Chitotriosidase enhances TGFβ-Smad signaling and uptake of β-amyloid in N9 microglia

Accepted Manuscript Title: Chitotriosidase enhances TGF␤-Smad signaling and uptake of ␤-amyloid in N9 microglia Authors: Xia Wang, Weihua Yu, Xue Fu, Meiling Ke, Qian Xiao, Yang Lu¨ PII: DOI: Reference:

S0304-3940(18)30641-4 https://doi.org/10.1016/j.neulet.2018.09.037 NSL 33826

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

27-5-2018 13-9-2018 20-9-2018

Please cite this article as: Wang X, Yu W, Fu X, Ke M, Xiao Q, Lu¨ Y, Chitotriosidase enhances TGF␤-Smad signaling and uptake of ␤-amyloid in N9 microglia, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.09.037 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.

Chitotriosidase enhances TGFβ-Smad signaling and uptake of βamyloid in N9 microglia Xia Wanga, Weihua Yub, Xue Fua, Meiling Keb, Qian Xiaoa, Yang Lüa, * a

Department of Geriatrics, The First Affiliated Hospital of Chongqing Medical

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University, Chongqing 400016, China Institutes of Neuroscience, Chongqing Medical University, Chongqing 400016,

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China

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* Corresponding author: Prof. Yang Lü,

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Tel: 86-23-89011622

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University, Chongqing 400016, China

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Department of Geriatrics, The First Affiliated Hospital of Chongqing Medical

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Fax: 86-23-68811487

Highlights:

Chitotriosidase acts as an important co-factor of TGFβ1 to augment TGFβ

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E-mail: [email protected]

receptors I expression and activation of Smad signaling in N9 microglia.

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Chitotriosidase enhances TGFβ1-induced uptake of β-amyloid in N9 microglia. Chitotriosidase may be a target for rescuing TGFβ-Smad signaling in Alzheimer's disease.

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Abstract TGFβ-Smad signaling is involved in the modulation of β-amyloid (Aβ) clearance in microglia. This signaling is impaired in the brain of Alzheimer's disease (AD). Chitotriosidase (CHIT1) is elevated in the cerebrospinal fluid and peripheral blood of

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AD patients, and has been reported to augment TGFβ signaling in fibroblast and T cells. In this study, we investigated the role of CHIT1 in TGFβ-Smad signaling and Aβ

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phagocytosis in N9 microglia. We found that CHIT1 significantly enhanced TGFβ1induced expression of TβRI (TGFβ receptor I) and activation of Smad signaling. CHIT1

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did not affect Aβ uptake in microglia by itself, but did enhance TGFβ1-induced

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phagocytosis of Aβ, which was blocked by pretreatment with SB431542 (TβRI

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inhibitor). These results indicate that CHIT1 may play a protective role in Aβ clearance

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by enhancing TGFβ signaling in microglia.

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Keywords: chitotriosidase, TGFβ1, microglia, Smad signaling

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1. Introduction β-amyloid (Aβ) is widely recognized as a central pathogenic factor of Alzheimer's disease (AD) via neurotoxic and inflammatory effects. Microglia, the resident phagocytes of the central nervous system, participate in the phagocytosis and

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proteolytic clearance of both soluble and fibrillary forms of Aβ [1]. Therefore, microglial recruitment promotes Aβ clearance and is neuroprotective in AD.

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Transforming growth factor-β1 (TGFβ1) plays a neuroprotective role in AD by

increasing Aβ uptake and decreasing Aβ-induced production of TNFα and nitric oxide

pathway is

impaired

in

AD

by

factors

that

include

Aβ,

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TGFβ-Smad

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in microglia. This process is mainly mediated by the Smad pathway [2-5]. However,

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hyperphosphorylated tau, aging, and persistent inflammatory activation [6-8]. The

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impairment of the TGFβ-Smad pathway has a negative effect on the neuroprotection of

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TGFβ1. Chitotriosidase (CHIT1) has been reported to interact with TGFβ1 to augment TGFβ receptor I (TβRI) expression and TGFβ signaling in fibroblasts and T cells [9,10].

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CHIT1 thus may be a target for rescuing TGFβ-Smad signaling in AD.

