The Synergistic Effect Study of Lipopolysaccharide (LPS) and A53T-α -synuclein: Intranasal LPS Exposure on the A53T-α -synuclein Transgenic Mouse Model of Parkinson's Disease

Aging and the interaction between genetic and environmental factors are believed to be involved in chronic Parkinson's Disease (PD) progression. Abnormal aggregated α -synuclein is the main component of Lewy body in PD patients. The intranasal route is believed to be a gate way to the brain which facilitates entry of environmental neurotoxin into the brain and account for the smell loss in early PD. In this study, we chronically applied intranasal lipopolysaccharides (LPS) exposure on 4-month-old, 8-month-old, 12-month-old and 16-month-old A53T-α -synuclein (A53T-α -Syn) transgenic C57BL/6 mice every other day for 2 months to evaluate the behavioral, pathological, biochemical changes as well as microglial activation in these mice. We observed intranasal LPS exposed A53T-α -Syn mice displayed a robust progressive olfactory disorder, hypokinesia, selective loss of dopaminergic neurons, reduction in striatal dopamine (DA) content, and accelerated α -synuclein aggregation in the SN in an age-dependent way. Furthermore, we found obvious NF-к B activation, Nurr1 inhibition, IL-1 β and TNF-α generation in microglia in the SN. By contrast, these PD-like changes were mild in WT and moderate in A53T-α -Syn mice in old-aged mice. This study demonstrated the synergistic effect of intranasal LPS and α -synuclein burden on PD progression. The potential mechanism was attributed to the inhibition of Nurr1 in microglial cells and the ampli�ed neuroin�ammation in the CNS. These multi-hit animals (aging, α -synuclein mutation and neuroin�ammation) might help us to investigate the mechanisms through which mutant α - synuclein and in�ammation work in concert to mediate PD neurodegeneration.


