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Published in final edited form as: J Neurochem. 2012 Apr 27;122(2):321–332. doi: 10.1111/j.1471-4159.2012.07754.x

Intravenous immunoglobulin (IVIg) protects neurons against amyloid beta-peptide toxicity and ischemic stroke by attenuating multiple cell death pathways

Alexander Widiapradja 1, Viktor Vegh 2, Ker Zhing Lok 1, Silvia Manzanero 1, John Thundyil 1, Mathias Gelderblom 3, Yi-Lin Cheng 1, Dale Pavlovski 1, Sung-Chun Tang 4, Dong-Gyu Jo 5, Tim Magnus 3, Sic L Chan 7, Christopher G Sobey 6, David Reutens 2, Milan Basta 8, Mark P Mattson 9, Thiruma V Arumugam 1,10
PMCID: PMC5146755  NIHMSID: NIHMS369241  PMID: 22494053

Abstract

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Intravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke and Alzheimer’s disease (AD) has been proposed, but little is known about the neuroprotective mechanisms of IVIg. In this study we investigated the effect of IVIg on downstream signaling pathways that are involved in neuronal cell death in experimental models of stroke and AD. Treatment of cultured neurons with IVIg reduced simulated ischemia- and amyloid βpeptide (Aβ)-induced caspase-3 cleavage, and phosphorylation of the cell death-associated kinases p38MAPK, JNK and p65, in vitro. Additionally, Aβ-induced accumulation of the lipid peroxidation product 4-HNE was attenuated in neurons treated with IVIg. IVIg treatment also upregulated the anti-apoptotic protein, Bcl2 in cortical neurons under ischemia-like conditions and exposure to Aβ. Treatment of mice with IVIg reduced neuronal cell loss, apoptosis and infarct size, and improved functional outcome in a model of focal ischemic stroke. Together, these results indicate that IVIg acts directly on neurons to protect them against ischemic stroke and Aβ-induced neuronal apoptosis by inhibiting cell death pathways and by elevating levels of the anti-apoptotic protein Bcl2.

Keywords: Stroke, IVIg, apoptosis, neurons, Aβ, Alzheimer’s Disease

INTRODUCTION

Intravenous immunoglobulin (IVIg) preparations are obtained by fractionating blood plasma from a pool of healthy donors. IVIg is used as a replacement therapy for patient with primary immune deficiencies. In addition, due to its immunomodulatory effects, IVIg has become a treatment of choice for a variety of immune disorders, including those that that affect the nervous system (Kotlan et al. 2009; Baerenwaldt et al. 2010). IVIg is also being used increasingly to treat autoimmune disorders, including those that that affect the nervous system (Baerenwaldt et al. 2010). Numerous mechanisms have been proposed to explain the clinical effects of IVIg preparations. IVIg can block the function of Fc receptors on phagocytes by saturating, altering or down-regulating the affinity of the Fc receptors (Aschermann et al. 2010; Anthony et al. 2011). IVIg can also impair leukocyte adhesion to endothelial cells, attenuate complement-mediated damage, modulate cytokine production by various cell types and inhibit apoptosis (Arumugam et al. 2007; Arumugam et al. 2008; Arumugam et al. 2009) and attenuate complement-mediated damage (Basta et al. 1989; Basta et al. 1989; Basta et al. 2003). Recently, we demonstrated that IVIg treatment significantly reduced brain infarct volume and mortality in a mouse model of stroke (Arumugam et al. 2007). We have also shown that human IgG levels were higher in samples obtained from the infarcted area as compared with the corresponding region in the contralateral non-injured brain hemisphere. We confirmed this by immunohistochemistry, which showed more intense and extensive staining for human IgG at the site of injury as compared with the contralateral side of the brain (Arumugam et al. 2007). Furthermore, dual staining for human IgG and blood vessels (collagen IV) allowed us to visualize the leakage of the BBB and the crossing of IgG into the parenchyma.

IVIg selectively neutralized complement component C3b and decreased the expression levels of endothelial and leukocyte adhesion molecules, neutrophil infiltration and microglial activation (Arumugam et al. 2007). However, it is not known whether the neuroprotective actions of IVIg in vivo are due only to effects on inflammatory cells, or might also involve direct actions on neurons.

