Neuregulin-1 protects mouse cerebellum against oxidative stress and neuroinflammation

Brain Research 1670 (2017) 32–43

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Neuregulin-1 protects mouse cerebellum against oxidative stress and neuroinflammation Junping Xu 1,2, Chengliang Hu 1,2, Shuangxi Chen 1,2, Huifan Shen 3, Qiong Jiang 4, Peizhi Huang 5, Weijiang Zhao 6,⇑ Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road, Shantou, Guangdong 515041, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 22 December 2016 Received in revised form 1 June 2017 Accepted 9 June 2017 Available online 13 June 2017 Keywords: Neuregulin-1b pErbB4 pErbB2 Oxidative stress Neuroinflammation

a b s t r a c t Cerebellum undergoes degenerative changes in neurodegenerative diseases. Two main factors including oxidative stress and neuroinflammation mediate neurodegeneration. Neuregulin-1 (Nrg1) has been implicated in many neurodegenerative diseases, while the underlying mechanisms are unknown. We hypothesized that Nrg1 prevents oxidative stress and neuroinflammation in neurodegeneration. We found a positive correlation between Nrg1 protein levels and ErbB4 and ErbB2 receptor phosphorylation in microarrays of normal human cerebellar tissue. In addition, Nrg1 was also co-localized with pErbB4 and pErbB2. Primary mouse cerebellar granule neurons (CGNs) were treated with H2O2 or LPS combined with recombinant Nrg1b (rNrg1b). Western blot analysis and immunofluorescence revealed that H2O2 and LPS-induced neuronal toxicity down-regulated the activation of ErbB receptors and Akt1, and the ratio of Bcl2/Bax, which was reversed by rNrg1b. In vivo studies showed that LPS-induced neuroinflammation in mouse cerebellum down-regulated pErbB4, pErbB2, pAkt1/Akt1 and Bcl2/Bax levels, whereas rNrg1b treatment reversed the changes. Immunohistochemistry and Western blot analysis showed that rNrg1b alleviates neuroinflammation by reducing the number of microglial cells and astrocytes and the expression of IL1b. Our results indicate that Nrg1 protects against oxidative stress and neuroinflammation in mouse cerebellum, suggesting potential therapeutic application in neuroinflammation associated with neurodegenerative diseases. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic Abbreviations: Nrg1, neuregulin-1; rNrg1b, recombinant Nrg1b; ErbB, epidermal growth factor receptor; LPS, lipopolysaccharides; CGNs, cerebellar granule neurons; AD, Alzheimer’s disease; PD, Parkinson’s disease; HD, Huntington’s disease; ALS, amyotrophic lateral sclerosis; SCA, spinocerebellar ataxia; Iba-1, ionized calcium binding adapter molecule 1; GFAP, glial fibrillary acidic protein; IL1b, interleukin 1b; TNFa, tumor necrosis factor a. ⇑ Corresponding author. E-mail addresses: [email protected] (J. Xu), [email protected] (C. Hu), [email protected] (S. Chen), [email protected] (H. Shen), [email protected] (Q. Jiang), [email protected] (P. Huang), [email protected] (W. Zhao). 1 Contributed equally. 2 Conceived and designed the experiments, performed the experiments, analyzed the data, contributed to reagents/materials/analysis tools, drafting the article and revising it critically and approving the final version to be published. 3 Performed the experiments and contributed reagents/materials/analysis tools. 4 Analyzed the data and contributed reagents/materials/analysis tools. 5 Performed the experiments, analyzed the data. 6 Conceived and designed the experiments, revised the article critically and approved the final version to be published. http://dx.doi.org/10.1016/j.brainres.2017.06.012 0006-8993/Ó 2017 Elsevier B.V. All rights reserved.

lateral sclerosis (ALS) and spinocerebellar ataxia (SCA) increase the healthcare burden worldwide (Mehta et al., 2016; Weuve et al., 2014). Neurodegenerative disorders are characterized by progressive loss of neuronal structure or function. The etiology of neuronal degeneration is poorly understood. The common pathological events include oxidative stress, neuroinflammation, mitochondrial dysfunction, neuronal apoptosis, protein aggregation and autophagy (Bhat et al., 2015; Ntsapi and Loos, 2016; Okouchi et al., 2007; Ross and Poirier, 2005). Oxidative stress and neuroinflammation are the two main factors underlying neurodegeneration (Carri et al., 2015; Fischer and Maier, 2015; Heneka et al., 2015). Oxidative stress is characterized by excessive production of reactive oxygen species (ROS) and lack of clearance, and is known to damage lipids, proteins and DNA, triggering excitotoxicity, and neuronal loss as well as axonal damage. In AD, the Ab-induced neuronal damage accelerates the generation of ROS, contributing to neurodegeneration, and inflammation by glial recruitment (Chiurchiu et al., 2016). Oxidative stress also leads to PD, ALS, and other diseases (Ganesan et al., 2015; Ghadge et al., 1997).