Introduction
Parkinson's disease (PD) is a chronic and slow-progressive neurodegenerative disorder, with a high incidence in aged people [1].The pathological changes of PD mainly include intracytoplasmic inclusions composed primarily of α-synuclein aggregates and the degeneration of dopaminergic (DAergic) neurons in the substania nigra (SN) [2].After 5-7 years of pre-clinical stage, clinical symptoms begin to appear when the loss reached at least 60% of pigmented DAergic neurons in the SN.It is critical and urgent to explore the trigger of the disease and the contributing factors in the process of chronic progressive degeneration.It is widely accepted that multifactorial factors contribute to PD pathogenesis, including aging, genetic predisposition and environmental exposures.Aging is the single most important risk factor for PD, and a sharp rise in incidence curve is observed in an age-dependent manner, especially in people aged over 50 [3][4][5].PD is always a matter of time.Apart from old age, genetic factors have been identi ed in familiar forms of PD, which contribute to approximately 10% of PD cases [6,7].Three missense mutations (A53T, A30P, E46K) and multiplication mutations of α-synuclein, which resulted consequent overexpression of the a-synuclein gene, caused autosomal dominant familial type of PD [8].
The α-synuclein gene locus also associates with the more common sporadic PD.Most importantly, numerous studies have identi ed environmental risk factors for PD, such as pesticides, solvents, metals, and pollutants recapitulate PD pathology in animal models.Thus, although genes are likely to play a role, the vast majority of PD cases cannot be ascribed exclusively to genetic factors, illustrating a multifactorial etiology with the important environmental contribution in PD pathogenesis [9][10][11][12].
Aggregated α-synuclein is the major component of Lewy body, a pathological marker of PD.An important reason for the damage of DAergic neurons may be attributed to the heavy burden of α-synuclein in DAergic neurons exceeds the degradation ability of DAergic neurons, resulting in abnormal aggregation and neurotoxic reactions.In view of the important role of abnormal aggregation of α-synuclein in the pathogenesis of PD, the transgenic model of α-synuclein has been widely used in experimental studies.However, disappointedly, most of the α-synuclein transgenic mice did not successfully replicate the degeneration of DAergic neurons [13,14,9].Inexplicable, even when a large amount of α-synuclein is deposited, the DAergic neurons can still resist the neurotoxicity of α-synuclein, which limits the application of these models.An important explanation for this phenomenon is that defecit in a single gene is not su cient to cause the loss of large numbers of DAergic neurons, but increase susceptibility to external factors, and environmental triggers may be necessary for PD degeneration.
A host of environmental toxins have been con rmed to increase the PD risk, including MPTP, pesticides such as paraquat, rotenone, heavy metals, industrial chemicals, air pollutants, etc., which can change gene expression linked to α-synuclein accumulation and DAergic degeneration and apoptosis [15,16].Most PD models are established by poisons, such as MPTP, 6-OHDA and paraquat models, which are acute or subacute models prepared with relatively high doses of poisons in young animals, and many lack the pathological changes of classical α-synuclein.The limitations of these toxic models make their applications restricted [17].
It is generally postulated that PD might be a primary disorder of olfaction, in which hyposmia precedes motor symptoms by years [18].According to Braak's staging, abnormal α-synuclein aggregation rst appeared in the olfaction bulb (OB) or medulla oblongata, gradually extended to the upper medulla oblongata, pons, SN and cortex in PD progression.The SN lesion may already be in the middle or late stage (stage 3 or 4) of PD.Prior to the onset of motor symptoms (stages 3 and 4), PD patients already exhibit some non-motor symptoms, such as an hyposmia or autonomic nervous system dysfunction (stages 1 and 2) [19].According to the Dual hit theory proposed by Professor Braak [20], the initiation of PD might be attributed to the invasion of some unknown pathogen (such as some infection sources and chemical substances, etc.) through nasal mucosa, OB and anterior olfactory nucleus into amygdala and olfactory tubercle.Intriguingly, airborne infectious, allergic and pollution agents could enter the brain via the olfactory tract and bypass the blood brain barrier, which activates the immune in ammation and neurotoxic reaction in the brain, and results in the damage of DAergic neurons.Therefore, the intranasal route is a direct communication between the environment and the brain.Thus, air pollutants may affect the brain either directly via transport of pollutants from the OB or through systemic in ammation that contributes to the neurodegenerative process.
Persistent chronic in ammatory responses may be a common feature of all degenerative diseases, including PD [21].Microglial activation and in ammation occur early in the disease process and continue throughout its course.Microglia can be activated by numerous factors such as α-synuclein aggregates, neuromelanin, brinogen or environmental LPS toxins, MPTP, pesticides, proteasome and heavy metals.
At the same time, the activation of microglia could encourage the activation of nuclear factor κB (NF-κB) and release of in ammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, leading ultimately to neuroin ammation, and destruction of DAnergic neurons [22,23].LPS, a potent stimulator of microglia, has been widely used to study in ammation-mediated DA neurodegeneration [24,25].The Nuclear receptor related 1 protein (NURR1), a member of the orphan nuclear receptor family, is critical for the generation, development, maturation and maintenance of DAergic neurons [26].Emerging evidence indicates that impaired NURR1 function might contribute to the pathogenesis of PD.
Nurr1 exerts anti-in ammatory effects via interaction with NF-κB to inhibit its nuclear translocation and suppress the expression of numerous in ammation-related genes regulated by NF-κB [27].
In this project, we planned to replicate the multiple causes of PD involving aging, α-synuclein gene mutation and environmental toxin by chronic nasal exposure of low-dose LPS in a transgenic mouse model overexpressing human A53T-α-synuclein mutation in an age-dependent manner, and observe the behavioral, pathological, biochemical changes as well as microglial activation in these mice.These multihit (aging, neuroin ammation and mutation of α-synuclein) mice not only provides better tool to advance our understanding of the role of neuroin ammation and α-synuclein dysfunction in the pathogenesis of PD, but also underscores synergistic effects of genetic predisposition and environmental exposures in the development of PD.

Animals
Human A53T α-Syn transgenic mice (B6; C3-Tg (PrnpSNCA*A53T)/Nju 20-22g, were purchased from the Jackson Laboratory.Homozygous A53T α-Syn mice and wildtype (WT) littermates were selected for our experiment.The animals were treated humanely and with regard for alleviation of suffering.All experiments were conducted in accordance with the guidelines of the International Council for Laboratory Animal Science.The study was approved by the Ethics Committee of Shanghai Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine, Shanghai, China.All mice were housed in controlled temperature, humidity rooms and pathogen-free conditions, on a 12h light/dark cycle with free access to food and water.At 2 month (m) old, 6m old,10m old,14m old, both the transgenic mice (n = 10) and their WT controls (n = 10) received normal saline (NS) or intranasal LPS (1mg/ml, Sigma-Aldrich, USA) dissolved in saline solution.After a slight anesthesia with ether, mice were held by the neck and were laid upside down with a nger under the neck to limit liquid ow down the trachea.10µl of LPS or saline solution was slowly introduced with a micropipette (over ~ 15s) into two nasal cavities separately.This procedure was repeated every other day for 2 months for next experiment [28].

Olfactory function
We test the olfactory function including social scent discrimination, non-social scent discrimination, and odor detection.The sni ng time of each mouse and the latency to nd the pellet were measured.