The potential therapeutic efficacy of IVIG has recently been tested in Alzheimer’s disease (AD) patients (Dodel et al. 2002; Relkin et al. 2009). Human clinical studies showed stabilization and even a mild improvement in cognitive function in the patients treated with IVIg (Dodel et al. 2004; Relkin et al. 2009). Furthermore, a recent study demonstrated the protective effects of IVIg against Aβ toxicity in primary mouse hippocampal neuronal cultures (Magga et al. 2010). However, the exact mechanisms by which IVIg elicits its neuroprotective effects are unknown. Hence, the main objective of our study was to explore the direct mechanisms involved in IVIg-induced neuroprotection in models of stroke and Aβ toxicity. We observed that IVIg promotes neuronal survival by inhibiting the activation of several stress-induced signaling pathways and up-regulating the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2).

MATERIALS AND METHODS

Primary cortical neuronal cultures

All animal experimental procedures performed were reviewed and approved by the University of Queensland Animal Care and Use Committee. The primary cortical neurons culture were obtained from 16-day C57B/6 mouse embryos as described previously (Okun et al. 2007). Cells were cultured on 35-60- or 100-mm diameter petri dishes containing Neurobasal medium containing 25 mM glucose and B-27 supplements (Invitrogen, USA), 2 mM L-glutamine, 0.001% gentamycin sulfate and 1 mM HEPES (pH 7.2) and maintained in 37 °C incubator. The neuronal purity of the cultures was approximately 95%, determined by immnunostaining using neuronal specific marker (MAP2) antibody and astrocyte specific marker (GFAP) antibody.

Glucose, oxygen-glucose deprivation and cell viability analysis

In order to induce glucose deprived (GD) condition, neuronal cultures were incubated in glucose-free Locke’s medium containing (in mmol/L) 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1 MgCl2 3.6 NaHCO3, 5 HEPES, pH 7.2, supplemented with gentamycin (5 mg/L; Invitrogen, USA) for 12 or 24 h. For oxygen and glucose deprivation (OGD), hypoxia was induced by saturating the Locke’s buffer with 95% N2/5% CO2, pH 7.4 gas mixture for 10 minutes before incubating the cultures in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for 12 h. Cell viability was determined by the trypan blue exclusion assay (Woodruff et al. 2011). In order to observe the effect of IVIg on GD- or OGD-induced cell death, the cultures were treated with different concentrations of IVIg (Sandoglobulin, CLS Biotherapy, Australia). Controls such as a vehicle- and a negative control (bovine serum albumin (BSA) (Sigma Aldrich, USA) were also included in the experiment.

Aβ toxicity experiments

Aβ1-42 (American Peptide, USA) was prepared according to the 1,1,1,3,3,3-Hexafluro-2-propanol (HFIP)-based protocol, as described previously (Stine et al. 2003). Vehicle control was made by adding the same volume of DMSO with 1X PBS as used to prepare the peptide solutions. The concentration of Aβ1-42 (10.0 μM) that was able to induce ~90% of neuronal cell death in vitro was used. To examine the effect of IVIg on Aβ-induced neuronal cell death, primary neurons were co-treated with 10.0 μ M o f oligomerized Aβ1-42 and different concentrations of IVIg (Sandoglobulin, CLS Biotherapy, Australia) for 24 h at 37oC. A vehicle control and a negative control were also included in the experiment: DMSO, and 10 mg/ml of bovine serum albumin (BSA) (Sigma Aldrich, USA), respectively.

Western blot analysis

20μg of protein samples were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and run in a Tris-glycine running buffer (BioRad, USA) at 80 Volts until the protein ladder (Lonza, USA) was spread optimally. The proteins were then transferred into nitrocellulose membranes (Bio-Rad, USA) using semi-dry transfer apparatus (Bio-Rad, USA) in transfer buffer (BioRad, USA) for 1.5 h at 350mA. The membranes were then incubated in blocking buffer (5% BSA (Bovine Serum Albumin) (Sigma Aldrich, USA) in Tris-buffered saline-T for one h at room temperature. The membranes were then incubated overnight at 4oC with primary antibodies against cleaved caspase 3, phospho-SAPK/JNK, phospho-p38, phospho-p65 NFκB, Bcl-2 (Cell Signaling, USA), 4-Hydroxynonenal (4-HNE) (Abcam, UK) and actin (Sigma Aldrich, USA). The membranes were washed with Tris-buffered saline-T before they were incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h. The membranes were then washed again with Tris-buffered saline-T and incubated with chemiluminescent substrate for enhanced chemiluminescence (Pierce Endogen, USA) for 2 minutes. The signals were visualized using X-ray films (Fujifilm Corporation, Japan).