J. Xu et al. / Brain Research 1670 (2017) 32–43

Chronic neuroinflammation is characterized by ongoing activation of microglia and astrocytes as well as the release of proinflammatory mediators. The activation of microglial cells around Ab has been reported in AD (Itagaki et al., 1989). Proinflammatory cytokines IL1b and TNFa are increased In substantia nigra (SN) of PD patients (Hunot et al., 1999). Inflammation has been considered one of the pathological characteristics of HD (Moller, 2010). It is reasonable to assume that anti-oxidative and anti-inflammatory treatments may represent an appropriate strategy against neurodegenerative diseases. Previous studies indicate that the cerebellum undergoes degenerative changes in AD, PD, ALS and HD (Burciu et al., 2015; Colloby et al., 2014; Tan et al., 2014; Wolf et al., 2015). Cerebellar granule neurons (CGNs) account for approximately 50% of the total number of neurons in the brain. Primary cultured CGNs have been widely used to study neuronal apoptosis in neurodegenerative disorders (Chen et al., 2009). Nrg1, the best characterized signaling protein of neuregulin family, has been implicated in neuronal migration, proliferation and differentiation (Erickson et al., 1997; Rio et al., 1997; Sanes and Lichtman, 2001; Vartanian et al., 1999). Nrg1 proteins contain an epidermal growth factor (EGF)-like domain (type a or b) (Falls, 2003). Nrg1b subtype is the most prominent Nrg1 in the nervous system. Nrg1 acts via receptor tyrosine kinases of the ErbB family, namely ErbB2 (Neu) (Schechter et al., 1984; Zhao and Schachner, 2013), ErbB3 (Kraus et al., 1989) and ErbB4 (Plowman et al., 1993). ErbB2 and ErbB4 are the two main receptors expressed in the mammalian central nervous system. Although ErbB2 fails to bind Nrg1, it forms ErbB2/ErbB4 complex. ErbB4 is activated by Nrg1 through either the homodimer ErbB4/ErbB4 or the heterodimer ErbB2/ErbB4 complex. Nrg1 has been investigated in schizophrenia, which is partially characterized by oxidative stress (Boskovic et al., 2011). Growing evidence supports the therapeutic role of Nrg1 in neurodegenerative diseases such as PD (Carlsson et al., 2011), AD (Jiang et al., 2016; Ryu et al., 2012) and ALS (Song et al., 2012). Nrg1 protects myocardial cells against oxidative stress (Xu et al., 2014) and plays a neuroprotective role against inflammatory responses in acute brain injuries (Li et al., 2015). These studies indicate that Nrg1 may be neuroprotective against oxidative stress and neuroinflammation via unknown mechanisms. In the present study, we found that Nrg1-ErbB exerted neuroprotective effect by inhibiting H2O2induced oxidative stress and LPS-induced neuroinflammation in the cerebellum. 2. Results 2.1. Nrg1 expression is positively correlated with phosphorylation of ErbB4 and ErbB2 in a human cerebellar tissue microarray Co-localization of Nrg1 with either pErbB4 or pErbB2 was used to search for a possible interaction between the Nrg1 protein levels and ErbB4 or ErbB2 phosphorylation. Each section revealing immunofluorescence staining of Nrg1 with either pErbB4 or pErbB2 was presented (Fig. 1A and C). We observed a significant correlation between Nrg1 and pErbB4 (r2 = 0.925, p < 0.0001; Fig. 1B) or Nrg1 and pErbB2 (r2 = 0.849, p < 0.0001; Fig. 1D). 2.2. Nrg1 and pErbB4, and Nrg1 and pErbB2 are co-localized in a human cerebellar tissue microarray To evaluate the expression and potential co-localization of Nrg1 with either pErbB4 or pErbB2, co-immunostaining of Nrg1 with either pErbB4 or pErbB2 was performed in 16 human cerebellar tissues. Representative immunofluorescence images of Nrg1 and