Social-scent discrimination
Social scents were assessed by a modi ed block test according to a previous study [29].Wooden blocks (1.8cm×1.8cm×1.8cm)were separately sealed with 5g of mice beddings from their home cages of test mice in 50ml of centrifugal tubes for 24h, so that the blocks took on the odors from mice respectively.In each trial, the test mouse was exposed to one block with its own odor (the familiar scent) and another block with another mouse's odor which was totally novel for the test mouse (the novel scent).The time of each mouse spent on sni ng each block was recorded in a 120s trial with a stopwatch (accurate to 0.01s).

Non-social scent discrimination
This test was performed in the home cage to alleviate the environmental stress.Before the test, scent solutions of cinnamon or paprika (100ng/ml) were freshly dissolved and ltered.Two plates were placed in the home cage.30µl of scent solution was dropped on a small piece of lter paper and put onto the plate for the test every time.Each mouse was habituated to cinnamon solution on one plate and distilled water (control) on the other plate for ve successive 5-min trials, separated by a 15-min interval.At the 6th trial, the mice were presented with a second novel paprika odorant replaced with cinnamon solution for 5min.The time spent on sni ng water or scent solution was respectively recorded, which was used as the ability to discriminate the odorants.The sni ng time from trial-1 to trial-5 would be decreased if there was no difference between the two odors, while the decreased sni ng time would be recovered if animals could detect the difference between the novel odorant and the familiar odorant.

Odor detection
Buried pellet test (BFT) was performed to evaluate odor detection.The housed mice received a foodrestricted diet (0.2g chow per mouse/24h) from 2 days before the test.Body weight was recorded during the experiment period and maintained at 80%~90% of the original body weight.The BFT was performed for 5 successive days and each mouse received one trial per day at the same time.1g food pellet was buried approximately 0.5cm below the surface of a 3cm-deep padding in the test cage (45cm×24cm×20cm).The location of the food pellet was changed daily at random.In each trial, test mouse was placed in the center of the cage.The latency to nd and eat the buried food pellet was recorded.If the mouse could not locate the food pellet within 5min, the latency was removed and recorded as 5min.The padding in the test cage was changed between trials.On the 6th day, a visual pellet test (VPT) was conducted in a similar way as the BFT except that the food pellet was located on the surface of the padding, aimed to make sure that the animals did not suffer from altered locomotor activity or motivation.

Open-eld test
Motor behavior was analyzed in an open-eld test.The apparatus consisted of a square (30*30cm) with a surrounding wall (height 15cm).The square oor was divided in 16 small squares (7.5*7.5cm) using transverse and longitudinal segments.Mice were placed in the center of the apparatus and their voluntary movement was analyzed for a period of 20min, the rst 5min serving as a period of habituation to reduce environmental stress.The apparatus was washed out after each test.

Pole Test
The climbing ability was measured by the pole test to evaluate the degree of bradykinesia [30].Brie y, the mouse was placed head-upward on the top of a vertical rough-surfaced pole (diameter 10mm; height 50cm) and the time until it descended to the oor (total time) was recorded with a maximum duration of 120s.If the animal fell the rest of the way, the time when it reached to the oor was recorded.If the animal could not turn downward and instead dropped from the pole, the total time was recorded as 120s (default value).Animals were trained three times every day for 3 days before test.During the test, every animal was tested for three times and the average value was used.
Mice were anesthetized by chloral hydrate and perfused through the left ventricle with saline solution, followed by 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 15min.The brains were removed and cryoprotected by soaking in 10%, 20% and 30% sucrose solution for each day in phosphate buffer until they sank.The brains were obtained and frozen in liquid nitrogen.Parallel series of 10µm-thick coronal sections were obtained in a freezing microtome.
The immuno uorescent intensity of TH in the striatum was determined based on optical density analysis by Image-Pro Plus software.Quantitative analysis of positive staining (the expression of p-NF-кB-p65, IL-1β, TNF-α and Nurr1 on CD11b + microglia) was determined based on number of double positive cells/mm 2 by Image-Pro Plus software.Images were taken from the same location in all animals.

Proteinase K (PK) treatment
For assessing accumulation of insoluble α-synuclein, a PK digestion step was included prior to immunostaining.Brie y, sections were mounted and dried on Permafrost + glass slides for at least 8h at 55°C.Sections were then brie y hydrated with TBS-T (10mM Tris-HCl, pH 7.8; 100mM NaCl; 0.05% Tween20), and digested with 50µg/ml PK (Invitrogen, Carlsbad, CA) in TBS-T (10mM Tris-HCl, pH 7.8; 100mM NaCl; 0.1% Tween20) for a period of 1.5h at 55°C.Sections were then xed for 10 min using 4% paraformaldehyde and then processed for α-synuclein immuno uorescence as described above.
After washing in TBST, the immunoblots were incubated with horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology) for 1h.The immunoblots were developed with an enhanced chemiluminescence (ECL) reagents (Millipore, USA), and measured with Quantity Software (Bio-Rad, CA).To compare protein loading, antibody directed against GAPDH was used.

