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Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson's disease model
Accepted Manuscript Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model Jianqing Lu, Feiyi Sun, Hong Ma, Hong Qing, Dr. Yulin Deng PII:
S0006-291X(15)30238-2
DOI:
10.1016/j.bbrc.2015.07.007
Reference:
YBBRC 34215
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 29 June 2015 Accepted Date: 1 July 2015
Please cite this article as: J. Lu, F. Sun, H. Ma, H. Qing, Y. Deng, Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model
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Jianqing Lu1, Feiyi Sun1, Hong Ma1, Hong Qing1, Yulin Deng1,*
School of life science, Beijing Institute of Technology, 5 South Zhongguancun Street,
*
Corresponding author: Dr. Yulin Deng
Tel: +86-10-68914907 E-mail: [email protected]
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Haidian District, Beijing, 100081, People's Republic of China
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Address: School of life science, Beijing Institute of Technology, 5 South Zhongguancun
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Street, Haidian District, Beijing, 100081, People's Republic of China
ACCEPTED MANUSCRIPT Abstract: Background and aims: Vector based over-expression of α-synuclein is a newly developed method to establish animal Parkinson’s disease (PD) model. In this paper, we inject the rat
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brain with recombinant adeno-associated virus (rAAV) to express α-synuclein wild-type and A53T mutation, and compared the degeneration of dopaminergic neurons between them. Method and results: The rAAV vectors were injected into the substantia nigra pars compacta
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(SNpc) of rat brain. In different time point, immunohistochemistry was used to detect the
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expression of α-synuclein. The expression level was lower in the 3rd and 6th week and increased from the 9th week. α-synuclein spread around the neurons in SNpc in the 12nd week. The loss of dopaminergic neurons was increasing along the expression of α-synuclein, and damage extent was more serious in the A53T group than the WT group. In the A53T
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group, there were more insoluble inclusions can be detected, and the phosphorylation of α-synuclein was also higher.
Conlusion: The result of comparison between the two types of α-synuclein showed that
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A53T mutated α-synuclein was more effective to establish PD model, and the model based
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A53T mutated α-synuclein was a suitable model to early-onset PD. Keywords: Parkinson’s disease; animal model; recombinant adeno-associated virus; α-synuclein
Abbreviations -LBs Lewy bodies -PD Parkinson’s disease
ACCEPTED MANUSCRIPT -PK Proterinase K - rAAV recombinant adeno-associated virus -SNpc substantia nigra pars compacta
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-WT wild-type
1. Introduction
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Parkinson’s disease (PD) is one of the most popular neurodegeneration diseases in the
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elders [1]. The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the appearance of protein aggregation named Lewy bodies (LBs) are two symptoms of PD [2]. There are many animal models established in the last dozens of years and most of them were based on the neurotoxins such as 6-OHDA and MPTP. However, there were few
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cases in these models can detect inclusions like LBs [3]. α-synuclein is the main composition of LBs and it is also considered as a genetic factor to PD. Some mutation of α-synuclein such as A53T, A30P and E46K were found in the familial PD patients [4]. In order to establish
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LBs formed PD model, α-synuclein over-expression based PD models were developed, and
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transgenic mice were investigated most well. In these α-synuclein transgenic mice, LBs were appeared in the SNpc with progressive loss of dopaminergic neurons [5]. Recently, vector based α-synuclein over-expression was developed as a new technology [6]. In these models, the viral vectors were injected to the SNpc and expressed α-synuclein in a small area of one side of brain [7]. The advantage of these models is that only dopaminergic neurons in the SNpc were transfected by the vector whereas other cells were not affected by α-synuclein over-expression [8]. The other advantage is the one-side destroyed of
ACCEPTED MANUSCRIPT dopaminergic neurons can be detected by behavior changes which showed rotation induced by amphetamine or apomorphine [9]. In these models, α-synuclein expressed in a high level and neurons lost along with the α-synuclein expression [10]. The insoluble inclusions were
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also detected in these models easily [11]. In our study, we chose α-synuclein widetype (WT) and A53T and over-express them in rat brain using recombinant adeno-associated virus (rAAV). After injection of these vectors,
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we detected the gene expression and neuron degeneration in the 3rd, 6th, 9th and 12nd week.
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The data showed that α-synuclein expression can induce a progressive degeneration of dopaminergic neurons. The A53T mutated α-synuclein can induce the loss of dopaminergic neurons more severely and enhance more inclusions formation. The model over-expressed
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A53T mutated α-synuclein can be chosen as the early-onset PD model.