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pErbB4 and pErbB2 are presented. Nrg1 and pErbB4 (Fig. 2A) or Nrg1 and pErbB2 (Fig. 2B) co-localize in CGNs at each tissue point. 2.3. rNrg1b activates Nrg1/ErbB signaling in cultured CGNs Western blot was conducted to investigate the role of rNrg1b in ErbB4 and ErbB2 phosphorylation after the cultured CGNs were treated with rNrg1b at concentrations ranging from 0 to 10 nM for 48 h (Liu et al., 2013; Yang et al., 2016b). Compared with the control group, ErbB4 and ErbB2 phosphorylation levels were increased in a dose-dependent manner with increasing concentration of rNrg1b. The maximal effect of rNrg1b on ErbB4 phosphorylation was observed at 5 nM (F(5,12) = 5.578, p < 0.01; Table 1A) and the maximal effect of rNrg1b on ErbB2 phosphorylation was observed at 10 nM (F(5,12) = 5.928, p < 0.01; Table 1A) compared with the vehicle control (Fig. 3A). We also observed that rNrg1b increased Akt1 phosphorylation levels, although the difference did not reach statistical significance (F(5,12) = 2.592, p = n.s.; Table 1A, Fig. 3B). These results indicate that Nrg1 activated ErbB4, ErbB2 in the CGNs. 2.4. rNrg1b protects cultured CGNs against H2O2-induced oxidative stress Western blot and immunofluorescence staining were used to analyze the phosphorylation levels of ErbB2, ErbB4 receptors and Akt1 in the primary cultured CGNs in response to treatment with 20 mM H2O2 for 2 h followed by rNrg1b treatment for 48 h. We found that compared with the vehicle control, the levels of pErbB4, pErbB2 and pAkt1/Akt1 were significantly reduced in cells treated with H2O2 (pErbB4: p = 0.02; pErbB2: p = 0.018; and pAkt1/Akt1: p = 0.034; respectively, versus vehicle control. Table 1B), which was reversed by rNrg1b treatment. No significant differences were found for pErbB4 (F(5,18) = 0.986, p = n.s.) whereas significant increase was observed for pErbB2 (F(5,18) = 5.513, p < 0.01; Table 1B). Similarly, phosphorylation of Akt1 was significantly increased (F(5,12) = 6.798, p < 0.01; Table 1B, Fig. 4A,B,D,E). Bcl2/ Bax ratio was significantly decreased in cells treated with H2O2 (p = 0.007 versus vehicle control). After treatment with rNrg1b, Bcl2/Bax was increased, although no significant differences were detected (F(5,12) = 1.998, p = n.s.; Table 1B, Fig. 4C). These results indicate that Nrg1 played a protective role against H2O2-induced oxidative stress through Akt1 activation and the increased ratio of Bcl2/Bax. 2.5. rNrg1b attenuates LPS-induced neurotoxicity in cultured CGNs CGNs were treated with rNrg1b at concentrations ranging from 0 to 10 nM, followed by addition of 1 mg/mL LPS 2 h later, for 48 h. Western blot and immunofluorescence staining were used to analyze the phosphorylation levels of ErbB2, ErbB4 and Akt1. We found that the levels of pErbB2 and pAkt1/Akt1 were significantly decreased (pErbB2: p = 0.025; pAkt1/Akt1: p = 0.001; respectively, versus vehicle control) and pErbB4 levels were significantly increased (p = 0.028 versus vehicle control) in response to LPS treatment. However, ErbB4, ErbB2 and Akt1 phosphorylation levels were significantly up-regulated in the cultured CGNs treated with rNrg1b (for pErbB4, F(5,12) = 6.026, p < 0.01; for pErbB2, F(5,12) = 16.396, p < 0.01; for pAkt1/Akt1, F(5,12) = 11.540, p < 0.01. Table 1C). Bcl2/Bax ratio was significantly decreased in response to LPS treatment (p = 0.016 versus vehicle control). Bcl2/Bax was slightly increased after treatment with rNrg1b, although no significant differences were detected (F(5,12) = 2.353, p = n.s.; Table 1C, Fig. 5C). These results indicate that Nrg1 protected CGNs against LPS-induced cytoxicity through Akt1 activation and the increased ratio of Bcl2/Bax.

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Fig. 1. Integrated fluorescence intensity (IFI) analyses of Nrg1 and pErbB4 or Nrg1 and pErbB2 in a human cerebellar tissue microarray. Nrg1 and pErbB4 (A), Nrg1 and pErbB2 (C) immunofluorescence staining of each tissue point; linear regression graph depicting the interaction between Nrg1 and pErbB4 (B) or Nrg1 and pErbB2 (D). The corresponding correlation coefficients (r) are presented.

2.6. rNrg1b activates Nrg1/ErbB signaling in LPS-induced neuroinflammation in mouse cerebellum

2.7. rNrg1b partly reverses LPS-induced neuroinflammation in mouse cerebellum

We injected LPS intraperitoneally with or without Nrg1b. Nrg1, pErbB4, pErbB2 and Akt1 phosphorylation levels in the cerebellum were detected using a Western blot. Five isoforms, namely 123, 70, 44, 36 and 33 kDa Nrg1 were detected. The 36 kDa isoform was significantly reduced (0.64 ± 0.10, p = 0.024 versus vehicle control) and the 123 kDa was significantly increased (1.17 ± 0.06, p = 0.04 versus vehicle control) in LPS-treated mice, there were no significant differences in 70 (1.15 ± 0.15), 44 (1.19 ± 0.11) and 33 (0.85 ± 0.12) kDa isoforms compared with vehicle control (Fig. 6A). The 33 kDa isoform was significantly increased after administration of rNrg1b (1.26 ± 0.18, p = 0.03 versus LPS group; Fig. 6A). rNrg1b caused no significant changes of 123 (1.25 ± 0.48), 70 (1.3 ± 0.30), 44 (1.56 ± 0.36) and 36 (0.97 ± 0.20) kDa isoforms compared with LPS-treated mice. The ErbB4 and Akt1 phosphorylation levels were significantly reduced in LPS-treated mice (pErbB4/ErbB4:0.67 ± 0.10, p = 0.03; pAkt1/Akt1: 0.68 ± 0.16, p = 0.039; respectively, versus vehicle control; Fig. 6B and C), there was no significant difference in pErbB2/ErbB2 level (0.68 ± 0.20) compared with vehicle control. The reduction of pErbB4/ErbB4 and pAkt1/Akt1 levels was reversed significantly after administration of rNrg1 (pErbB4/ErbB4: 0.93 ± 0.09, p = 0.028; and pAkt1/ Akt1: 1.32 ± 0.31, p = 0.033; respectively, versus LPS group; Fig. 6B and C). A significant decrease in Bcl2/Bax ratio in response to LPS treatment was observed (0.30 ± 0.11, p = 0.008 versus vehicle control), which could be fully reversed by rNrg1b treatment (1.38 ± 0.47, p = 0.018 versus LPS group; Fig. 6D). These results indicate that chronic inflammation inhibits Akt1 activation, whereas Nrg1b can inhibit the effect of LPS in cerebellum.