The measurement of striatal biogenic amines
Brains were used to dissect the two striata.The striatum samples were weighed and homogenized in 10 volumes of 0.2M perchloric acid containing 100µM EDTA-2Na and 100ng/ml isoproterenol (the internal standard for the measurement of catecholamine and 5-HT contents) in a homogenizer at a maximum setting for 20s on ice.After 30min in the mixture of ice and water, the homogenate was centrifuged at 15000×g for 20min at 4°C.The solution of 1 M sodium acetate was added to adjust the pH value to approximately 3.0.After ltration (0.45µm), the samples were injected into a HPLC system.DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) were detected using a HPLCelectrochemical detection system.Brie y, 20µL of the homogenate sample was injected into the HPLCelectrochemical detection system by using an L-2200 autosampler (HITACHI, Tokyo, Japan) at 4°C, separated at 25°C on a reverse-phase analytical column (EICOMPAK SC-5ODS, 3.0mm×150mm; Eicom, Kyoto, Japan), eluted at a ow rate of 0.5 ml/min at 30°C with 0.1 M sodium acetate/citric acid (pH 5.4) containing 17% methanol, 190mg/L sodium l-octanesulfonate, and 5 mg/L EDTA-2Na, and determined by an 2465 electrochemical detector (Waters, Milford, MA, USA).The chromatograms were recorded and analyzed using a computer with Millennium32 Chromatography Manager Software (Waters).

Statistical analysis
For all statistical comparisons we rst analyzed using one-way analysis of variance (ANOVA).P value less than 0.05 was considered signi cant.If the ANOVA was signi cant, all post-hoc tests were conducted using Tukey's multiple comparison test.(Graph Prism 9, GraphPad Software, Inc.).Data were presented as mean ± S.E.M.

Results
Impaired Olfactory discrimination in A53T α-Syn mice after LPS challenge To examine the olfactory activity after LPS instillation, social-scent discrimination, non-social scent discrimination and odor detection (Buried pellet test, BFT) was performed in the 4m-old, 8m-old, 12m-old and 16m-old A53T α-Syn mice.In the social scent discrimination task, the WT and A53T α-Syn mice spent more time to sniff the novel scent block (novel scent) than their own scent block (familiar scent) at all ages, displaying a normal function in the social scent discrimination.While in the intranasal LPS exposed A53T α-Syn mice, no difference was found in the time spent to sniff the novel scent block and their own scent block at all ages compared to age-matched A53T α-Syn mice, indicating an impaired social-scent discrimination in these mice (4m: familiar scent vs novel scent: 6.75s vs 12.75s, P 0.001 in WT mice, 5.88s vs 9.63s, P 0.01 in A53T α-Syn mice; 8m: familiar scent vs novel scent: 7.38s vs 11.13s, P 0.05 in WT mice, 6.86s vs 10s, P 0.05 in A53T α-Syn mice; 12m: familiar scent vs novel scent: 7s vs 10.5s, P 0.05 in WT mice, 6.67s vs 9.67s, P 0.05 in A53T α-Syn mice; 16m: familiar scent vs novel scent: 5.64s vs 8.73s, P 0.05 in WT mice, 6s vs 9.17s, P 0.05 in A53T α-Syn mice) (Fig. 1A).
In the BFT, no difference was found in the latency to nd the buried pellet between WT and A53T α-Syn mice at all ages, while a signi cant difference was found in the intranasal LPS exposed 8m-old, 12m-old and 16m-old A53T α-Syn mice in comparison to A53T α-Syn mice without LPS exposure, indicating an impaired odor detection in these mice.In the VPT which was set to exclude the possible in uence of motor function or motivation, the two groups behaved similarly (8m: 51.66s in A53T α-Syn mice, 91.84s in A53T α-Syn mice exposed to intranasal LPS, P 0.01; 12m: 67.04s in A53T α-Syn mice, 152.7s, in A53T α-Syn mice exposed to intranasal LPS, P 0.01; 16m: 113.6s in A53T α-Syn mice, 184.9s in A53T α-Syn mice exposed to intranasal LPS, P 0.05) (Fig. 1C).