2. Materials and methods 2.1 Vector preparation
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The EGFP, α-synuclein WT and A53T gene were constructed into the shuttle plasmid
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using HindⅢ and XhoⅠ with CAG promoter and WPRE enhancer. The shuttle plasmid and helper plasmid pFd6 and pH22 were co-transfected to HEK293 cell with calcium phosphate assay. The rAAV was packaged in the HEK293 cell for 3 days and then lyse the cells using 0.5% sodium deoxycholate. The vector was purified by HiTrap Heparin HP (GE Healthcare, USA) and concentrated using 100kD ultrafiltration tube (Merck-Millipore, USA). The titer of the rAAV was determined by realtime qPCR of WPRE enhancer, the forward primer: TGGCGTGGTGTGCACTGT, the reverse primer: GTTCCGCCGTGGCAATAG. The titer of
ACCEPTED MANUSCRIPT all of the three vectors was diluted to 1×1013 gc/mL by PBS. 2.2 Animal and surgery Adult male Wistar rats (Vital River, China) were kept in five per cage with adequate food
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and water. The light/dark cycle was 12h which controlled by program. All procedures were conducted in authorization by Animal Ethics Committee of Beijng Institute of Technology. The weight of these rats were 270~320g at the time of surgery. After deeply anesthetized
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by pentobarbital sodium at 50mg/kg i.p., the rats were put on a stereotaxic frame and fixed
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their skull with the ear bars and the incisor bar. Then 4µL viral solution was injected to SNpc through a hole above it. The coordinates of the SNpc: antero-posterior -5.3mm from bregma, medio-lateral +2.2mm from midline, and dorso-ventral -7.7mm below dura. The injection was performed using 10µL Hamilton syringe with a thin needle (outer diameter of 0.2mm,
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Hamilton, USA), and the infusion rate was 1µL/min controlled by a syringe pump (model UMP3, WPI, USA). After injection, the needle was kept in the brain for 3 min to make the solution diffuse completely.
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2.3 Behavior testing
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After injection, the apomorphine induced rotation test was carried out at the 3rd, 6th, 9th and 12nd week. These rats were injected 0.5mg/mL apomorphine (dissolved in 0.9% saline) i.p., and then put them to a big cage. The rotation behavior was captured by a digital video camera for 45min.
2.4 Immunohistochemistry and immunofluorescence At the same time point of behavior testing, the rats in corresponding group were deeply anesthetized by pentobarbital sodium at 50mg/kg i.p., and then perfused through their heart
ACCEPTED MANUSCRIPT with 500mL saline. After perfusion, the brains were removed quickly and immerse to ice cold 4% paraformaldehyde for at least 24h, and then transfer them to 25% sucrose for cryoprotection. Following the brains were cut to 30µm sections by freezing microtome
solution (30% sucrose, 30% ethylene glycol in PBS) until using.
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(CM1900, Leica, Gemany).The sections in striatum and midbrain were stored in anti-freeze
For immunohistochemistry, the sections were picked from anti-freeze solution and
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washed with PBS-T (0.3% Triton X-100 in PBS). In order to quench the endogenous
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peroxidase, these sections were transferred to 1% H2O2 for 15 min at room temperature (RT) and then wash with PBS-T 3 times, 5 min each. Following these sections were blocked by 1% normal goat serum for 30 min and then incubated with primary antibody diluted with 1.5% normal goat serum in PBS-T: α-synuclein (1:2000, mouse monoclone, Abcam, UK), TH
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(1:1000, rabbit polyclone, Merck-Millipore, USA), phosphor-Ser-129 α-synuclein (rabbit, 1:1000, Abcam, UK) at 4℃ overnight with gentle shake. The next day these sections were picked from primary antibody to wash for 3 times and then incubated them with biotinylated
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second antibody diluted with PBS-T (1:200, Vector Laboratories, USA) for 2 hours at RT.
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After incubation, these sections were washed incubated with ABC complex solution diluted with PBS-T (1:100, Vector Laboratories, USA, combine reagent A to reagent B for 1:1) for 2 hours. At last these sections were developed with DAB and mounted to slides with PVP mounting medium and acquire the images using digital scanner or microscope (IX71, Olympus, Japan). For immunofluorescence, the procedure was similar with immunohistochemistry without the 1% H2O2 incubation. The primary antibody was the mixture of α-synuclein and TH (the
ACCEPTED MANUSCRIPT dilution was the same with above) and the next day they were incubated with the mixture of fluorescence second antibody: Alexa Fluor 488-labeled goat anti-mouse IgG (1:200, Zhongshan Goldbridge, China), TRITC-labeled goat anti rabbit IgG (1:100, Zhongshan
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Goldbridge, China). After incubation, these sections were washed and mounted with anti-fade PVP mounting medium and acquire the images using laser confocal microscope (TCS SP5,
2.5 Proteinase K digestion assay Proterinase
K
(PK)
solution
incubation
was
carried
out
before
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The
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Leica, Germany)
immunohistochemistry for α-synuclein. After the first wash step, these sections were incubated with 10µg/mL PK for 10 min at 37 ℃ and then post-fixed with 4% paraformaldehyde for 10 min at RT. The following procedure was the same with 2.4 but the
2.6 Statistical analysis
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only difference is using PBS instead of PBS-T.
All results are expressed as means ± SD, and the statistical significance between two
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groups were evaluated using T-test analysis. P< 0.05 was considered statistically significant.
3. Results
3.1 α-synuclein expression in dopaminergic system The expression of α-synuclein is slow and progressive (Fig.1). There was only slight express in SNpc at the 3rd week and 6th week, and there was no significant different between the WT group and A53T group (Fig. 1A). As the assemble of projection fibers of dopaminergic neurons in SNpc, the expression of α-synuclein in striatum was similar with
ACCEPTED MANUSCRIPT SNpc, but the level was higher in the A53T group than the WT group at the 6th week(Fig. 1B ). The expression level increased more higher from the 9th week. In the SNpc, there was large area of α-synuclein expression and the A53T group was larger than the WT group (Fig.
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1A). In the striatum, the stained area was distributed to all dopaminergic fibers in A53T group from the 9th weeks, whereas the WT group reached the same extent at the 12nd weeks (Fig. 1B).