Middle cerebral artery occlusion and reperfusion

Three-month-old C57BL/6 male mice were used for in vivo experiments. The focal cerebral ischemia/reperfusion (I/R) model was similar to that described previously (Arumugam et al. 2004). In the sham group, these arteries were visualized but not disturbed. Mice were either administered with 2 g/kg IVIg or vehicle by direct infusion into the femoral vein (200 μL) either 30 min before the onset of ischemia or 3 h after the ischemic period. Cerebral blood flow was measured by placing the animal’s head in a fixed frame after it had been anesthetized and prepared for surgery. A craniotomy was performed to access the left MCA and was extended to allow positioning of a 0.5-mm Doppler probe (Moor LAB, Moor Instruments, UK) over the underlying parietal cortex approximately 1 mm posterior to bregma and 1 mm lateral to the midline. The functional consequences of focal cerebral I/R injury were evaluated using a 5-point neurological deficit score and were assessed in a blinded fashion (Bederson et al. 1986).

Immunocytochemistry and Immunohistochemistry

The cortical neurons were cultured in 6-well plates containing microscope coverslips, treated with glucose free Locke’s buffer and IVIg and then were fixed in 4% paraformaldehyde. The cells were then incubated for 1 h with primary cleaved caspase-3 (Cell signaling, USA) and MAP2 (Millipore, USA) antibody followed by 2 h incubation with Alexa fluor 488 (Invitrogen, USA) conjugated secondary antibody and Alexa fluor 568 (Invitrogen, USA) conjugated secondary antibody at room temperature respectively. Frozen brain sections were incubated for 1 h with primary MAP2 (Millipore, USA), GFAP (Sigma Aldrich, USA) and Iba1 (Abcam, UK) antibodies followed by 2 h incubation with Alexa fluor 488 (Invitrogen, USA) conjugated secondary antibody at room temperature. Both cells and frozen sections were stained with nuclear stain DAPI for 15 minutes before mounted with VectaShield (Vector, USA). Images were acquired using fluorescence microscope (Olympus, Japan).

TUNEL assay

The tissue slides were dried at room temperature for 24 h and washed in 1X PBS for 15 min. The TUNEL assay was done according to the manufacturer’s instruction (Roche, Switzerland). Once the assay procedure was completed, and the coverslip was mounted on a slide. The slides were viewed under the fluorescence microscope and TUNEL positive cells were counted.

In vivo magnetic resonance imaging method

In vivo mouse brain data was acquired on a Bruker 16.4T (700 MHz) small animal MRI scanner, connected to an AVANCE spectrometer running ParaVision 5.1. Multiple echo time T2-weighted and multiple b-value diffusion-weighted images were acquired for the study. Two-dimensional contiguous slices were obtained with an in-plane field-of-view of 15 mm; the number of slices was 9 with a slice thickness of 0.9 mm and without acceleration. The standard T2 relaxometry sequence was used with the following parameters: matrix size = 256, TE=14 ms, 28 ms, 42 ms, 56 ms, 70 ms, and 84 ms, TR = 2000 ms, number of averages = 1, and bandwidth = 50000 Hz. The standard diffusion sequence was used with the following parameters: matrix size = 128 128, TE = 20 ms, T2 = 1500 ms, number of averages = 1, bandwidth = 50000 Hz and b-value = 151 ms, 705 ms, 1169 ms, 1618 ms. Individual mouse brains were segmented based on a combination of T2-weighted images, and using our in-house developed region growing MATLAB (version 7.10.0 R2010a) program.

T2-map and apparent diffusion coefficient (ADC) computation

T2 values were calculated by masking individual brain slices and performing a fit in a voxel-by-voxel manner across all TE images of the T2-weighted acquisition. The standard model was used to obtain the T2 map: S=S0e−TE∕T2+k, where S is the measured image voxel signal, S0 is the TE=0ms signal (unknown), TE (in ms) is the echo time used to acquire the image and k (unknown) is the offset applied to improve the quality of fit. T2 values calculated at each voxel in the brain were used to form the T2 map. Similarly, the apparent diffusion coefficient was computed using the following standard model: log(S) = log(S0) – b × ADC, where b is the b-value, and ADC is the apparent diffusion coefficient (unknown). A brain volume corresponding to the region affected by stroke was calculated by thresholding T2 and ADC values and by counting the number of voxels that were larger than the set value. This count was multiplied by voxel volume to obtained infarct volume for both the T2 and diffusion-based analyses.