The mouse cerebellum intraperitoneally injected with of LPS with or without Nrg1b was stained immunohistochemically for Iba-1, GFAP, IL1b and TNFa. The levels of Iba-1, GFAP and IL1b were increased in the cerebellum of mice with LPS-induced neuroinflammation, which was reversed by Nrg1b treatment, (Fig. 7A-F). By contrast, no apparent changes in TNFa were observed (Fig. 7G-H). The number of Iba-1 positive cells was increased in LPS treated mice (593 ± 130) compared with control (146 ± 42, p = 0.005), and reduced in Nrg1b treated mice (333 ± 81, p = 0.042 versus LPS group; Fig. 7I). Western blot were used to analyze the protein levels of IL1b and TNFa. We found that the level of IL1b was significantly increased after LPS treatment (4.33 ± 0.54, p = 0.009 versus vehicle control). However, IL1b level was significantly down-regulated after treatment with Nrg1b (2.55 ± 0.93, p = 0.045 versus the group treated with LPS; Fig. 7J). No significant changes were found for TNFa levels in both groups (Fig. 7J). These results indicate that Nrg1b attenuated the activation of microglia and astrocytes in chronic neuroinflammation. 3. Discussion The aim of this study was to evaluate the neuroprotective effect and mechanism of exogenous rNrg1b against neuronal oxidative stress and neuroinflammation in mouse cerebellum. Multiple assessments demonstrate concentration-dependent neuroprotective effects of rNrg1b against H2O2-induced neuronal oxidative stress and LPS-induced apoptosis. These results indicate that rNrg1b-induced phosphorylation of Nrg1 receptors (ErbB4 and

J. Xu et al. / Brain Research 1670 (2017) 32–43

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Fig. 2. Representative immunofluorescence staining of Nrg1 and pErbB4 (A); and Nrg1 and pErbB2 (B) in the human cerebellar tissue microarray. Co-staining of Nrg1 (green) with pErbB4 or pErbB2 (red) in the cerebellar granule neurons in the human cerebellar tissues; scale bar represents 20 mm.

ErbB2) may activate Akt1 signaling pathways, which are linked to neuronal cell survival and protection. To elucidate the protective role of Nrg1/ErbB signaling in the cerebellum in neurodegenerative disease, we first investigated the co-localization of Nrg1 with either pErbB4 or pErbB2 on the human cerebellar tissue microarray. We observed co-localization of Nrg1, as well as linear correlation of Nrg1 with the phosphorylation of ErbB4 and ErbB2 receptors in human cerebellar tissue

microarray, suggesting a positive interaction between Nrg1 and pErbB4/pErbB2 in the cerebellum. Consistent with previous studies (Dang et al., 2016), we demonstrated that Nrg1b activates ErbB4 and ErbB2 receptors as well as Akt1 both in vitro and in vivo. Akt1 plays a key role in reducing apoptosis, neuronal proliferation and migration. Nrg1 mediates neurodevelopment via Akt1 signaling pathway (Fukazawa et al., 2003; Guo et al., 2010). Nrg1 activates Akt1 by binding to ErbB4/

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Table 1 Duration of pErbB4, pErbB2, pAkt1/Akt1 and Bcl2/Bax for rNrg1b (A), H2O2 with or without rNrg1b (B), and LPS with or without rNrg1b (C) treated groups. A pErbB4 pErbB2 pAkt1/ Akt1 B pErbB4 pErbB2 pAkt1/ Akt1 Bcl2/Bax C pErbB4 pErbB2 pAkt1/ Akt1 Bcl2/Bax