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In order to investigate if the rAAV can specific infect the dopaminergic neurons, double
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immunofluorescence stain for α-synuclein and TH was performed in SNpc. The green fluorescence was EGFP or α-synuclein and the red one was TH which is the marker of dopaminergic neurons, and the result of merge the two images together showed that all green fluorescence was co-local with red one (Fig. 1C). This indicated that the genes which rAAV
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carried (EGFP or α-synuclein) were expressed in the dopaminergic neurons. 3.2 Apomorphine induced rotation
When dopaminergic system was damaged at one side, the rats can display rotation to the
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healthy side induced by apomorphine, and the speed of rotation show positive correlation
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with the damage extent. There were no detectable rotation in the control group and EGFP group, and this showed that the dopaminergic system may be not affect by single injection of rAAV. However, the rotation was detected when α-synuclein expressed. In the α-synuclein WT group, the rats showed no rotation in the first 9 weeks, and reached 2.68±1.74 turning/min at the 12nd week. Although the α-synuclein expression was the same level in A53T group with WT group at the 3rd week, the rotation in A53T group was 1.76 turning/min, and kept the similar level to 1.83±1.08 at the 6th week. As the expression level
ACCEPTED MANUSCRIPT reached higher from the 9th week, the rotation speed increased to 2.97±1.99 turning/min at the 9th week and even higher to 3.91±1.62 turning/min at the 12nd week (Fig. 2). These result suggested that the A53T mutation in α-synuclein may enhance the toxic of the protein
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and make the symptom appear earlier. 3.3 α-synuclein expression induced loss of dopaminergic neurons
The immunohistochemistry of TH can stain the dopaminergic neurons (Fig. 3). In the
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SNpc, there was no detectable loss in the control group and EGFP group, this result was
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consistent with the behavior result. It indicated that rAAV infection cannot contribute to the loss of dopaminergic neurons. When α-synuclein expressed in SNpc, there were significant loss of dopaminergic neurons in the SNpc at one side. In the α-synuclein WT group, the loss of dopaminergic neurons can be detected very late only at the 12nd week, and it was the same
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time of the appearance of rotation in this group. There was no significant loss of dopaminergic neurons in the A53T mutation group at the 3rd week and 6th week although the rotation can be detected, and this may be concern to the rotation speed was not very high.
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From the 9th week, the rotation speed reached over 2 turning/min, and the loss of
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dopaminergic neurons can be detected in the injected side. The extent of this damage was increasing with time in the A53T mutation group (Fig.3A). The performance of dopaminergic fibers in the striatum was the same condition with the SNpc. The control group and EGFP group showed symmetry between the two sides, and this meant there was no damage in the striatum. In the α-synuclein WT group, there was no change of the fibers density until the 9th week, and the density decreased at the 12nd week. As the loss of dopaminergic neurons in SNpc, the fibers density decreased from the 9th week
ACCEPTED MANUSCRIPT in the A53T group. The reduction of this density in the A53T group was more than the WT group (Fig 3B). 3.4 α-synuclein expression led to aggregation and phosphorylation
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From the results above, the toxic of α-synuclein WT was different from A53T mutation. In order to investigate the aggregated pattern of these two kinds of α-synuclein, PK digestion assay was performed with immunohistochemistry of α-synuclein in the 12nd week. In the
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SNpc, the α-synuclein stain distributed a large area around the SNpc before PK treated (Fig.
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4A and 4C). However, if incubated with PK, there were different results between the WT and A53T mutation. In the WT group, there were only the dopaminergic neuron shape area can be stained, and the other area was still white (Fig. 4B). In the A53T group, α-synuclein aggregated inclusions spread as the same area with untreated section, and there was no visible
(Fig. 4D).
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neuron shape as the WT group. The inclusions in the A53T group were like fibers in the SNpc
In the striatum, α-synuclein was expressed in all the dopaminergic fibers before PK
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treated, and there was no significant different between the WT group and A53T group (Fig.
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4E and 4G). After incubated with PK, the inclusions of α-synuclein were appeared in the sections, and the inclusion number in the A53T group was more than the WT group (Fig. 4F and 4H). Unlike the fiber shape of α-synuclein inclusions in the SNpc, the inclusions in the striatum were spheric, and this shape was very similar to the LBs (Fig.4K). As α-synuclein phosphorylation is the character of LBs, the immunohistochemistry of Ser-129 phosphorylated α-synuclein was performed in the SNpc. The results showed that there were only little stained dots in the SNpc area in the WT group, and the number
ACCEPTED MANUSCRIPT increased in the A53T group nearly by double (Fig. 4I and 4J).