Behavioral de cit in A53T α-Syn mice after intranasal LPS exposure
To examine the motor activity after LPS exposure, open-eld and pole test were performed in mice.As shown in Fig. 2, all of the mice basically showed a progressive decreased locomotor activity in an agedependent fashion, characterized by reduction in ambulatory motor activity (Fig. 2a) and more time needed to climb to the top of the pole (Fig. 2b).No obvious difference was found between WT mice and A53T α-Syn mice in ambulatory motor activity, but in comparison to WT mice, the 12m-old A53T α-Syn mice showed lower climbing ability (time to climb to the top of the pole, 9s in WT α-Syn mice, 15.3s in A53T α-Syn mice, P 0.01) (Fig. 2b).It is worth noting that LPS-administered A53T α-Syn mice developed a more serious reduction in ambulatory motor activity at the age of 8m, 12m and 16m in the open eld test (voluntary movement, mean ± SEM; 8m: 311s in A53T α-Syn mice, 202s in A53T α-Syn mice exposed to LPS, P 0.01; 12m: 246s in A53T α-Syn mice, 153s in A53T α-Syn mice exposed to LPS, P 0.05, respectively; 16m: 199.9s in A53T α-Syn mice, 133.4s in A53T α-Syn mice exposed to LPS, P 0.05, respectively) (Fig. 2a) and needed more time to climb to the top of the pole at all ages compared with age-matched A53T α-Syn mice without LPS administration (time to climb to the top of the pole, 4m: 8.3s in A53T α-Syn mice, 17s in LPS exposed A53T α-Syn mice, P 0.01; 8m: 7.8s in A53T α-Syn mice, 13.3s in LPS exposed A53T α-Syn mice, P 0.01, 12m: 15.3s in A53T α-Syn mice, 25.8s in LPS exposed A53T α-Syn mice, P 0.05, 16m: 19.3s in A53T α-Syn mice, 28.3s in LPS exposed A53T α-Syn mice, P 0.05,respectively) (Fig. 2b).Our model exhibits a basic feature of human PD, chronic progressive bradykinesia.
Increased α-synuclein accumulation in OB of A53T α-Syn mice after LPS exposure α-synuclein pathology is also present in OB of PD.Pathological studies have observed that the OB is commonly lled with misfolded α-synuclein and that Lewy bodies in the OB precede those in the intracerebral basal ganglia structures [31].After the olfactory discrimination test, the α-synuclein levels were analyzed in the OB in this study by western blot analysis.