Vehicle control

rNrg1b 0.5 nM

rNrg1b 1 nM

rNrg1b 2.5 nM

rNrg1b 5 nM

rNrg1b 10 nM

n

1 ± 0.00 1 ± 0.00 1 ± 0.00

2.32 ± 0.31 1.33 ± 0.23 1.93 ± 0.65

3.43 ± 0.43 1.66 ± 0.58 2.58 ± 1.38

3.88 ± 0.99* 1.81 ± 0.55 2.78 ± 0.48

4.45 ± 1.68** 2.42 ± 0.69 2.74 ± 0.83

4.21 ± 1.24* 3.51 ± 1.13** 2.97 ± 0.79

3 3 3

Vehicle control

H2O2

H2O2 + rNrg1b0.5 nM

H2O2 + rNrg1b1 nM

H2O2 + rNrg1b2.5 nM

H2O2 + rNrg1b5 nM

H2O2 + rNrg1b10 nM

n

1 ± 0.00 1 ± 0.00 1 ± 0.00

0.55 ± 0.20# 0.64 ± 0.15# 0.52 ± 0.16#

1.03 ± 0.53 1.01 ± 0.53 0.66 ± 0.28

1.23 ± 1.00 1.31 ± 0.25 1.11 ± 0.10

1.40 ± 0.61 1.76 ± 0.64 1.48 ± 0.56

1.20 ± 0.54 1.91 ± 0.55* 2.24 ± 1.02

1.06 ± 0.11 2.37 ± 0.81** 2.96 ± 0.95**

4 4 3

1 ± 0.00

0.42 ± 0.09#

0.66 ± 0.17

0.68 ± 0.35

0.85 ± 0.26

1.05 ± 0.51

1.04 ± 0.24

3

Vehicle control

LPS

LPS + rNrg1b0.5 nM

LPS + rNrg1b1 nM

LPS + rNrg1b2.5 nM

LPS + rNrg1b5 nM

LPS + rNrg1b10 nM

n

1 ± 0.00 1 ± 0.00 1 ± 0.00

1.48 ± 0.14# 0.57 ± 0.12# 0.59 ± 0.02##

2.58 ± 0.34 0.52 ± 0.21 0.72 ± 0.26

2.99 ± 1.14 1.06 ± 0.33 1.04 ± 0.20

3.82 ± 1.20* 1.30 ± 0.20 1.29 ± 0.10*

4.47 ± 0.28** 1.94 ± 0.44** 1.49 ± 0.25**

3.23 ± 0.47 2.52 ± 0.53*** 1.53 ± 0.25**

3 3 3

1 ± 0.00

0.43 ± 0.13#

0.37 ± 0.06

0.59 ± 0.28

0.64 ± 0.15

0.74 ± 0.18

0.85 ± 0.30

3

Note: Data were presented as mean ± SD. Independent Student’s t-test was used to compare H2O2 or LPS treated group with vehicle control. One-way ANOVA with Tukey’s post hoc test was used to compare rNrg1b treated groups with vehicle control, H2O2 + rNrg1b treated groups with H2O2 treated group, or LPS + rNrg1b treated groups with LPS treated group. #p < 0.05; ##p < 0.01; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. ErbB4, ErbB2 and Akt1 phosphorylation levels are increased in cultured mouse cerebellar granule neurons in response to rNrg1b treatment for 48 h at various concentrations: 0, 0.5, 1, 2.5, 5, and 10 nM. pErbB4 and pErbB2 (A) levels, and Akt1 phosphorylation levels (B) in CGNs following 48 h treatment with rNrg1b at the indicated concentrations. Values represent mean ± SD (3 independent experiments); *p < 0.05, **p < 0.01 compared with the control group.

ErbB2 to protect cells against a variety of toxic effects. Following transient focal cerebral ischemia, Nrg1 protected neurons against apoptosis by regulating the expression of Akt and its downstream targets Bad and Bcl2 (Guo et al., 2010). In the hippocampus of rats exposed to chronic unpredictable mild stress, the activation of ErbB receptors and Akt was attenuated, whereas the enhancement of Nrg1/ErbB signaling partially normalized the stress-induced behavioral changes (Dang et al., 2016). Our results established that Nrg1/ErbB induces Akt1 signaling in CGNs, indicating that Nrg1/ ErbB/Akt1 signaling attenuates neurodegeneration. In order to further elucidate the therapeutic role of Nrg1 in neuronal degeneration, we treated CGNs with rNrg1b, after inducing neuronal oxidative stress and apoptosis using H2O2 and LPS. H2O2-induced oxidative stress attenuated the activation of both ErbB receptors and suppressed the downstream Akt1 phosphorylation. LPS-induced apoptosis inhibited ErbB2 and Akt1 phosphorylation, whereas the pErbB4 level was slightly elevated. The primary human umbilical venous endothelial cells treated with

LPS showed an increased phosphorylation level of ErbB4 (Bueter et al., 2006). ErbB4 tyrosine 1056 phosphorylation enables ErbB4 activation via ErbB4/ErbB4 coupling to the PI3kinase/Akt signaling pathway (Mill et al., 2011). The increased ErbB4 Tyr1056 phosphorylation and the decreased ErbB2 Tyr1248 phosphorylation suggest that LPS-induced apoptosis potentiates ErbB4/ErbB4 signaling, but reduces the ErbB4/ErbB2 signaling. We hypothesize that activation of ErbB4/ErbB4-based signaling may partially compensate the toxic effects of LPS (Schmiedl et al., 2011). The purity of the cultured neurons is 90%-95% (data not shown). Although publications have shown that in vitro cultured pure neurons can be affected by H2O2 or LPS (Baptista et al., 2015; Calvo-Rodriguez et al., 2017; Lu et al., 2014; Zou et al., 2016), the glial cells in H2O2 or LPS induced cell-toxicity may also played a role in our study. Nevertheless, our results indicated a beneficial role of Nrg1 in reversing H2O2 or LPS induced cell-toxicity. In the in vivo study of LPS-induced chronic inflammation, we detected changes in several Nrg1 isoforms, suggesting that LPS