4. Discussion
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α-synuclein is the first genetic factor found related with PD, and A53T is an important mutation in the pathogenesis of PD [12]. LBs as the hallmark of PD contain abundance aggregated α-synuclein, and over-expression of α-synuclein is thought to be a genetic way to
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establish PD model. In transgenic mice model, α-synuclein expression induced different
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effects because of the promoter and genetic background[13]. Several line expressed human α-synuclein were established using the PDGF-β promoter, and the results showed that only the Line which expressed more α-synuclein can induced significant loss of dopaminergic neurons with the most inclusions formation[14]. The investigation of effect of α-synuclein
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mutation also performed in the transgenic mice. In their experiment, the PrP promoter was used and these model express high level of α-synuclein. However, they only compared the WT, and mutated A30P and A53T genotype in the number of lumbar motor neurons, and the
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result showed that the A53T mutations showed the most damage. A53T mutated α-synuclein
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also increased the degeneration and apoptosis of neurons to high level [15]. In our experiment, we compared the WT and A53T mutation using rAAV based α-synuclein overexpression model. There are many investigations had been carried out used this method. In most of these experiments, WT and A53T were not compared. The results showed that after injected the rAAV to the SNpc, there were high level of α-synuclein expressed in the dopaminergic neurons of SNpc, and α-synuclein also can be detected in the striatum [16]. However, they only showed the end point of the expression but not the progress.
ACCEPTED MANUSCRIPT Recently, a study using rAAV2/7 to express α-synuclein in SNpc showed the immunofluorescence of α-synuclein, but the results only showed the data of 5 day and 8 week, and it’s cannot illustrate the expression level of α-synuclein [17]. In our study, we used the
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immunohistochemistry to stain α-synuclein in SNpc and the results showed the progress of α-synuclein expression. The expression level in the first 6 weeks was not very high, and the protein only expressed in the neurons body. However, the results in the 9th week and 12nd
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showed an enlarged area of expression, and α-synuclein spread from the neurons formed
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fiber-like state. The expression in the A53T group was a little higher than the WT group, and this may be result of the mutated α-synuclein is aggregated more easily. The loss of dopaminergic neurons was detected in most rAAV-based α-synuclein over-expression model, but the extents were varied. The behavior test was carried out in some
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of these models, and the result showed the progressive increasing of apomorphine induced rotation [18]. However, there was no comparison between the WT and A53T group. In our experiment, the data showed that although the expression level was no significant different in
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the two group, the A53T group displayed rotation earlier and more serious. The
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immunohistochemistry of TH in SNpc and striatum showed the loss of dopaminergic neurons in the A53T group was more than the WT group. The neurons loss progress agreed with the α-synuclein expression level. This result showed that the mutated A53T α-synuclein is more toxic to dopaminergic neurons, and induced the degeneration of neurons very early. The A53T over-expression model may be a suitable model of the early-onset PD. α-synuclein aggregation is a hallmark of PD, and it’s easily to be detected in the α-synuclein over-expression model. The insoluble aggregates were found in the SNpc of
ACCEPTED MANUSCRIPT α-synuclein A53T over expressed model using PK digest assay [19]. However, the aggregates in that experiment was similar with the result of our WT group, and the damage extent also reached the similar level. In our experiment, the A53T group showed fragment like mode,
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and spread to a large area around the SNpc. The inclusions in the striatum of the A53T group were also more than WT group, and the phosphorylation of these inclusions in the SNpc showed the same tendency between the two groups. These results showed that the A53T
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mutation in α-synuclein can enhance protein aggregation and LBs-like inclusions formation.
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In conclusion, the results in our study showed the progressive expression of α-synuclein via rAAV injection with the degeneration of dopaminergic neurons. The A53T mutated α-synuclein was more toxic to the neurons than WT and enhanced aggregation of this protein. The results were useful to the researchers who want to choose rAAV-α-synuclein to establish
Acknowledgements
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PD model.
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20435020).
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We thank the National Natural Science Foundation of China supported our study (No.
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ACCEPTED MANUSCRIPT Figure legends:
Fig.1 The expression of α-synuclein in the SNpc and striatum. (A, B) The expression of
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α-synuclein in SNpc and striatum in the 3rd, 6th, 9th and 12nd week detected using immunohistochemistry of α-synuclein. (C) The double immunofluorescence of α-synuclein and TH in the 12nd week. The green fluorescence was EGFP or α-synuclein and the red
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fluorescence was TH. When merge together, the yellow stained was the neurons expressed
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α-synuclein. (Scale bar =100µm)
Fig. 2 Apomorphine induced rotation test. The control and EGFP group didn’t show any rotation, so there were only the data of α-synuclein WT and A53T group showed in the figure.
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* p＜0.05
Fig. 3 The loss of dopaminergic neurons in the SNpc and striatum. (A, B) The results of
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immunohistochemistry of TH detected in the SNpc and striatum. The rows were different
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time point and the lines were the different groups of rAAV injection. The arrows showed the significant loss of dopaminergic neruons or fibers.
Fig. 4 The insoluble and phosphorylated inclusions in the SNpc and striatum. (A~D) The PK resistant inclusions in the SNpc. The right sections were not treat with PK, and the right were treated with 10µg/mL PK for 10min.After treated with or without PK, all sections were stained with immunohistochemistry of α-synuclein.
(E~F) The PK resistant inclusions in
ACCEPTED MANUSCRIPT the striatum, and the arrows showed the inclusions. The treating progress of the sections was same with A~D.
(I, J) The phosphorylated inclusions in the SNpc were stained with
immunohistochemistry of phosphor-Ser-129 α-synuclein. The arrows were showed the
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phosphorylated inclusions. (Scale bar =200µm)
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ACCEPTED MANUSCRIPT Highlights 1. We compared the rAAV based α-synuclein over-expression in different time point. 2. We demonstrated the A53T mutated α-synuclein can induce degenerated of
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dopaminergic neurons more severely and early. 3. The A53T mutated α-synuclein can enhance insoluble inclusions formation.