LPS exposure
The characteristic pathological event of PD model is the loss of DAergic neurons and accumulation and aggregation of α-synuclein in the SN.Next, we observed the pathological changes in these mice with intranasal LPS exposure.Firstly, we observed the DAergic neuronal loss and α-synuclein expression in the SN using double-labeled immuno uorescence, co-localization of tyrosine hydroxylase-immunoreactive (TH-ir) neurons (green) and α-synuclein expression (Fig. 4A).TH-ir neurons were counted stereologically from anterior to posterior of the SN and the numbers were shown in Fig. 4B.The data showed all groups including WT, A53T, LPS exposed A53T mice showed a progressive DA neuronal loss in an age-dependent manner.By the time the WT mice were 16ms of age, approximately 25.6% of DAergic neurons were lost in the SN compared to that of 4ms age (5575 at 4m, 5567 at 8m 5067 at12m 4150 at 16m in WT mice, 4m vs 16m, P<0.05, respectively).By the time the A53T α-Syn mice were 8ms and 16ms of age, signi cant 20.5% and 40% of DAergic neurons were lost in the SN compared to that of 4ms age (5467 at 4m, 5067 at 8m, 4433 at 12m 3333 at 16m in A53T α-Syn mice, 4m vs 8m, P<0.01, 4m vs 16m, P<0.01, respectively).In addition, no signi cant difference in loss of DAergic neurons was found between WT mice and A53T α-Syn mice at the age of 4,8,8m, indicating that the expression of α-synuclein was not su cient to cause neurodegeneration in A53T mice at that age.However, the 16m-old A53T α-Syn mice developed a more serious loss of TH-ir neurons compared to the WT mice (P<0.05).It's worth noting that after LPS instillation, accelerated and robust DA neuronal loss (39%, 46.6%, 46.2%, 63.7% ) was observed compared with age-matched A53T α-Syn mice at the age of 4m, 8m, 12m and 16m (3400 at 4m, 2975 at 8m, 3000 at 12m 2025 at 16m in A53T α-Syn mice exposed to LPS, P<0.01, respectively) .In addition, by the time the LPS exposed A53T α-Syn mice were 16ms of age, signi cant DAergic neurons were lost in the SN compared to that of 4ms age (4m vs 16m, P<0.05).
In addition to developing progres sive neurodegeneration, immuno uorescent staining also showed the expression of α-synuclein was obviously increased in A53T α-Syn mice compared with WT mice, while further enhanced α-synuclein aggregates were found in TH-ir neurons in the SN in intranasal LPS treated A53T α-Syn mice at the age of 4m, 8m, 12m and 16m (Fig. 4A).
Considering insoluble aggregation of α-synuclein is pathogenic, we examined PK resistant α-synuclein immunostaining on A53T α-Syn mice challenged with LPS.A few PK resistant Lewy-body-like α-synuclein aggregates were observed in the perinuclear compartment of DAergic neurons in A53T α-Syn mice, indicating most of α-synuclein deposits in the SN were soluble.By contrast, LPS-exposed A53T α-Syn mice accumulated more PK resistant α-synuclein in perinuclear locations in the SN than did in A53T α-Syn mice.These mice displayed age-related accumulation of insoluble α-synuclein, indicating that insoluble α-synuclein may participate in the pathogenesis of DA degeneration in intranasal LPS-treated mice.The abnormal accumulation of α-synuclein in DAergic neurons may trigger the downstream pathogenic cascades leading to the loss of DAergic neurons in A53T mice (Fig. 5).
Next, we also examined the double-labeled immuno uorescent intensity of TH-ir (green) and α-synuclein-ir (red) in the striatum to determine whether dopamine terminals were affected (Fig. 6A).The photomicrographs illustrating TH immunoreactivity in striatum was shown in Fig. 6B.To quantify this nding, we conducted optical density analysis.Similar to the SN, all groups including WT, A53T, LPS exposed A53T mice showed a progressive TH loss in an age-dependent manner (100% at 4m, 100.3% at 8m 89.8% at 12m 81.9% at 16m in WT mice; 108% at 4m, 89.7% at 8m 80.4% at 12m 68.5% at 16m in A53T α-Syn mice, 70.6% at 4m, 59.8% at 8m, 56.6% at 12m 43.9% at 16m in LPS exposed A53T α-Syn mice respectively).The results did not show the mice develop obvious loss of TH in the A53T α-Syn mice compared to age-matched WT mice at the age of 4m, 8m, 12m, while an obvious loss of TH (68.5%) in the A53T α-Syn mice compared to age-matched WT mice (81.9%) at the age of 16m in the striatum (P<0.05).However, the intensity of TH-ir staining was obviously reduced in the A53T α-Syn mice after LPS instillation compared with age-matched A53T α-Syn mice at the age of 4m, 8m, 12m and 16m (P<0.01,respectively).
