Fig. 5B-D
). As microglial internalization of -synuclein was attenuated by CD, -synuclein-induced production of extracellular O2•– and iROS was dose-dependently decreased by CD (Fig. 5E
), indicating that internalization of -synuclein via phagocytosis was required for microglial activation with burst of ROS products.
-Synuclein-mediated dopaminergic neurotoxicity was dependant on activation of NADPH oxidase
We determined the role of activation of NADPH oxidase in microglial activation and neurotoxicity by comparing the effects of -synuclein on neuron-glia mixed cultures prepared from NADPH oxidase-deficient (PHOX–/–) and wild-type (PHOX+/+) mice. The results indicated that PHOX–/– mice were much more resistant to -synuclein-induced dopaminergic neurotoxicity, as assessed by DA uptake (Fig. 6
A), when primary neuron-glia cultures were treated with -synuclein (50–250 nM) for up to 10 days.
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In addition to resisting neurotoxicity induced by -synuclein, primary microglia cultures derived from PHOX–/– mice produced much less extracellular O2•– and iROS compared with PHOX+/+ mice when challenged with -synuclein (Fig. 6B, C
). Notably, however, -synuclein-produced oxidative stress, though attenuated, was not totally prevented in mice lacking PHOX, particularly when a higher dosage of -synuclein was used, indicating that other sources were contributing to ROS productions in microglia treated with -synuclein. Nonetheless, ROS production by activated microglia is critical in its enhancement of dopaminergic toxicity, as inclusion of SOD and catalase in the coculture media largely abolished microglia-mediated neurotoxicity (not shown), a result entirely consistent with our earlier observation (25
, 28)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-synuclein, the major component of Lewy bodies in patients with PD or DLB, activated microglia leading to enhanced dopaminergic neurotoxicity. Microglial phagocytosis of -synuclein and activation of NADPH oxidase appeared to be pivotal in aggregated -synuclein-induced microglial activation and neurotoxicity.
Although detailed mechanisms remain to be defined, aggregated -synuclein has been shown to be cytotoxic by many investigators (33
, 34)
. A prevalent hypothesis suggests that mutated -synuclein, overexpressed -synuclein, or oxidatively damaged -synuclein tend to form soluble oligomers or eventually insoluble fibrils such as in Lewy bodies that are refractory to the UPS-mediated proteolysis, resulting in cell death (23
, 24
, 34)
. Evidence supporting this hypothesis includes 1) -synuclein is a substrate for the UPS (36
, 37)
and recent data indicate that the 20S proteasome is primarily responsible for -synuclein metabolism and degradation (37)
; 2) mutations in -synuclein cause the protein to misfold and aggregate, resist proteolysis, and inhibit proteasomal function (38)
; 3) misfolded and/or aggregated -synuclein directly induces mitochondrial dysfunction and increases oxidative stress (39
, 40)
, the two most consistent findings in PD patients; and 4) -synuclein null mice display functional deficits in the nigrostriatal DA system (41)
and are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic degeneration, indicating that normal -synuclein function is important to dopaminergic neuron viability (42)
. Consistent with these observations, our study clearly demonstrates that aggregated -synuclein is capable of producing selective dopaminergic neurotoxicity in primary neuron-glia cultures (Fig. 1)
. What makes dopaminergic neurons more susceptible to aggregated -synuclein-induced neurotoxicity is not completely understood at this time, although the neurotransmitter DA and the oxidative stress associated with its metabolism (43
, 44)
, as well as the higher density of microglia in the SN than in the other brain regions (45)
, likely play an essential role.
The enhancement of -synuclein-induced dopaminergic neurotoxicity by activated microglia resembles our earlier findings showing that ß-amyloid peptide-induced neurotoxicity was enhanced in the presence of microglia (28)
. The potency of -synuclein in causing microglial activation is remarkable as it was 10-fold more potent in activating microglia and producing neurotoxicity than aggregated ß-amyloid. It appears that, though less potent than LPS, aggregated -synuclein was able to activate microglia more readily than rotenone (25
, 26)
. The fact that aggregated -synuclein activated microglia signifies an important mechanism by which PD progresses. It is likely that regardless of the etiology as to what sets -synuclein aggregation in motion, dying or dead dopaminergic neurons in the SNpc may release aggregated -synuclein in the nigral parenchyma, which consequently activates microglia and pathologically reinforces dopaminergic neurodegeneration.