4. The A53T mutated α-synuclein based model can considered as an early-oneset PD
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model.
S0006-291X(15)30238-2
DOI:
10.1016/j.bbrc.2015.07.007
Reference:
YBBRC 34215
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 29 June 2015 Accepted Date: 1 July 2015
Please cite this article as: J. Lu, F. Sun, H. Ma, H. Qing, Y. Deng, Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison between α-synuclein wild-type and A53T mutation in a progressive Parkinson’s disease model
1
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Jianqing Lu1, Feiyi Sun1, Hong Ma1, Hong Qing1, Yulin Deng1,*
School of life science, Beijing Institute of Technology, 5 South Zhongguancun Street,
*
Corresponding author: Dr. Yulin Deng
Tel: +86-10-68914907 E-mail: [email protected]
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Haidian District, Beijing, 100081, People's Republic of China
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Address: School of life science, Beijing Institute of Technology, 5 South Zhongguancun
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Street, Haidian District, Beijing, 100081, People's Republic of China
ACCEPTED MANUSCRIPT Abstract: Background and aims: Vector based over-expression of α-synuclein is a newly developed method to establish animal Parkinson’s disease (PD) model. In this paper, we inject the rat
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brain with recombinant adeno-associated virus (rAAV) to express α-synuclein wild-type and A53T mutation, and compared the degeneration of dopaminergic neurons between them. Method and results: The rAAV vectors were injected into the substantia nigra pars compacta
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(SNpc) of rat brain. In different time point, immunohistochemistry was used to detect the
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expression of α-synuclein. The expression level was lower in the 3rd and 6th week and increased from the 9th week. α-synuclein spread around the neurons in SNpc in the 12nd week. The loss of dopaminergic neurons was increasing along the expression of α-synuclein, and damage extent was more serious in the A53T group than the WT group. In the A53T
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group, there were more insoluble inclusions can be detected, and the phosphorylation of α-synuclein was also higher.
Conlusion: The result of comparison between the two types of α-synuclein showed that
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A53T mutated α-synuclein was more effective to establish PD model, and the model based
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A53T mutated α-synuclein was a suitable model to early-onset PD. Keywords: Parkinson’s disease; animal model; recombinant adeno-associated virus; α-synuclein
Abbreviations -LBs Lewy bodies -PD Parkinson’s disease
ACCEPTED MANUSCRIPT -PK Proterinase K - rAAV recombinant adeno-associated virus -SNpc substantia nigra pars compacta
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-WT wild-type
1. Introduction
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Parkinson’s disease (PD) is one of the most popular neurodegeneration diseases in the
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elders [1]. The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the appearance of protein aggregation named Lewy bodies (LBs) are two symptoms of PD [2]. There are many animal models established in the last dozens of years and most of them were based on the neurotoxins such as 6-OHDA and MPTP. However, there were few
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cases in these models can detect inclusions like LBs [3]. α-synuclein is the main composition of LBs and it is also considered as a genetic factor to PD. Some mutation of α-synuclein such as A53T, A30P and E46K were found in the familial PD patients [4]. In order to establish
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LBs formed PD model, α-synuclein over-expression based PD models were developed, and
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transgenic mice were investigated most well. In these α-synuclein transgenic mice, LBs were appeared in the SNpc with progressive loss of dopaminergic neurons [5]. Recently, vector based α-synuclein over-expression was developed as a new technology [6]. In these models, the viral vectors were injected to the SNpc and expressed α-synuclein in a small area of one side of brain [7]. The advantage of these models is that only dopaminergic neurons in the SNpc were transfected by the vector whereas other cells were not affected by α-synuclein over-expression [8]. The other advantage is the one-side destroyed of
ACCEPTED MANUSCRIPT dopaminergic neurons can be detected by behavior changes which showed rotation induced by amphetamine or apomorphine [9]. In these models, α-synuclein expressed in a high level and neurons lost along with the α-synuclein expression [10]. The insoluble inclusions were
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also detected in these models easily [11]. In our study, we chose α-synuclein widetype (WT) and A53T and over-express them in rat brain using recombinant adeno-associated virus (rAAV). After injection of these vectors,
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we detected the gene expression and neuron degeneration in the 3rd, 6th, 9th and 12nd week.
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The data showed that α-synuclein expression can induce a progressive degeneration of dopaminergic neurons. The A53T mutated α-synuclein can induce the loss of dopaminergic neurons more severely and enhance more inclusions formation. The model over-expressed
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A53T mutated α-synuclein can be chosen as the early-onset PD model.