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CHIT1 is a member of the glycosyl hydrolase family 18, and is mainly synthesized by activated macrophages, including microglia [11,12]. CHIT1 is

mainly secreted as

a 50-kDa active enzyme, but some of it is processed to a 39-kDa form that accumulates

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in lysosomes [13]. Elevated levels of CHIT1 expression have been observed in the peripheral blood of AD patients [14]. The activity of CHIT1 is also increased in the cerebrospinal fluid (CSF) of patients with AD compared to that in controls and patients with mild cognitive impairment [15]. Moreover, the level of CHIT1 is increased in the

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brain of APP/PS1 double transgenic mice in a time-dependent manner [16]. However, the exact function of CHIT1 in AD has not been fully defined. This study was undertaken with the hypothesis that CHIT1 augments the TGFβ-Smad signaling

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pathway and has an impact on the phagocytosis of Aβ in microglia.

2. Materials and Methods

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2.1. Cell culture and treatments

N9 microglia share many phenotypical characteristics with primary microglia, and

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have been widely used to investigate the Aβ-mediated inflammatory response in AD.

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Furthermore, N9 microglia possess phagocytic capability [17]. The murine microglial

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cell line N9 was a gift from the Department of Anesthesia, the Affiliated Children’s

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Hospital of Chongqing Medical University. Cells were grown in Dulbecco’s modified

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Eagle medium: Nutrient Mixture F-12 (DMEM/F12; Hyclone, USA) supplemented with 10% fetal calf serum (Hyclone, USA) and 1% penicillin/streptomycin (Hyclone,

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USA) at 37 °C in a humidified 5% CO2 atmosphere.

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Microglia were randomly divided into five groups. The control group was cultured with serum-free medium. The TGFβ1 group was treated with 1 ng/ml recombinant human TGFβ1 (PeproTech, USA) as described previously [3]. The CHIT1 group was

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treated with 100 ng/ml recombinant mouse CHIT1 (R&D Systems, USA) as described previously [10]. The TGFβ1+CHIT1 group was treated with 1 ng/ml TGFβ1 and 100 ng/ml CHIT1. The SB431542+TGFβ1+CHIT1 group was pretreated with 10 μM

SB431542 (TβRI inhibitor; Selleck, USA) for 1 h, followed by treatment with 1 ng/ml

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TGFβ1 and 100 ng/ml CHIT1. After 24 h incubation at 37 °C, total RNA and protein of were isolated.

2.2. RNA isolation and quantitative RT-PCR

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RNA was extracted from N9 microglia using RNAiso Plus (Takara Bio, Japan), and reverse transcribed to cDNA with the PrimeScript®RT kit (Takara Bio, Japan)

5’-TTATGAGAGAATGCTGGTATGCC-3’ (forward),

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according to the manufacturer’s instructions. The following primers were used: TβRⅠ: 5’-CCTTCCTGTTGGCTGA

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GTTGT-3’ (reverse); GAPDH: 5’-GTGCTGAGTATGTCGTGGAGTCT-3’ (forward),

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5’-AGTCTTCTGGGTGGCAGTGA-3’ (reverse). For PCR, 1 μl cDNA of each sample

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and 5 μl SYBR Green (Bimake, USA), 0.5 μl forward primer (10 μM), and 0.5 μl

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reverse primer (10 μM) were added to the reaction system and diluted with RNase-free

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water to 10 μl. The cycling conditions were 95 °C for 30 s, 50 cycles of 95 °C for 5 s, and 60 °C for 45s. The comparative Ct for quantitative gene expression of TβRI and

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GAPDH was analyzed using Bio-Rad CFX Manager. The results were calculated using

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the 2[-ΔΔCT] equation.

