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-Synucleinopathy in an A53T Transgenic Mouse Model
From the Center for Neurodegenerative Disease Research,* Institute on Aging, Department of Pathology and Laboratory Medicine, and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
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Abstract |
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-synuclein transgenic mice (M83), which develop -synuclein neuropathology, were treated with the pesticides paraquat and maneb (either singly or together), and their effects were analyzed. Immunohistochemical and biochemical analyses showed that chronic treatment of M83 transgenic mice with both pesticides (but not with either pesticide alone) drastically increased neuronal -synuclein pathology throughout the central nervous system including the hippocampus, cerebellum, and sensory and auditory cortices. -Synuclein-associated mitochondrial degeneration was observed in M83 but not in wild-type -synuclein transgenic mice. Because -synuclein inclusions accumulated in pesticide-exposed M83 transgenic mice without a motor phenotype, we conclude that -synuclein aggregate formation precedes disease onset. These studies support the notion that environmental factors causing nitrative damage are closely linked to mechanisms underlying the formation of -synuclein pathologies and the onset of Parkinson’s-like neurodegeneration.
-synuclein (-syn) inclusions and neuron loss. -Syn is an abundant, soluble protein primarily expressed at synaptic terminals. Although the exact functions of -syn are unknown, evidence suggests a role in synaptic activities (reviewed in Ref. 1
), including the ability to act as a molecular chaperone that assists in refolding proteins during the process of synaptic release.2
-Syn became an intense focus of research when it was identified as the building block of the pathological hallmarks of PD known as Lewy bodies and Lewy neurites.3,4
The mechanisms that convert the normally soluble -syn into insoluble filamentous aggregates in Lewy bodies and Lewy neurites of PD are unknown, but environmental factors have been implicated (reviewed in Refs. 5, 6
). For example, pesticides may contribute to the risk for PD7-13
through oxidative and nitrative mechanisms by generating reactive oxygen species, alkylating reduced thiols, and inhibiting mitochondrial complex I (reviewed in Refs. 5, 6, 14
). Of particular importance in this regard are paraquat (PQ) and maneb (MB). PQ resembles the metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) known as MPP+ because PQ is preferentially toxic to dopaminergic neurons. PQ exposure produces nigral degeneration, behavioral deficits, synaptic alterations, and decreased dopamine levels,15-19
while it also accelerates the fibrillization of recombinant -syn in vitro.18,20
Furthermore, MB decreases motor activity and increases the neurotoxicity of MPTP,21,22
possibly by way of mitochondrial23
and/or proteasomal inhibition.24
The toxic effects of PQ and MB are synergistic in mice exposed to both pesticides.25-27
To gain insights into the role of PQ and MB in nitrative and oxidative damage and the onset of -syn pathology versus the development of a motor and other clinical phenotypes, we studied the effects of PQ and MB treatment on transgenic mice that overexpress human A53T -syn and model PD-like neurodegenerative synucleinopathies.28
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Materials and Methods |
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Tg mice expressing wild-type (WT; M7 line) or A53T (M83 line) human -syn protein under the control of the prion protein promoter were generated and characterized as described previously.28
In these studies, homozygous M7 Tg and M83 Tg mouse lines were used in addition to non-Tg (NTg) mice with the same background.
Biochemical Extraction and Protein Assessment
The cerebellum, brain stem, hippocampus, and cortex of euthanized NTg, M7 Tg, and M83 Tg mice were thoroughly homogenized in high salt (HS) buffer (50 mmol/L Tris, pH 7.5, 750 mmol/L NaCl, 5 mmol/L ethylenediamine tetraacetic acid, and protease inhibitor cocktail) and centrifuged at 100,000 x g for 25 minutes. Pellets were re-extracted in 1% Triton X-100 and recentrifuged, and myelin was removed by homogenization in 1 mol/L sucrose in HS buffer. After recentrifugation, pellets were extracted in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 5 mmol/L ethylenediamine tetraacetic acid, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). After another sedimentation step, pellets were sonicated in 70% formic acid. After centrifugation at 100,000 x g for 25 minutes, formic acid was removed by lyophilization, and the dried material was resuspended in sodium dodecyl sulfate sample buffer by sonicating and heating the sample to 100°C for 5 minutes. The bicinchoninic acid assay (Pierce, Rockford, IL) was used to determine protein concentrations, and 10 µg of total protein from HS and RIPA fractions and 5 µl of formic acid fractions were loaded on separate lanes of 15% polyacrylamide gels. The expression and distribution of human -syn, as well as nitrated/oxidized proteins, were analyzed by Western blot analysis. In addition, differences in levels of human -syn were determined by Western blots using 125I-labeled anti-mouse secondary antibody. The membranes were dried and exposed to a PhosphorImager plate (GE Healthcare, Piscataway, NJ), and the radioactive signal was quantified using ImageQuant software (GE Healthcare).