2. Materials and methods 2.1 Vector preparation
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The EGFP, α-synuclein WT and A53T gene were constructed into the shuttle plasmid
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using HindⅢ and XhoⅠ with CAG promoter and WPRE enhancer. The shuttle plasmid and helper plasmid pFd6 and pH22 were co-transfected to HEK293 cell with calcium phosphate assay. The rAAV was packaged in the HEK293 cell for 3 days and then lyse the cells using 0.5% sodium deoxycholate. The vector was purified by HiTrap Heparin HP (GE Healthcare, USA) and concentrated using 100kD ultrafiltration tube (Merck-Millipore, USA). The titer of the rAAV was determined by realtime qPCR of WPRE enhancer, the forward primer: TGGCGTGGTGTGCACTGT, the reverse primer: GTTCCGCCGTGGCAATAG. The titer of
ACCEPTED MANUSCRIPT all of the three vectors was diluted to 1×1013 gc/mL by PBS. 2.2 Animal and surgery Adult male Wistar rats (Vital River, China) were kept in five per cage with adequate food
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and water. The light/dark cycle was 12h which controlled by program. All procedures were conducted in authorization by Animal Ethics Committee of Beijng Institute of Technology. The weight of these rats were 270~320g at the time of surgery. After deeply anesthetized
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by pentobarbital sodium at 50mg/kg i.p., the rats were put on a stereotaxic frame and fixed
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their skull with the ear bars and the incisor bar. Then 4µL viral solution was injected to SNpc through a hole above it. The coordinates of the SNpc: antero-posterior -5.3mm from bregma, medio-lateral +2.2mm from midline, and dorso-ventral -7.7mm below dura. The injection was performed using 10µL Hamilton syringe with a thin needle (outer diameter of 0.2mm,
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Hamilton, USA), and the infusion rate was 1µL/min controlled by a syringe pump (model UMP3, WPI, USA). After injection, the needle was kept in the brain for 3 min to make the solution diffuse completely.
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2.3 Behavior testing
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After injection, the apomorphine induced rotation test was carried out at the 3rd, 6th, 9th and 12nd week. These rats were injected 0.5mg/mL apomorphine (dissolved in 0.9% saline) i.p., and then put them to a big cage. The rotation behavior was captured by a digital video camera for 45min.
2.4 Immunohistochemistry and immunofluorescence At the same time point of behavior testing, the rats in corresponding group were deeply anesthetized by pentobarbital sodium at 50mg/kg i.p., and then perfused through their heart
ACCEPTED MANUSCRIPT with 500mL saline. After perfusion, the brains were removed quickly and immerse to ice cold 4% paraformaldehyde for at least 24h, and then transfer them to 25% sucrose for cryoprotection. Following the brains were cut to 30µm sections by freezing microtome
solution (30% sucrose, 30% ethylene glycol in PBS) until using.
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(CM1900, Leica, Gemany).The sections in striatum and midbrain were stored in anti-freeze
For immunohistochemistry, the sections were picked from anti-freeze solution and
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washed with PBS-T (0.3% Triton X-100 in PBS). In order to quench the endogenous
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peroxidase, these sections were transferred to 1% H2O2 for 15 min at room temperature (RT) and then wash with PBS-T 3 times, 5 min each. Following these sections were blocked by 1% normal goat serum for 30 min and then incubated with primary antibody diluted with 1.5% normal goat serum in PBS-T: α-synuclein (1:2000, mouse monoclone, Abcam, UK), TH
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(1:1000, rabbit polyclone, Merck-Millipore, USA), phosphor-Ser-129 α-synuclein (rabbit, 1:1000, Abcam, UK) at 4℃ overnight with gentle shake. The next day these sections were picked from primary antibody to wash for 3 times and then incubated them with biotinylated
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second antibody diluted with PBS-T (1:200, Vector Laboratories, USA) for 2 hours at RT.
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After incubation, these sections were washed incubated with ABC complex solution diluted with PBS-T (1:100, Vector Laboratories, USA, combine reagent A to reagent B for 1:1) for 2 hours. At last these sections were developed with DAB and mounted to slides with PVP mounting medium and acquire the images using digital scanner or microscope (IX71, Olympus, Japan). For immunofluorescence, the procedure was similar with immunohistochemistry without the 1% H2O2 incubation. The primary antibody was the mixture of α-synuclein and TH (the
ACCEPTED MANUSCRIPT dilution was the same with above) and the next day they were incubated with the mixture of fluorescence second antibody: Alexa Fluor 488-labeled goat anti-mouse IgG (1:200, Zhongshan Goldbridge, China), TRITC-labeled goat anti rabbit IgG (1:100, Zhongshan
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Goldbridge, China). After incubation, these sections were washed and mounted with anti-fade PVP mounting medium and acquire the images using laser confocal microscope (TCS SP5,
2.5 Proteinase K digestion assay Proterinase
K
(PK)
solution
incubation
was
carried
out
before
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The
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Leica, Germany)
immunohistochemistry for α-synuclein. After the first wash step, these sections were incubated with 10µg/mL PK for 10 min at 37 ℃ and then post-fixed with 4% paraformaldehyde for 10 min at RT. The following procedure was the same with 2.4 but the
2.6 Statistical analysis
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only difference is using PBS instead of PBS-T.
All results are expressed as means ± SD, and the statistical significance between two
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groups were evaluated using T-test analysis. P< 0.05 was considered statistically significant.
3. Results
3.1 α-synuclein expression in dopaminergic system The expression of α-synuclein is slow and progressive (Fig.1). There was only slight express in SNpc at the 3rd week and 6th week, and there was no significant different between the WT group and A53T group (Fig. 1A). As the assemble of projection fibers of dopaminergic neurons in SNpc, the expression of α-synuclein in striatum was similar with
ACCEPTED MANUSCRIPT SNpc, but the level was higher in the A53T group than the WT group at the 6th week(Fig. 1B ). The expression level increased more higher from the 9th week. In the SNpc, there was large area of α-synuclein expression and the A53T group was larger than the WT group (Fig.