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Fig. 4. rNrg1b reverses pErbB4, pErbB2 and pAkt1/Akt1 levels in H2O2-induced oxidative stress in primary cultured CGNs. Western blot of pErbB4 and pErbB2 (A), pAkt1/Akt1 (B) and Bcl2/Bax (C) levels in CGNs pretreated with 20 mM H2O2 for 2 h prior to 48 h treatment with rNrg1b. (n = 4 for pErbB4 and pErbB2, n = 3 for pAkt1/Akt1 and Bcl2/Bax, independent Student’s t-test or one-way ANOVA with Tukey’s post hoc test; data were expressed as means ± SD); #p < 0.05 and ##p < 0.01 vs. vehicle control and *p < 0.05 and ** p < 0.01 vs. H2O2-treated group. Representative immunofluorescence images of pErbB4 (D) and pErbB2 (E); scale bar represents 20 mm.

has diverse effects on various Nrg1 isoforms. The role of each isoform in neurodegenerative diseases warrants further study. In LPSinduced neuronal toxicity and neuroinflammation, the levels of pErbB4, pErbB2 and pAkt1/Akt1 were all decreased, which were reversed by Nrg1b. In vivo, both microglia and astrocytes show LPS-induced inflammation, in which the toxicity due to proinflammatory cytokines affects CGNs, which may partially explain the changes in pErbB4 levels between in vitro and in vivo studies. Under inflammation, Akt1 is activated in microglial cells to promote the release of pro-inflammatory cytokines (Chong et al., 2005; Lee et al., 2016; Yang et al., 2016a). However, Akt1 activation plays a protective role against cellular inflammation and death in neurons (Kang et al., 2003). Atorvastatin protects motor neurons against free radical-induced neuronal damage via Akt1 and Erk signaling pathways (Lee et al., 2016). Therefore, the decrease in the phosphorylation level of Akt1 in mouse cerebellum after LPSinduced inflammation is attributed to Akt1 activity in both glial cells and neurons. Bcl2 family proteins are major regulators of apoptosis belonging to the mitochondria-dependent pathway (Kroemer, 1997). The Bcl2 family includes both anti-apoptotic proteins such as Bcl2, Bcl-xL and pro-apoptotic proteins such as Bax, Bak (Besbes et al., 2015). In the present study, the downregulation of Bcl2/Bax in LPS-administered mouse cerebellum was conteracted by rNrg1b, suggesting that treatment with rNrg1b may inhibit neuronal apoptosis. IL1b has been implicated in inflammation associated with neuronal damage (Ghosh et al., 2013; Lyman et al., 2014). Nrg1b down-regulates IL1b mRNA levels without affecting TNFa mRNA

levels in acute brain injuries (Li et al., 2015). Nrg1b reduces IL1binduced cellular permeability of vascular endothelial cells and attenuates microvascular changes (Wu et al., 2016). Nrg1b also reverses the elevation of IL1b and TNFa levels in astrocytes with oxygen and glucose deprivation (Wang et al., 2012). In this study, we used LPS to induce chronic inflammation and treated mice with rNrg1b. We found that rNrg1b attenuated LPS-induced microglial and astrocytic activation as well as IL1b expression. These effects protect neurons from injury via pro-inflammatory cytokines and attenuate cerebellarin neurodegeneration. The results also indicate that Nrg1b prevents inflammation in neurodegenerative disorders. Other regions in the brain such as hippocampus and frontal cortex are more sensitive to neurodegenerative diseases. Nrg1b may also be neuroprotective against oxidative stress and inflammation by stimulating Akt1 in these affected brain regions, although the underlying mechanisms have yet to be determined. 4. Experimental procedure 4.1. Animals Female and male C57BL/6 mice (age, 3 months; weight,25 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). Two female and one male C57BL/6 mice were mated in each cage for six cages and the offspring on postnatal day 7 (P7) were used for primary culture of CGNs. Nine male C57BL/6 mice were used for the in vivo study of LPS induced inflammation. Animals were maintained at 25 °C on a 12 h

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Fig. 5. rNrg1b protects CGNs against LPS-induced neurotoxicity by increasing pErbB4, pErbB2 and pAkt1/Akt1 levels. Western blot of pErbB4 and pErbB2 (A), pAkt1/Akt1 (B) and Bcl2/Bax (C) levels in CGNs treated with rNrg1b followed by addition of 1 mg/mL LPS 2 h later, for 48 h. (n = 4, independent Student’s t-test or one-way ANOVA with Tukey’s post hoc test; data were expressed as means ± SD). #p < 0.05, ##p < 0.01 vs. vehicle control and *p < 0.05, **p < 0.01 and ***p < 0.001 vs. LPS-treated group. Representative immunofluorescence images of pErbB4 (D) and pErbB2 (E); scale bar represents 20 mm.