PQ and MB Treatment of Mice
NTg, M7 Tg, and M83 Tg mice at 3, 8, or 12 months of age were injected intraperitoneally with saline, PQ, MB, or PQ and MB (referred to as PQ/MB hereafter) twice a week for 3 consecutive weeks. PQ was used at a dose of 10 mg/kg in mice of 8 or 12 months of age and a dose of 5 mg/kg in mice of 3 months of age. MB was used at a dose of 30 mg/kg in 8- or 12-month-old mice or a dose of 15 mg/kg in 3-month-old mice. Mice were monitored for behavioral changes and weight loss and then sacrificed by intraperitoneal xylazine and ketamine administration, followed by perfusion with phosphate-buffered saline. Brains were fixed in 70% ethanol/150 mmol/L NaCl for histochemical analysis or frozen for biochemical analysis; many brains were hemi-sectioned for both histochemical and biochemical analyses.
Antibodies
LB509 is a mouse monoclonal antibody (mAb) specific for human -syn,29
and Syn303 is a mouse mAb raised against oxidized human -syn that specifically labels pathological -syn inclusions.30
N808 and NSyn14 are mouse mAbs raised against nitrated -syn; N808 recognizes all nitrated proteins (B.I. Giasson, unpublished data), whereas NSyn14 specifically recognizes nitrated -syn.31
The neurofilament (NF) antibody is a polyclonal rabbit antibody.32
UB1B4 (D. Sampuathu, unpublished data) and UB1510 (Chemicon International, Inc., Temecula, CA) are mouse mAbs that recognize ubiquitin protein. HSP90 is a mouse mAb from Stressen Bioreagents (Ann Arbor, MI). Other antibodies include Lamp-2 (ABL-93; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA).
Immunohistochemistry
Mice were perfused with saline and subsequently with 70% ethanol/150 mmol/L NaCl. Brains were removed and postfixed for approximately 24 hours and then separated into four 
3-mm-thick sections. Brain sections then were processed and dehydrated through a series of graded ethanol solutions to xylene at room temperature, infiltrated with paraffin at 60°C, and cut into 6-µm-thick sections.33
Immunohistochemistry was performed as described previously34
using the avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). Double fluorescent immunohistochemistry with Syn303 and NF antibodies was performed as described previously.31
Staining was analyzed with an Olympus BX51 microscope (Olympus, Tokyo, Japan).
Conventional and Immuno-Electron Microscopy
NTg, M7 Tg, and M83 Tg mice with saline or PB/MB treatment were fixed with 2% paraformaldehyde and 1.5% glutaraldehyde in 0.1 mol/L phosphate-buffered saline (pH 7.4) for conventional EM. Brains were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L phosphate-buffered saline for immuno-electron microscopy (immuno-EM). Brains were fixed overnight and then cut into 50-µm-thick sections using a Vibratome 3000 (Vibratome, St. Louis, MO). Immuno-EM sections were processed with Syn303, Lamp-2, or cytochrome c or for silver enhancement as described previously.35 Subsequently, sections processed for immunostaining and for conventional EM were dehydrated by a graded series of ethanol, postfixed with 1% osmium tetra-oxide, and embedded in EPON812. Ultra-thin sections were cut, stained with uranium acetate, and examined under an electron microscope (JEM-1010; JEOL, Tokyo, Japan).