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1A). In the striatum, the stained area was distributed to all dopaminergic fibers in A53T group from the 9th weeks, whereas the WT group reached the same extent at the 12nd weeks (Fig. 1B).
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In order to investigate if the rAAV can specific infect the dopaminergic neurons, double
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immunofluorescence stain for α-synuclein and TH was performed in SNpc. The green fluorescence was EGFP or α-synuclein and the red one was TH which is the marker of dopaminergic neurons, and the result of merge the two images together showed that all green fluorescence was co-local with red one (Fig. 1C). This indicated that the genes which rAAV
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carried (EGFP or α-synuclein) were expressed in the dopaminergic neurons. 3.2 Apomorphine induced rotation
When dopaminergic system was damaged at one side, the rats can display rotation to the
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healthy side induced by apomorphine, and the speed of rotation show positive correlation
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with the damage extent. There were no detectable rotation in the control group and EGFP group, and this showed that the dopaminergic system may be not affect by single injection of rAAV. However, the rotation was detected when α-synuclein expressed. In the α-synuclein WT group, the rats showed no rotation in the first 9 weeks, and reached 2.68±1.74 turning/min at the 12nd week. Although the α-synuclein expression was the same level in A53T group with WT group at the 3rd week, the rotation in A53T group was 1.76 turning/min, and kept the similar level to 1.83±1.08 at the 6th week. As the expression level
ACCEPTED MANUSCRIPT reached higher from the 9th week, the rotation speed increased to 2.97±1.99 turning/min at the 9th week and even higher to 3.91±1.62 turning/min at the 12nd week (Fig. 2). These result suggested that the A53T mutation in α-synuclein may enhance the toxic of the protein
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and make the symptom appear earlier. 3.3 α-synuclein expression induced loss of dopaminergic neurons
The immunohistochemistry of TH can stain the dopaminergic neurons (Fig. 3). In the
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SNpc, there was no detectable loss in the control group and EGFP group, this result was
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consistent with the behavior result. It indicated that rAAV infection cannot contribute to the loss of dopaminergic neurons. When α-synuclein expressed in SNpc, there were significant loss of dopaminergic neurons in the SNpc at one side. In the α-synuclein WT group, the loss of dopaminergic neurons can be detected very late only at the 12nd week, and it was the same
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time of the appearance of rotation in this group. There was no significant loss of dopaminergic neurons in the A53T mutation group at the 3rd week and 6th week although the rotation can be detected, and this may be concern to the rotation speed was not very high.
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From the 9th week, the rotation speed reached over 2 turning/min, and the loss of
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dopaminergic neurons can be detected in the injected side. The extent of this damage was increasing with time in the A53T mutation group (Fig.3A). The performance of dopaminergic fibers in the striatum was the same condition with the SNpc. The control group and EGFP group showed symmetry between the two sides, and this meant there was no damage in the striatum. In the α-synuclein WT group, there was no change of the fibers density until the 9th week, and the density decreased at the 12nd week. As the loss of dopaminergic neurons in SNpc, the fibers density decreased from the 9th week
ACCEPTED MANUSCRIPT in the A53T group. The reduction of this density in the A53T group was more than the WT group (Fig 3B). 3.4 α-synuclein expression led to aggregation and phosphorylation
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From the results above, the toxic of α-synuclein WT was different from A53T mutation. In order to investigate the aggregated pattern of these two kinds of α-synuclein, PK digestion assay was performed with immunohistochemistry of α-synuclein in the 12nd week. In the
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SNpc, the α-synuclein stain distributed a large area around the SNpc before PK treated (Fig.
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4A and 4C). However, if incubated with PK, there were different results between the WT and A53T mutation. In the WT group, there were only the dopaminergic neuron shape area can be stained, and the other area was still white (Fig. 4B). In the A53T group, α-synuclein aggregated inclusions spread as the same area with untreated section, and there was no visible
(Fig. 4D).
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neuron shape as the WT group. The inclusions in the A53T group were like fibers in the SNpc
In the striatum, α-synuclein was expressed in all the dopaminergic fibers before PK
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treated, and there was no significant different between the WT group and A53T group (Fig.
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4E and 4G). After incubated with PK, the inclusions of α-synuclein were appeared in the sections, and the inclusion number in the A53T group was more than the WT group (Fig. 4F and 4H). Unlike the fiber shape of α-synuclein inclusions in the SNpc, the inclusions in the striatum were spheric, and this shape was very similar to the LBs (Fig.4K). As α-synuclein phosphorylation is the character of LBs, the immunohistochemistry of Ser-129 phosphorylated α-synuclein was performed in the SNpc. The results showed that there were only little stained dots in the SNpc area in the WT group, and the number
ACCEPTED MANUSCRIPT increased in the A53T group nearly by double (Fig. 4I and 4J).