The other interesting finding in our study is that phagocytosis of -synuclein is required for microglial activation and the subsequent dopaminergic neurotoxicity. This finding is similar to a recent report from our laboratory showing that internalization of diesel exhaustion particles by microglia is required for producing dopaminergic neurotoxicity induced by the particles (46)
. It is noteworthy that -synuclein exerts different patterns of microglial activation from that produced by LPS at low or moderate concentration (1–10 ng/mL) (11
, 35)
. This conclusion is supported by observations showing that the pattern of reactive species released from microglia treated by -synuclein is different from that of those treated with LPS and that blockage of -synuclein internalization by microglia substantially prevented production of extracellular O2•– and iROS mediated by -synuclein (Fig. 5B, C
).
The events leading to oxidative bursts after microglial phagocytosis of -synuclein remain elusive; however, activation of NADPH oxidase certainly is one of the major steps, as neuron-glia cultures derived from PHOX–/– mice were much more resistant to -synuclein-induced neurotoxicity associated with reduced production of extracellular O2•– and iROS. Notably, attenuation of oxidative stress induced by aggregated -synuclein in microglia-enriched cultures obtained from PHOX–/– mice, though significant, was not complete, indicating there are other sources for ROS production in this model. Other potential sources for ROS production in microglia are many, including but not limited to, cyclooxygenase (COX), peroxidase, and mitochondria. ROS generation by mitochondria is particularly important, given that internalized aggregated -synuclein can damage mitochondria (39)
and that damaged mitochondria are well known to produce a significant amount of oxidative stress (5
, 47)
. Nonetheless, even though suppression of O2•– and iROS production mediated by -synuclein was not complete in microglia-enriched cultures derived from PHOX–/– mice, when a higher dosage of -synuclein was used, knocking out NADPH oxidase clearly protected dopaminergic neurotoxicity regardless of the dosage of -synuclein (Fig. 6A
compared with Fig. 6B, C
). These results further indicate that generation of O2•– by activated NADPH oxidase is a critical step in -synuclein-mediated neurotoxicity.
Our results are consistent with earlier observations by others that astroglia are largely neurotrophic (13
, 48)
, as increasing the percentage of astroglia in neuron-enriched cultures and microglia depleted neuron-glia cultures, which were resistant to -synuclein-induced dopaminergic neurotoxicity (Fig. 3A, C
, respectively). The biological mechanisms underlying the protection afforded by astroglia are incompletely understood. Nonetheless, this protection can be attributed at least partially to the growth factors produced by astroglia—glial cell line-derived neurotrophic factor (GDNF)—now on clinical trial as one of the neuroprotective agents (49)
.
In summary, using different primary mesencephalic cultures we have demonstrated that aggregated -synuclein activated microglia, which in turn further enhanced -synuclein-mediated neurotoxicity. It appeared that phagocytosis of -synuclein, with subsequent activation of NADPH oxidase, plays a central role in the pathogenesis of microglial activation and associated neurotoxicity induced by aggregated -synuclein. Although detailed mechanisms remain to be defined, these observations suggest a novel mechanism by which PD progresses (i.e., via a self-propelling pathway). These results further support the concept that microglia, particularly NADPH oxidase, should be a therapeutic target in preventing PD progression.
Received for publication July 12, 2004. Accepted for publication October 14, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Z. Qin, D. Hu, S. Han, S. H. Reaney, D. A. Di Monte, and A. L. Fink Effect of 4-Hydroxy-2-nonenal Modification on {alpha}-Synuclein Aggregation J. Biol. Chem., February 23, 2007; 282(8): 5862 - 5870. [Abstract] [Full Text] [PDF]
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-globulin); d: 44 kDa (ovalbumin); e: 17 kDa (myolobin); f: 1.3 kDa (vitamin B12). It appeared that the majority of 