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Results |
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-Syn Mice Exacerbates -Syn Pathology
Cohorts of 3-, 8-, and 12-month-old NTg, M7 Tg, and nonsymptomatic M83 Tg mice were exposed to saline, PQ, MB, or PQ/MB. NTg (n = 5) and M7 Tg (n = 14) mice did not present a phenotype or -syn neuropathology in any brain regions examined when treated with PQ and/or MB at any age, and they were similar to saline-treated mice in all respects (NTg, n = 5; M7 Tg, n = 5) (Figure 1, E, K, P, and U
for NTg and D, J, O, and T for M7 Tg). Furthermore, no noticeable cell loss in the substantia nigra or other brain regions was observed in NTg, M7 Tg, or M83 Tg mouse lines at any age after PQ and/or MB treatment. Three-month-old M83 Tg mice did not develop -syn inclusions from chronic pesticide exposure (n = 6); however, the majority of the 8- and 12-month-old asymptomatic M83 Tg mice (n = 7 of 9 treated) exposed to PQ/MB developed -syn-positive inclusions of varying sizes in the granule layer of the cerebellar cortex, molecular layer of hippocampus, and deep layers of the somatosensory and auditory cortices (Figure 1, A, B, F, G, L, M, Q, and R)
. However, these mice did not show inclusion formation after treatment with either PQ or MB alone or with saline (Figure 1, C, H, N, and S)
. Thus, PQ/MB exposure in M83 Tg mice induced -syn neuropathology in brain regions not observed in these mice as reported previously.28
-Syn aggregates were also found in the brain stem, including the raphe nucleus, arcuate nucleus, striatum, and thalamus in seven of the nine treated M83 Tg mice (Figure 1, F and G
; data not shown). Although untreated M83 Tg mice eventually develop -syn inclusions in the brain stem, striatum, and thalamus,28
the proportion of affected mice is much higher than expected at this age. The -syn-positive inclusions noted here, including cell body and punctated structures, were essentially colocalized with NF staining (Figure 1, V, W, and X)
. Interestingly, however, the small punctated inclusions in the hippocampus were not localized with NF (Figure 1, Y, Z, and ZA)
, NeuN, or glial fibrillary acidic protein (data not shown), and none of the -syn-positive inclusions were Thioflavin-S positive. In addition, the saline-treated, asymptomatic M83 Tg mice at 8 and 12 months of age did not accumulate -syn in any brain region, including the brain stem (Figure 1, C, H, N, and S)
. These results suggest that PQ/MB exposure induced -syn pathology in these brain areas.
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-syn in these brain regions. Treated M83 Tg mice displayed only minor weight loss, hind limb weakness, and diminished spontaneous movements but no paralysis, tremors, or hunched posture (data not shown). Therefore, -syn pathology precedes phenotype and symptom onset in PQ/MB-treated mice. Pesticide Exposure Leads to Disorganized and Degenerated Mitochondria and Accumulation of Lysosomes
EM studies were performed on brain sections collected from NTg, M7 Tg, and M83 Tg mice exposed to either saline or PQ/MB (n = 1 to 3 mice per group). In both brain stem and hippocampal sections of saline-treated NTg, M7 Tg, and M83 Tg mice, the majority of mitochondria had healthy appearances (data not shown). In contrast, PQ/MB-treated mice easily revealed membrane structure disorganization (Figure 2, A–D)
. In particular, samples from treated M83 Tg mice showed membranous lamination (Figure 2D)
, whereas treated NTg and M7 Tg mice failed to do so (Figure 2C)
. Immuno-EM observations using Syn303 did not show -syn-positive immunolabeling associated with normal mitochondria from NTg, M7 Tg, or M83 Tg mouse brains. Positive -syn labeling was only present in mitochondria with disrupted membranes (arrowheads in Figure 2, E and G
), and this observation was unique to PQ/MB-treated M83 Tg mice.