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2.3. Western blot Total proteins from N9 microglia were isolated using radioimmunoprecipitation

lysis buffer (Beyotime, China) supplemented with phosphatase and protease inhibitors (Beyotime, China) at 4 °C. The supernatant and debris were collected and centrifuged at 12000 rpm for 15 min to obtain the protein containing supernatant. Proteins were

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mixed with loading buffer, boiled for 5 min, and then maintained at -20 °C until use. Equal amounts of proteins (20-30 μg) were electrophoretically separated by 10% SDSPAGE and transferred onto 0.22-μm polyvinylidene difluoride membranes. Blots were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline containing 0.05 Tween-

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20 (TBST) at room temperature, and incubated with primary antibodies at 4 °C overnight. Primary antibodies against TβRI (1:200, Santa Cruz Biotechnology, USA),

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Smad2/3 (1:1000, Cell Signaling Technology, USA), phospho-Smad2/3 (1:1000, Cell

Signaling Technology, USA), and GAPDH (1:500, Hangzhou Goodhere Biotechnology,

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China) were used (Table 1). The membranes were then incubated with goat anti-rabbit

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IgG horseradish peroxidase-conjugated secondary antibody (1:5000, ABclonal, USA)

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for 1 h at 37 °C. The bands were visualized by enhanced chemiluminescence substrate

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(Beyotime, China), photographed, and analyzed using a Fusion-FX7 imaging system

2.4. Aβ uptake assay

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(Vilber Lourmat, Marne-la-vallée, France).

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Carboxyfluorescein (FAM)-labeled Aβ1-40 (Anaspec, USA) was initially

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resuspended in 1% NH4OH and then phosphate buffered saline (PBS) was added to 1mg/ml. The mixture was incubated at 37 °C for 12 h to promote fibril formation, as

previously described [18]. Following incubation, the mixture was diluted in

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DMEM/F12 medium to 1 ug/ml before it was applied to N9 cells. After 24 h stimulation with 1 ng/ml TGFβ1 and/or 100 ng/ml CHIT1, with or without pretreatment with 10 μM SB431542 for 1 h, cells were rinsed and incubated with FAM-labeled fAβ1-40 in DMEM/F12 for 3 h. The cells were washed with PBS and fixed with 4%

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paraformaldehyde at room temperature for 15 min. The fixed cells were stained with 4', 6-diamidino-2-phenylindole at room temperature for 5 min. After washing and mounting, Aβ uptake were visualized using a PM20 automatic microscope (Olympus, Japan), and images were obtained. Aβ1-40 average signal intensity in individual cells

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were quantified and expressed as fold change compared with control cells (at least 30

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cells were scored per group).

2.5. Statistical analyses

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Statistical analyses were performed using SPSS 19.0 software (SPSS, Inc., USA).

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Data represent the mean ± standard deviation (SD) of at least three independent

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experiments. One-way analysis of variance (ANOVA) followed by Turkey’s post hoc

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test were used to test the difference among different groups. A P-value < 0.05 was

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3. Results

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considered statistically significant.

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3.1. Effect of CHIT1 and TGFβ1 on TβRI and Smad signaling We examined the expression of TβRI in N9 microglia after different treatments.

The qRT-PCR and western blot examinations demonstrated that both mRNA and

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protein levels of TβRI significantly increased in TGFβ1 treated N9 microglia compared to that in the control group (P<0.05). CHIT1 treatment alone had no significant impact on the expression of TβRI, while co-treatment with CHIT1 and TGFβ1 resulted in a significant increase in the mRNA and protein levels of TβRI compared to that in the

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TGFβ1 treatment group (P<0.05). In contrast, when cells were pretreated with SB431542, an inhibitor of TβRI, the expression of TβRI recovered

to a significantly

lower level (P<0.05; Fig. 1) We next investigated the effects of CHIT1 treatment on Smad signaling in N9

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microglia. Smad2/3 and pSmad2/3 proteins were expressed (Fig. 2). The levels of total Smad2/3 were not altered by different treatments. TGFβ1 treatment resulted in

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increased protein levels of pSmad2/3 when compared to the control group (P<0.05), while CHIT1 by itself did not have a significant effect on the protein levels of pSmad2/3

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(P>0.05). However, in the presence of TGFβ1, CHIT1 significantly enhanced TGFβ1-

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induced pSmad2/3 protein expression (P<0.05), which was inhibited by pretreatment

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with SB431542 (P<0.05).