In addition to developing progres sive neurodegeneration, Immuno uorescent staining also showed the expression of α-synuclein was obviously increased in A53T α-Syn mice compared with WT mice, while further enhanced α-synuclein expression were found in TH-ir neurons in the striatum in intranasal LPS treated A53T α-Syn mice at the age of 4m, 8m, 12m and 16m (Fig. 6A).

Reduction of striatal DA level and increase of DAergic metabolite turnover in A53T α-Syn mice after intranasal LPS insult
There are indications that an increase in DA turnover might be an early compensatory mechanism for DA degeneration in PD [32].For examination of DAergic neurotransmitter and metabolite levels, striatal HPLC was performed and analyzed for DA and the metabolites DOPAC and HVA (Fig. 8).As shown in Fig. 8a, all groups including WT, A53T, LPS exposed A53T mice showed a progressive decrease in DA release in an age-dependent manner (100% at 4m, 90% at 8m 78% at 12m 67% at 16m in WT mice; 99.9% at 4m, 80.3% at 8m 55.9% at 12m 51 % at 16m in A53T α-Syn mice, 61.9% at 4m, 41% at 8m, 31.2% at 12m 21.6% at 16m in LPS exposed A53T α-Syn mice respectively).A signi cant reduction of striatal DA level in A53T α-Syn mice relative to the WT mice was observed at the age of 12m.An obvious reduction of striatal DA level was found in A53T α-Syn mice treated with intranasal LPS relative to A53T α-Syn mice.
Intriguingly, the co-expression of CD11b/p-NF-кB and CD11b/IL-1β was up-regulated in A53T α-Syn at the age of 12 and 16m, and the co-expression of CD11b/TNF-α was up-regulated at the age of 16ms which indicated over-expression of α-synuclein could aggravate the in ammation in the SN (P 0.05).As expected the co-expression of CD11b/p-NF-кB, IL-1β, TNF-α were greatly enhanced in A53T α-Syn mice after LPS treatment at all ages (Fig. 9D, P<0.01 or P<0.05, respectively).Therefore, LPS-exposed A53T α-Syn mice exhibited a high level of p-NF-kB expression and its downstream cytokines including IL-1β, TNFα on microglia.
Nurr1 inhibition in A53T α-Syn mice after intranasal LPS insult Impaired Nurr1 function have been implied to contribute to the pathogenesis of PD.Nurr1 has been reported to be expressed in microglia, binding to NF-кB responsive elements in the promoter region of proin ammatory genes to reduce the expression of these genes, inhibiting the generation of proin ammatory mediators and consequently protects against in ammation-mediated neuronal death.
In this study, we determined the Nurr1 expression on microglia in SN by immuno uorescent staining by co-expression of CD11b and Nurr1.As a result, we found a tendency of Nurr1 to be suppressed in A53T α-Syn in an age-dependent manner, but it did not reach a signi cant difference.However, we found the expression of CD11b and Nurr1 was robustly inhibited in A53T α-Syn mice exposed to intranasal LPS compared with A53T α-Syn mice at all ages (CD11b/Nurr1-ir numbers; 285 at 4m, 295.6 at 8m 279.7 at 12m 241.6 at 16m in WT mice; 286.7 at 4m, 261.6 at 8m 256 at 12m 208 at 16m in A53T α-Syn mice, 165.7at 4m, 171 at 8m, 164.3 at 12m 129.1 at 16m in LPS exposed A53T α-Syn mice respectively) (Fig. 10A and 10B, P<0.05, respectively).These results suggest that the Nurr1 was suppressed in A53T α-Syn mice after LPS instillation, which might participate in LPS-induced in ammation and DA neuronal death.