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. The amount of lysosome induction was higher in treated Tg mice than in treated NTg mice, and membrane-like components within the lysosomes resembled damaged mitochondria as reported recently.36
Anti-Lamp-2 antibody was used in immuno-EM studies to identify lysosomes and identified various lysosomal structures (Supplemental Figure 1A, http://ajp.amjpathol.org). Immuno-EM revealed that these components in the lysosomes frequently associated with -syn (Figure 2H)
. Furthermore, degraded membrane-like components in the lysosomes were labeled for cytochrome c in both PQ/MB-treated M7 and M83 Tg mice (Figure 2H)
. The mitochondria and lysosomes associated with -syn immunoreactivity may have contributed to the enhancement of immunoreactivity in PQ/MB-treated M83 Tg mice that were noted in the light microscopy level. These results suggest that PQ/MB treatment leads to mitochondrial damage and lysosomal degradation in all mice used in this study, yet there was significantly more mitochondrial damage in treated M83 Tg mice than M7 Tg or NTg mice.
Exposure to PQ/MB Promotes the Formation of Filamentous -Syn Aggregates in Axon Terminals
Additional immuno-EM studies were performed using the Syn303 antibody on brain stem and hippocampal sections collected from NTg, M7 Tg, and M83 Tg mice exposed to either saline or PQ/MB. Among the mice used in the current study, PQ/MB-treated mice were the only group of mice that showed exacerbated -syn pathology. Plastic-embedded semithin sections of hippocampi from saline-treated M83 Tg mice indicated the presence of modest -syn-positive immunolabeling that was closely associated with neuronal cell bodies (Figure 3A)
, whereas their PQ/MB-treated counterparts showed an enhanced staining and a greater number of punctated inclusions with -syn-positive immunoreactivity (Figure 3B)
. Ultrastructural analysis of these sections confirmed that the saline- and PQ/MB-treated M83 Tg mice had positive -syn labeling surrounding synaptic vesicles at axon terminals (Figure 3, C and D)
. These positively labeled terminals were found widely in the neurophil, including the area adjacent to neuronal cell bodies.
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-syn revealed clusters of filamentous aggregates composed of fibrils with a diameter of approximately 10 to 20 nm (Figure 3, E and F)
. The range of diameter in its individual filament was comparable with that of -syn filaments in our previous study.37
The degree of filament density and the size of aggregates (100 to 500 nm) varied greatly. These distinct filaments were decorated with -syn-positive silver grains (arrow in Figure 3F
). The -syn-positive filamentous aggregates were more abundant in sections from the hippocampus than those from the raphe nucleus. These filamentous aggregates were clearly located within a membrane boundary. Various sizes of vesicles and mitochondria were located in proximity to each other (Figure 3E)
. At the outer limbs of the membrane-bound structure, a number of presynaptic terminals were detected. Judging from the location and structure, it was presumed that these aggregates were located in neuronal processes. NTg and M7 Tg mice with or without PQ/MB treatment failed to demonstrate such filamentous structures.
Effects of Pesticide Exposure on the Levels and Solubility of Human -Syn
Quantitative biochemical analysis of human -syn expression in the cerebellum, brain stem, hippocampus, and cortex of treated and untreated M83 Tg mice (n = 3 control and n = 6 treated mice per group) showed a dramatic increase in levels of HS-soluble -syn in the cerebellum and brain stem (Figure 4A)
. Furthermore, quantification of extracts from these same brain regions of treated and untreated M83 Tg mice showed a significant increase in RIPA-soluble -syn protein levels in the cerebellum, brain stem, and cortex (Figure 4B)
. In addition, levels of nitrated proteins increased significantly in HS fractions obtained from the cerebellum and brain stem from PQ/MB-treated M83 Tg mice (Figure 4, C and E)
, and the nitrated -syn-specific antibody NSyn14 used in immunohistochemistry showed that these inclusions were composed of nitrated -syn protein (Figure 4D)
, thereby providing support for nitrative damage due to chronic pesticide exposure. Finally, biochemical and histochemical comparisons of treated versus nontreated mice showed no significant changes in the levels of HSP90 (Supplemental Figure 1, B and C) and ubiquitin (Supplemental Figure 1, D and E) in pesticide-exposed mice.
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Discussion |
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-syn, mitochondrial dysfunction, and nitrative/oxidative damage play significant roles in this process (reviewed in Refs. 5, 6
). Mitochondrial impairments have been found in human PD cases because there is a 30 to 40% decrease in complex I activity and immunoreactivity in the substantia nigra.38-42
Mitochondrial deficits may lead to the production of reactive oxygen species and hence oxidative stress, which has also been linked to PD pathogenesis (reviewed in Refs. 5, 43
).