light/12 h dark cycle and provided food and water ad libitum. The Ethics Committee of Shantou University Medical College approved the experimental protocols. All the animal experiments were in accordance with the guidelines issued by the Chinese Animal Welfare Agency. All efforts were made to minimize the suffering of animals and reduce the number of animals used in the experiments. 4.2. Recombinant human neuregulin-1b (rNrg1b) The recombinant human neuregulin-1b (Thermo Fisher Scientific, MA) derived from E. coli was dissolved in phosphatebuffered saline (PBS) at pH 7.4 and used for cell culture experiments in vitro and for intraperitoneal (i.p.) injection in mice.

conjugated to DylightTM 594 (1:1000; Jackson Immuno Research Laboratories, PA) for 1.5 h at room temperature. The samples were mounted using ProLongÒ Gold Antifade reagent with DAPI (Thermo Fisher Scientific) and finally examined using an Olympus confocal system (FV-1000; Olympus, Japan). Integrated fluorescence intensity (IFI) was used to evaluate the Nrg1 protein levels, and ErbB4 and ErbB2 phosphorylation levels in human cerebellar tissue samples. IFI of each tissue sample was determined using the FluorChem HD2 gel imaging system (Alpha Innotech, CA). The images were analyzed using Image Tool II software (University of Texas Health Science Center, TX). IFI was evaluated using a grayscale ranging from 0 to 255, and the correlation between Nrg1 and pErbB4 or pErbB2 was analyzed using bivariate correlation analysis.

4.3. Human cerebellar tissue microarray and immunofluorescence staining

4.4. Primary culture and treatment of mouse CGNs

A human brain tissue microarray containing cerebellar tissues was purchased from Chaoying Biotechnology (Xian, China). Immunofluorescence staining of the tissues was performed as described previously (Chen et al., 2015). Briefly, human cerebellar tissue microarray sections were rehydrated and antigen retrieval was performed. The sections were blocked with 10% normal donkey serum (NDS) and incubated with primary antibody mixtures of mouse anti-Nrg1 antibody, rabbit anti-pErbB4 antibody, or rabbit anti-pErbB2 antibody (1:400; Santa Cruz Biotechnologies, CA) overnight at 4 °C. The samples were rinsed with PBS and incubated with donkey anti-mouse secondary antibody conjugated to DylightTM 488, and donkey anti-rabbit secondary antibody

Cerebellar tissues were obtained from postnatal C57BL/6 mice on P7. After dissection, they were digested with 0.125% trypsin diluted in Hank’s balanced salt solution for 30 min at 37 °C followed by trituration. Dissociated cells were purified and resuspended in DMEM/F-12 containing 10% FBS and 1% penicillinstreptomycin solution (Solarbio Biotech Corp, Beijing, China). The finely separated CGNs were seeded onto poly-D-lysine-coated 48-well or 24-well plates at a concentration of 1  105 cells per well. After 4 h incubation, the medium was replaced with Neurobasal-A medium supplemented with 2% B-27 (Thermo Fisher Scientific) and 1% penicillin-streptomycin solution. The cells were incubated at 37 °C in 5% CO2 atmosphere for 12 h. The medium

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Fig. 6. rNrg1b triggers Nrg1/ErbB signaling in LPS-induced neuroinflammation in mouse cerebellum. Western blot of Nrg1 isoforms (A), pErbB4 and pErbB2 (B), pAkt1/Akt1 (C), and Bcl2/Bax (D) after intraperitoneal injection of LPS and rNrg1b for 7 days (n = 3, independent Student’s t-test; data were expressed as means ± SD); #p < 0.05 vs. vehicle control and *p < 0.05 vs. LPS-treated group.

was replaced with B-27-free Neurobasal-A medium containing rNrg1b, H2O2 (20 mM) or LPS (1 mg/mL). We routinely obtain homogeneous cultures comprised of 90%-95% CGNs. Since the cultures

were used within three days, the cytosine b-d-arabinofuranoside (Ara-C, commonly used for inhibition of non-neuronal cell growth) was not added.

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Fig. 7. rNrg1b partly reverses LPS-induced neuroinflammation in mouse cerebellum. Representative images of cerebellar tissues of mice treated by LPS co-administered with or without rNrg1b (A-H). Immunohistochemical analysis of Iba-1 (A, B), GFAP (C, D), IL1b (E, F), and TNFa (G, H) after intraperitoneal injection of LPS and rNrg1b for 7 days (n = 3). Numbers of Iba-1 positive cells after intraperitoneal injection of LPS and rNrg1b for 7 days (I). Western blot analysis of IL1b and TNFa (J) after intraperitoneal injection of LPS and rNrg1b for 7 days (n = 3, independent Student’s t-test; data were expressed as means ± SD); #p < 0.05, ##p < 0.01 vs. vehicle control and *p < 0.05 vs. LPS-treated group. Scale bar represents 60 mm.