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3.2. Effect of CHIT1 and TGFβ1 on the uptake of Aβ We then examined the functional effects of TGFβ1 and CHIT1 on the phagocytic

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potential of N9 microglia. Microscopy analysis showed that TGFβ1 treatment led to

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significantly increased internalization of fAβ1-40 in microglia compared to that in the control microglia (P<0.05). CHIT1 treatment alone did not affect fAβ1-40 uptake by microglia. However, TGFβ1 and CHIT1 co-treatment significantly increased TGFβ1-

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induced phagocytosis of fAβ1-40 (P<0.05). The TβRI inhibitor SB431542 reduced induction of phagocytosis by TGFβ1 and CHIT1 (P<0.05; Fig. 3).

4. Discussion

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The present results demonstrate that CHIT1 acts as an important co-factor of TGFβ1 to augment TβRI expression and Smad signaling in microglia. Furthermore, we demonstrate that CHIT1 enhances the TGFβ1-induced uptake of Aβ by microglia. CHIT1 is the best-characterized true chitinase that binds and cleaves chitin. It

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contains both a chitin-binding domain and an enzymatically active domain that catalyze the hydrolysis of β (1→4) glycosidic bonds in carbohydrate [19]. CHIT1 plays a pivotal

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role in the defense against chitin-containing human pathogens, such as fungi and insects [20]. CHIT1 is upregulated during both innate and acquired immunity, and has been

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implicated in the activation and polarization cascades of macrophages [21]. Chitinase

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enzyme activity in CSF is powerful as a single marker to discreminate patients with AD

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from those without dementia, even in comparison with standard clinical markers like

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tau and Aβ42, and combined analysis of chitinase with other markers reportedly

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increases the accuracy to a maxium of 91% [22]. Our previous study shows that CHIT1 is beneficial in AD. For it can polarize microglia into an M2 phenotype and resist the

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deposition of Aβ oligomers [23]. CHIT1 may play important roles in the diagnosis and

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treatment of AD. A recent study correlated CSF CHIT1 concentrations with amyotrophic lateral sclerosis disease progression and severity, but not survival time [24]. However, whether the levels of CSF CHIT1 correlate with AD disease progression

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or prognosis is unknown, and additional studies are required. Our study shows that CHIT1 interacts with TGFβ1 to increase TβRI expression

and activation of Smad signaling in microglia. A recent study shows that CHIT1 enhances TGFβ-dependent regulatory T cells conversion by increasing T cell TβRI

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expression and signaling [10]. These results indicate that CHIT1 acts as a co-factor of TGFβ1 in different cells. Impairment of TGFβ-Smad signaling can compromise the neuroprotective role of TGFβ1 in AD. Thus CHIT1 may be beneficial in AD by rescuing TGFβ-Smad signaling. The expression of CHIT1 and TGFβ1 is increased in

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the CSF of AD patients [15,25], which may be a compensatory response to the impaired Smad signaling. However, the increased production of CHIT1 and TGFβ1 is not

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sufficient to prevent the progression of neurodegeneration in AD. Therefore, overexpression of CHIT1 in AD may be a target for treatment.

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In the present study, we further demonstrate that CHIT1 alone is not able to affect

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the uptake of Aβ in N9 microglia. Multiple cell surface receptors are involved in Aβ

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uptake by microglia, including scavenger receptors A (SR-A) and CD36 [26,27].

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CHIT1 overexpression has no effect on the expression of SR-A and CD36 in

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macrophages [11]. These results indicate that CHIT1 itself has no effect on scavenger receptor expression and Aβ phagocytosis. However, co-treatment with CHIT1 and

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TGFβ1 significantly enhanced the phagocytosis of Aβ in N9 microglia, which was

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inhibited by the TβRI inhibitor SB431542. These results indicate that CHIT1 can augment TGFβ1-induced Aβ phagocytosis by microglia, which is partially mediated by

TGFβ signaling. TGFβ1 stimulation can increase the expression of SR-A through a

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Smad3-dependent mechanism in microglia [5]. Therefore, it is reasonable to suppose

that CHIT1 may act synergistically with TGFβ1 to enhance the expression of SR-A through the Smad3 pathway, thus increasing the uptake of Aβ. Promotion of Aβ clearance by microglia may be a target for AD treatment. The

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present data show that CHIT1 and TGFβ1 play a positive role in Aβ clearance via microglia, and hence may have a neuroprotective effect in AD. We previously described that chitinase may weaken the deposition of Aβ oligomers in the brain of AD rats by reducing chitin [23]. Chitin is an important component of the highly insoluble amyloid

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fibrils that characterize AD [28]. Chitin or chitin-like polysaccharides may provide scaffolding for protein accumulation and fibril formation, and increased CHIT1 activity

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may protect the brain from the deposition of chitin-like substance and formation of

amyloid fibrils. As Aβ accumulation in AD brains is a result of both excessive

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production and impaired clearance, reduction in Aβ production and promotion of Aβ

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clearance are possible treatment strategies of AD. Therefore, CHIT1 may play a

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neuroprotective role in AD by increasing the clearance and decreasing the deposition

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of Aβ.