Discussion
The development of PD is a long process.It is widely accepted that PD arises from a combination of an individual's genetic predispositions (one hit) and a long time of environmental exposures (two hit), in the context of aging (three hit).However, there is little experimental evidence that directly supports this idea.It is di cult to model accurately the duration, route, and overall burden of exposure to neurotoxicants in PD [33,34].In this study, we aimed to examine whether human mutant α-synuclein potentiates the effect of the environmental toxicants LPS and replicates the multiple causes of PD.We observed the typical motor and non-motor features of PD, progressive nigrostriatal degeneration manifested by nigral cell death and striatal dopamine depletion, and the presence of α-synuclein deposition by genetically A53T α-Syn engineered mice.We took into account factors such as aging, genetics, environment, real exposure pathways, and chronic progression to maximumly simulate the real course of disease.
Aging is an important factor in neurodegeneration.PD could be a consequence of the neuronal degeneration which are particularly vulnerable to aging process.In this study, aging was taken into rst consideration.We studied olfactory activity, behavioral alterations, the dopamine degeneration, αsynuclein deposition, the changes of the striatal biogenic amines, microglial activation, Nurr1 suppression in the nigrostriatal pathway in WT mice, A53T α-Syn mice and intranasal LPS exposed A53T α-Syn mice at 4 (young age), 8 (middle age), 12 (middle and old age), 16months (old age).As a result, we found all of the mice developed a progressive de cit in the odor discrimination ability and motor function, loss of DAnergic neurons, α-synuclein deposition, microglial activation, Nurr1 suppression in the SN, as well as reduction in TH and dopamine release and increase in DA turnover in the striatum in an age-dependent manner, especially at 12 and 16 months.Although mild in WT mice, moderate in A53T α-Syn mice, the process of neurodegeneration is inevitable, and we speculate that neurons in older mice may be more vulnerable to damage.
Our study observed there were obvious α-synuclein deposits in the OB, striatum and the SN in A53T α-Syn mice, most of which were soluble and can be degraded by PK.Different from most of the previous studies which did not successfully replicate the degeneration of DAergic neurons in α-synuclein transgenic mice, according to the increase of age, our study found the A53T mice showed statistically signi cant lower climbing ability, decreased TH and reduction in DA release in the striatum at 12m-old age, signi cant loss of DAergic neurons in the SN, decrease in TH and increase in HVA release in the striatum at 16m-old age, as well as increase in DA turnover (HVA/DA) in the striatum at 8m,12m and 16m-old age, compared to age-matched WT mice.The degeneration of DAergic neurons was consistent with the decrease of TH in the striatum at 16m-old age.Longitudinal in ammation activation included NF-кB activation and its downstream cytokines including IL-1β, TNF-α at 8m,12m and 16m-old age compared to age-matched WT mice.These data indicated that α-synuclein deposition alone could cause moderate, but was not su cient to cause serious behavioral de cit and neuronal death.The over-expressed α-synuclein might enhance the sensitivity and decrease the injury threshold of DAergic neurons.We speculated that the αsynuclein gene increased the genetic susceptibility exposure to environmental toxins.
In our further study in intranasal LPS and α-synuclein multi hit mice, in comparison to A53T α-Syn mice, olfactory discrimination showed impaired social-scent discrimination, non-social scent discrimination and impaired odor detection.Motor activity implied a more serious reduction in voluntary movement and climbing ability.Pathological examination further con rmed the dopamine depletion and α-synuclein accumulation in the OB and the nigrostriatal system.The PK-resistant α-synuclein aggregates were formed in the SN.Therefore, in the context of in ammation, these mice displayed a progressive hypokinesia, selective loss of DAergic neurons, and reduction in striatal dopamine DA content, as well as accelerated α-synuclein aggregation in the SN.Furthermore, we observed more obvious NF-кB activation, Nurr1 inhibition, IL-1β and TNF-α generation in microglia in the SN.These changes also behaved in an age-depended fashion.
Chronic in ammation can directly or indirectly contribute to the etiology and progression of PD [35,36].
The causes of in ammation in PD brain may include age advancement, infectious agents (bacteria or viruses), exotoxins (pesticides or MPTP, etc.), or deposition of insoluble protein brils (α-synuclein, etc.).LPS, a potent glial activator, is often suspended in the air as a component of the air pollutant PM2.5 or as part of house dust, which can be inhaled into nose.The inhaled LPS can enter the brain via the olfactory tract and bypass the blood brain barrier to activate the in ammation in the brain and damage the sensitive DAergic neurons in the SN [37,38].
Our previous study also found that intranasal LPS could activate microglia in the OB and SN in mice [28,39].In ammatory activation, including NF-κB and downstream cytokines including IL-1β, IL-6 and TNF-α release are increased in PD brain [40].Recent studies have also found that Nurr1 was expressed in glia and exerted anti-in ammatory effects by inhibiting the transcription of NF-κB target genes in microglia and astrocytes.Nurr1 can suppress the production of TNF-α via interaction with NF-κB and can inhibit its nuclear translocation [41,27].Therefore, inhibition of Nurr1 can lead to the ampli cation of in ammatory responses.Moreover, neuroin ammation has been suggested to induce alteration and aggregation of α-synuclein.It has also been hypothesized that secreted extracellular α-synuclein can immediately activate glial cells and subsequently induce neuronal in ammation [42].Studies have con rmed that degenerative DAnergic neurons could release abnormally aggregated α-synuclein into the intercellular space, which was further phagocyted by microglia, leading to the microglial activation and release of in ammatory mediators, thus further ampli ed the in ammatory reaction by α-synuclein, thereby promoting the internalization and degradation of this protein [43].Further in vivo and in vitro studies showed that aggregated and nitrated α-synuclein induced by in ammation could signi cantly activate microglia to NF-κB activation [44,45].A recent study found intranasal LPS could induce microglial activation, in ammatory cytokine expression in the OB and regulate α-synuclein pathology in the OB, striatum and SN via IL-1β/IL-1R1 signaling [46].Furthermore, NURR1 was found to be downregulated in midbrain DA neurons that express high levels of αsynuclein.Studies con rmed that the expression of α-synuclein and Nurr1 in the DAergic neurons of PD patients was negatively correlated [47,48].Reduction of Nurr1 was also found in both A53T-α-Syn transgenic mice and rat models overexpressed AAV-mediated α-synuclein, which even occurred before the occurrence of pathological changes.α-synuclein could exert a toxic role by reducing the level of Nurr1 [49][50][51].Therefore, α-synuclein probably ampli ed the in ammatory reaction via Nurr1/NF-κB pathway.
In our intranasal LPS-A53T α-Syn multi hit mice, Nurr1 inhibition and NF-κB was found in these mice at all ages.We speculated that Nurr1/NF-κB pathway is involved in the neurovegetative process of the mice.
LPS induced inhibition of Nurr1 and activation of NF-κB caused α-synuclein aggregation.The aggregated α-synuclein further ampli ed the in ammatory reaction.Furthermore, nigral cell death in this model was found to be progressive (from 39% at 4m to 63.7% at 16m), suggesting that a potential relationship between sustained α − synuclein expression and neurotoxicity within the SN.Thus, ongoing neurodegeneration and chronic neuroin ammation cause a vicious self-propelling cycle that makes it possible for neurodegeneration to become a chronic progressive process.

Conclusion
In conclusion, the intranasal LPS-A53T α-Syn mice with PD-related α-synuclein gene defects chronically exposed to subtoxic doses of environmental toxicant LPS successfully implicated the synergistic effects of neuroin ammation (Nurr1/NF-κB pathway is involved in glial activation in the neurovegetative process) and α-synuclein dysfunction in driving chronic PD neurodegeneration.Thus, we replicate the multiple causes of PD involving aging, genetic mutations and exposure to environmental toxins and provided valuable tools for studying the mechanisms of PD progression and developing disease-

Figure 7 Western
Figure 7