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collected using an epifluorescence microscope (Axio Imager Z1, Zeiss, Germany). Confocal images of tissue sections were acquired using an Olympus confocal system. 4.7. Immunohistochemical staining and quantification of the number of Iba-1 expressing cells Procedures for immunohistochemistry have been described in detail (Yang et al., 2016b). In brief, mice were perfused with a 4% paraformaldehyde solution. Cerebellar sections (8-mm thick) were prepared using a cryostat microtome (LeicaCM1860, Leica Biosystems, Germany). Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0) at 95 °C for 40 min. Sections were incubated in 3% H2O2 to clear endogenous peroxidase, blocked and incubated overnight with primary antibodies targeting the following proteins: rabbit anti-Iba-1, rabbit anti-GFAP, rabbit anti-IL1b and rabbit anti-TNFa (1:200; Boster, China). After washing in PBS, the mixture was treated with biotinylated secondary antibody and streptavidin-peroxidase conjugate. The enzymeactivity was developed. The antigen-antibody complexes were visualized using an AEC kit (ZSGB Biotechnology Co., Beijing, China) according to the manufacturer’s protocols. The cerebella of the three groups (control, LPS, LPS + rNrg1b; n = 3 mice/group) at the similar size were picked for detecting Iba-1 positive cells using immunohistochemistry. The number of Iba-1 expressing cells was counted in one half of the cerebella which was cut into two symmetrical halves by midline incision. Data were represented as number of Iba-1 positive cells/mm2. The quantification was performed using the ImageJ image analysis software. 4.8. Western blot Fig. 7 (continued)

4.5. LPS and rNrg1b treatment Nine adult male C57BL/6 mice weighing 18–21 g were divided into three groups: control group, LPS group and LPS/Nrg1 group. Each group consists of three mice. All the regents were administered using i.p. injections for seven consecutive days. At 9:00 AM each day, mice in the control group received a daily dose of 100 mL normal saline (pH 7.4). The other two groups were administered 100 mL LPS (Sigma-Aldrich, Rehovot, Israel) dissolved in normal saline at a dose of 250 lg/kg. At 11:00 AM each day, the control and the LPS groups were treated with 100 mL normal saline, while the LPS/Nrg1 group was administered 100 mL rNrg1b containing a dose of 35 mg/kg. At 7:00 PM on day 7, the mice were anesthetized and euthanized by decapitation. Brains were rapidly removed and cut into two symmetrical halves by midline incision. One half was fixed in 10% formol saline for histochemical studies, and the other half was stored at 80 °C for Western blot analysis. 4.6. Immunofluorescence CGNs were cultured for 48 h on PDL-coated glass slides and fixed in 4% paraformaldehyde. The mouse cerebellar sections (8lm thick) and cultured CGNs were blocked with 10% normal donkey serum in PBS at room temperature for 1 h. Samples were incubated overnight at 4 °C with a mixture of anti-b-III tubulin antibody and various primary antibodies. After rinsing with PBS, immunoreactivities were visualized by incubation with donkey anti-mouse secondary antibody conjugated to Dylight 488 (1:500) and donkey anti-rabbit secondary antibody conjugated to Dylight 594 (1:1000). DAPI was used to stain the nuclei in the cerebellar sections. Fluorescence images of CGN samples were

The whole cell lysates and tissue lysates were collected in a RIPA buffer mixture (Solarbio Biotech) supplemented with PMSF (1:200; Solarbio Biotech), combined with 25% LDS Sample Buffer (Invitrogen, CA) and heated at 95 °C for 15 min. Protein samples were separated via 10% SDS-PAGE gel and electroblotted onto polyvinylidenedifluoride (PVDF) membranes (Millipore, MA). Non-specific protein binding sites were blocked with 5% nonfat dry milk or BSA dissolved in Tris–HCl saline buffer containing 0.1% Tween-20 (TBST, pH 7.4). Membranes were incubated overnight at 4 °C with one of the following antibodies: mouse antiNrg1, rabbit anti-pErbB4, rabbit anti-pErbB2, mouse anti-pAkt1, mouse anti-Akt1, mouse anti-Bcl2, rabbit anti-Bax or mouse antib-Actin (1:1000; Santa Cruz Biotechnologies); rabbit anti-IL1b and rabbit anti-TNFa (1:500; Boster). After washing with 0.1% TBST three times for 5 min each, horseradish peroxidase-conjugated goat anti-mouse (1:2000; Boster) and goat anti-rabbit secondary antibodies (1:1000; Boster) in 5% nonfat dry milk or BSA diluted in TBST were used. Membranes were washed three times in TBST for 5 min each at room temperature. The immunoreactive signals were visualized with enhanced chemiluminescence (ECL) solution (Beyotime Institute of Biotechnology, Haimen, China). The signal intensity was quantified by densitometry using Image J software (National Institutes of Health, MD). 4.9. Statistical analysis Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (Version 17.0, IBM Corporation, NY). Data were expressed as the mean ± standard deviation (SD). Two-tailed Student’s t-test was used to compare the two treatment groups, and one-way analysis of variance (ANOVA) was used for multiple group comparisons followed by Tukey post hoc tests. P < 0.05 indicated statistically significant difference. Both

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