4. Discussion
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α-synuclein is the first genetic factor found related with PD, and A53T is an important mutation in the pathogenesis of PD [12]. LBs as the hallmark of PD contain abundance aggregated α-synuclein, and over-expression of α-synuclein is thought to be a genetic way to
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establish PD model. In transgenic mice model, α-synuclein expression induced different
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effects because of the promoter and genetic background[13]. Several line expressed human α-synuclein were established using the PDGF-β promoter, and the results showed that only the Line which expressed more α-synuclein can induced significant loss of dopaminergic neurons with the most inclusions formation[14]. The investigation of effect of α-synuclein
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mutation also performed in the transgenic mice. In their experiment, the PrP promoter was used and these model express high level of α-synuclein. However, they only compared the WT, and mutated A30P and A53T genotype in the number of lumbar motor neurons, and the
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result showed that the A53T mutations showed the most damage. A53T mutated α-synuclein
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also increased the degeneration and apoptosis of neurons to high level [15]. In our experiment, we compared the WT and A53T mutation using rAAV based α-synuclein overexpression model. There are many investigations had been carried out used this method. In most of these experiments, WT and A53T were not compared. The results showed that after injected the rAAV to the SNpc, there were high level of α-synuclein expressed in the dopaminergic neurons of SNpc, and α-synuclein also can be detected in the striatum [16]. However, they only showed the end point of the expression but not the progress.
ACCEPTED MANUSCRIPT Recently, a study using rAAV2/7 to express α-synuclein in SNpc showed the immunofluorescence of α-synuclein, but the results only showed the data of 5 day and 8 week, and it’s cannot illustrate the expression level of α-synuclein [17]. In our study, we used the
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immunohistochemistry to stain α-synuclein in SNpc and the results showed the progress of α-synuclein expression. The expression level in the first 6 weeks was not very high, and the protein only expressed in the neurons body. However, the results in the 9th week and 12nd
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showed an enlarged area of expression, and α-synuclein spread from the neurons formed
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fiber-like state. The expression in the A53T group was a little higher than the WT group, and this may be result of the mutated α-synuclein is aggregated more easily. The loss of dopaminergic neurons was detected in most rAAV-based α-synuclein over-expression model, but the extents were varied. The behavior test was carried out in some
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of these models, and the result showed the progressive increasing of apomorphine induced rotation [18]. However, there was no comparison between the WT and A53T group. In our experiment, the data showed that although the expression level was no significant different in
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the two group, the A53T group displayed rotation earlier and more serious. The
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immunohistochemistry of TH in SNpc and striatum showed the loss of dopaminergic neurons in the A53T group was more than the WT group. The neurons loss progress agreed with the α-synuclein expression level. This result showed that the mutated A53T α-synuclein is more toxic to dopaminergic neurons, and induced the degeneration of neurons very early. The A53T over-expression model may be a suitable model of the early-onset PD. α-synuclein aggregation is a hallmark of PD, and it’s easily to be detected in the α-synuclein over-expression model. The insoluble aggregates were found in the SNpc of
ACCEPTED MANUSCRIPT α-synuclein A53T over expressed model using PK digest assay [19]. However, the aggregates in that experiment was similar with the result of our WT group, and the damage extent also reached the similar level. In our experiment, the A53T group showed fragment like mode,
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and spread to a large area around the SNpc. The inclusions in the striatum of the A53T group were also more than WT group, and the phosphorylation of these inclusions in the SNpc showed the same tendency between the two groups. These results showed that the A53T
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mutation in α-synuclein can enhance protein aggregation and LBs-like inclusions formation.
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In conclusion, the results in our study showed the progressive expression of α-synuclein via rAAV injection with the degeneration of dopaminergic neurons. The A53T mutated α-synuclein was more toxic to the neurons than WT and enhanced aggregation of this protein. The results were useful to the researchers who want to choose rAAV-α-synuclein to establish
Acknowledgements
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PD model.
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20435020).
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We thank the National Natural Science Foundation of China supported our study (No.
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ACCEPTED MANUSCRIPT Figure legends:
Fig.1 The expression of α-synuclein in the SNpc and striatum. (A, B) The expression of
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α-synuclein in SNpc and striatum in the 3rd, 6th, 9th and 12nd week detected using immunohistochemistry of α-synuclein. (C) The double immunofluorescence of α-synuclein and TH in the 12nd week. The green fluorescence was EGFP or α-synuclein and the red
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fluorescence was TH. When merge together, the yellow stained was the neurons expressed
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α-synuclein. (Scale bar =100µm)
Fig. 2 Apomorphine induced rotation test. The control and EGFP group didn’t show any rotation, so there were only the data of α-synuclein WT and A53T group showed in the figure.
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* p＜0.05
Fig. 3 The loss of dopaminergic neurons in the SNpc and striatum. (A, B) The results of
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immunohistochemistry of TH detected in the SNpc and striatum. The rows were different
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time point and the lines were the different groups of rAAV injection. The arrows showed the significant loss of dopaminergic neruons or fibers.
Fig. 4 The insoluble and phosphorylated inclusions in the SNpc and striatum. (A~D) The PK resistant inclusions in the SNpc. The right sections were not treat with PK, and the right were treated with 10µg/mL PK for 10min.After treated with or without PK, all sections were stained with immunohistochemistry of α-synuclein.
(E~F) The PK resistant inclusions in
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(I, J) The phosphorylated inclusions in the SNpc were stained with
immunohistochemistry of phosphor-Ser-129 α-synuclein. The arrows were showed the
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phosphorylated inclusions. (Scale bar =200µm)
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ACCEPTED MANUSCRIPT Highlights 1. We compared the rAAV based α-synuclein over-expression in different time point. 2. We demonstrated the A53T mutated α-synuclein can induce degenerated of
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dopaminergic neurons more severely and early. 3. The A53T mutated α-synuclein can enhance insoluble inclusions formation.
4. The A53T mutated α-synuclein based model can considered as an early-oneset PD
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model.