Several recently published studies have shown that treatment with pesticides and other complex I inhibitors can mimic PD pathology in some animal models. For example, chronic administration of MPTP can lead to nigral degeneration, behavioral impairments, and proteinaceous inclusions that are immunoreactive for -syn and ubiquitin in WT mouse brains, whereas mice lacking -syn do not undergo behavioral deficits or neuronal degeneration under the same conditions.44
Other studies also showed that the pesticide rotenone, which is a classical inhibitor of mitochondrial complex I, can lead to nigral degeneration associated with the formation of -syn-positive inclusions.45-48
Finally, intraperitoneal injections of PQ into NTg and Tg mice expressing human WT and A53T -syn under the control of the tyrosine hydroxylase promoter resulted in an up-regulation of -syn expression and the formation of -syn-positive inclusions; however, nigral degeneration induced by PQ was only found in NTg mice, suggesting that human -syn expression in these Tg mice may play a protective role against pesticide-induced damage.15,18
Other investigators have shown that overexpression of -syn in mice leads to alterations in mitochondria, increased free radical production, and neuronal death.49
Taken together, these and other studies suggest a link between oxidative/nitrative stress and -syn aggregation in PD pathogenesis. To investigate these mechanisms further, transgenic mice expressing human WT or A53T -syn under the control of the prion protein promoter were challenged with PQ and MB.
Previous studies demonstrated that M83 A53T human -syn Tg mice, but not M7 WT -syn Tg mice, recapitulate key features of a PD-like neuronal synucleinopathy.28
Here, we showed that exposure to PQ and MB augmented the formation of neuronal -syn pathologies in M83, but not M7 Tg or NTg mice, throughout the CNS, including regions of the brain stem, hippocampus, cerebellum, cortex, thalamus, and striatum. Similar to previous studies,15
these pesticide-treated mice show elevated levels of -syn in the brain stem and do not undergo nigral degeneration, suggesting a potential protective role for -syn in this brain region. The accumulation of -syn in the HS-insoluble/RIPA-soluble biochemical fractions reflects the increased formation of inclusions in the respective brain regions. However, biochemical analysis of mouse brains also revealed an up-regulation of aqueous-soluble -syn protein in the cerebellum and brain stem of PQ/MB-treated mice. Although the mechanism of this stress-induced -syn up-regulation remains to be elucidated, it may be linked to the impairment of -syn degradation by lysosomes and autophagosomes.50
PQ/MB exposure also resulted in significant increases in the levels of nitrated proteins. Therefore, the formation of -syn-positive aggregates due to PQ/MB exposure could be a combined result of increased levels of -syn and nitrative and oxidative challenges. However, the formation of aggregates in the cortex without increased levels of RIPA-soluble -syn indicates that nitrative stress may be a more important factor. These results suggest that the A53T mutation in -syn confers susceptibility to nitrative damage, which can then lead to increased -syn accumulation and aggregate formation. This finding is consistent with the effect of the A53T mutation in increasing the propensity of -syn to fibrillize.37,51,52
In summary, these studies strengthen the link between pesticide exposure and PD and support the concept that nitrative damage plays a role in mechanisms underlying the formation of -syn pathologies and the onset of PD-like neurodegeneration. In addition, genetic mutations, as exemplified by the A53T mutation in -syn, as well as environmental toxins can synergistically lead to neurodegenerative synucleinopathies. Genetic mutations in the -syn gene may prime neurons to develop pathology, but a secondary insult such as an environmental toxin may catalyze or enhance this process by damaging mitochondria and/or reducing the amount of -syn degradation. Finally, the experimental model described here recapitulates some of the features of PD and provides important insights into disease mechanism.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants AG09215, AG10124, and NS044233. J.Q.T. is the William Maul Measey-Truman G. Schnabel, Jr., Professor of Geriatric Medicine and Gerontology. V.M.-Y.L. is the John H. Ware Third Chair of Alzheimer’s disease research.
E.H.N. and K.U. contributed equally to the manuscript.
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication October 19, 2006.
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