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The limitation of our study is that we only studied the role of CHIT1 in vitro. Further studies should aim to determine the effects of CHIT1 and TGFβ1 on mouse

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models of AD. We speculate that the knock-out of CHIT1 in such models may have a

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negative effect on TGFβ-Smad signaling and further impair the cognitive function of AD mice, while overexpression of CHIT1 may have a protective role against AD. In conclusion, we have shown that CHIT1 interacts with TGFβ1 to increase Aβ

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clearance by microglia which is partially mediated by the enhancement of TβRI expression and activation of Smad signaling. Thus, CHIT1 might be neuroprotective in AD.

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Funding This study was supported by grants from Key-project of Social undertakings and people's livelihood security of Chongqing Science & Technology Commission (cstc2017shms-zdyfX0009), Sub-project under Science and Technology Benefit Plan

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of Chongqing Science & Technology Commission (cstc2015jcsf10001-01-01),

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National Key Clinical Specialties Construction Program of China (No. [2013]544).

Disclosure statement

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The authors declare no conflict of interest.

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Table 1. Primary antibodies for western blots manufacturer

Rabbit polyclonal

Santa Cruz Biotechnology,

antibody TβRI

concentration

time

temperature

1:200

24h

4 °C

1:1000

24h

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Primary antibodies

USA

antibody Smad2/3

USA

Rabbit monoclonal

Cell Signaling Technology,

antibody P-Smad2/3 Rabbit polyclonal

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Cell Signaling Technology,

1:1000

24h

4 °C

24h

4 °C

USA Hangzhou Goodhere

A

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PT

ED

M

A

Biotechnology, China

N

1:500

antibody GAPDH

18

4 °C

U

Rabbit monoclonal

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Figures

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Fig. 1. CHIT1 enhanced TGFβ1-induced TβRI expression (a) qRT-PCR showed increased TβRI

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mRNA levels in N9 microglia after TGFβ1 treatment. Co-treatment with TGFβ1 and CHIT1

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significantly increased TβRI mRNA levels, which were inhibited by pretreatment with SB431542.

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(b) Western blotting showed increased TβRI protein levels in N9 microglia after TGFβ1 treatment. Co-treatment with TGFβ1 and CHIT1 increased protein levels of TβRⅠ, which were inhibited by

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pretreatment with SB431542. *P<0.05.

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IP T SC R U N A

M

Fig. 2. CHIT1 enhanced TGFβ-Smad signaling. (a) Western blotting showed the levels of Smad2/3

ED

were not altered in N9 microglia by different treatments. However, increased pSmad2/3 protein levels were observed after TGFβ1 treatment. Co-treatment with TGFβ1 and CHIT1 increased

PT

protein levels of pSmad2/3, which were inhibited by pretreatment with SB431542. (b-c)

CC E

Quantification of western blots of Smad2/3 and pSmad2/3 expression. Data were given as means ±

A

standard error from three independent experiments, *P<0.05.

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IP T SC R U N A M ED PT CC E

Fig. 3. CHIT1 enhanced TGFβ1-induced uptake of fAβ1-40 by microglia. (a) TGFβ1 increased fAβ1by microglia. Co-treatment with TGFβ1 and CHIT1 increased TGFβ1-induced uptake of

A

40 uptake

fAβ1-40. Pretreatment with SB431542 inhibited the induction of phagocytosis by TGFβ1 and CHIT1.

FAM-labeled fAβ1-40 (green), DAPI (blue). Scar bar=50 μm (b) Quantification of fAβ1-40 average signal intensity in individual cells and expressed as fold change compared with control cells, *P<0.05. 21