Data analysis

All the results are reported as the means ± S.D. The overall significance of the data was examined by one-way analysis of variance (ANOVA). The differences between the groups were considered significant at P<0.05 with the appropriate Bonferroni correction made for multiple comparisons. Neurological behavior scores were analyzed by using a nonparametric Kruskal-Wallis test and Dunn’s Multiple Comparison Test.

RESULTS

Modulation of in vitro ischemic stroke-induced neuronal cell death by IVIg

We first evaluated the effect of different concentrations of IVIg in a cell culture model of ischemic neuronal injury in which primary mouse cortical neurons were subjected to conditions mimicking in vivo occlusion of a cerebral artery, including glucose deprivation (GD), and combined oxygen and glucose deprivation (OGD). Treatment with low concentrations of IVIg (0.1 mg/mL, 0.3 mg/mL) significantly increased GD-induced neuronal cell death whereas high concentrations of IVIg (3 mg/mL, 5 mg/mL, 10 mg/mL) significantly decreased GD-induced neuronal cell death (Fig. 1a). Treatment with high concentration of IVIg (5 mg/mL, 10 mg/mL) also significantly decreased OGD-induced neuronal cell death (Fig. 1b). We next analyzed levels of cleaved caspase-3, an apoptotic protease, following GD and OGD conditions. Treatment with a low concentration of IVIg (0.1 mg/mL) increased the levels of cleaved caspase-3 (Fig. 1c, d) whereas, higher concentrations of IVIg (2.5 mg/mL and 5 mg/mL) significantly reduced the level of cleaved caspase-3 (Fig. 1d) in primary neuronal cells subjected to GD for 12 h. Similarly, in cultured neurons subjected to OGD conditions for 12 h, the level of cleaved caspase-3 increased in response to a low concentration of IVIg treatment (0.1 mg/mL) and significantly decreased in response to higher concentrations of IVIg treatment (2.5 mg/mL, 5 mg/mL) (Fig. 1e, f).

Figure 1. IVIg protects cultured neurons against simulated ischemia.

Figure 1

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a. Treatment with low concentrations of IVIg (0.1 mg/ml, 0.3 mg/ml) significantly increased GD-induced neuronal cell death, whereas high concentrations of IVIg (3 mg/mL, 5 mg/mL, 10 mg/mL) significantly decreased GD-induced neuronal cell death. ***, P < 0.05 compared to the vehicle + GD group; +++, P < 0.05 compared to the GD group. b. Treatment with high concentrations of IVIg (5 mg/mL, 10 mg/mL) also decreased OGD-induced neuronal cell death. ***, P < 0.05 compared to the normal group; +++, P < 0.05 compared to the vehicle + OGD group. Treatment with a low concentration of IVIg (0.1 mg/mL) increased the expression level of cleaved caspase 3 (c and d), whereas high concentrations of IVIg (2.5 mg/mL, 5 mg/mL) significantly reduced the expression level of cleaved caspase-3 (D) in primary neuronal cells subjected to GD conditions for 12 h. *, P < 0.05 compared to GD + vehicle group; ++, P < 0.01 compared to GD + vehicle. e and f. Similarly, the level of cleaved caspase-3 increased with low concentrations of IVIg (0.1 mg/mL) and decreased with high concentrations of IVIg treatment (2.5 mg/mL, 5 mg/mL) in primary neuronal cells subjected to OGD conditions for 12 h. n = 3 or more biologically different cultures in each group. *, P < 0.05 compared to GD + vehicle group; ++, P < 0.01 compared to GD + vehicle.

Modulation of Aβ-induced neuronal death by IVIg

To evaluate the effect of IVIg on Aβ-induced neuronal cell death in vitro, a dose titration of Aβ was first performed to identify a toxic concentration of oligomerized Aβ to be used in subsequent experiments. Various concentrations of Aβ ranging from 0.1 μM to 10 μM significantly increased the percentage of neuronal cell death compared to vehicle-treated neurons. It was observed that the most toxic dose of oligomerized Aβ on primary neuron cultures was 10 μM (Fig. 2a). Furthermore, the addition of IVIg treatment to Aβ-treated neurons significantly reduced cell death (Fig. 2b). This protective effect was concentration-dependent, with 10 mg/mL IVIg having the maximum protection, reducing cell death to 30% (Fig. 2b). Since cleavage of caspase-3 is implicated in Aβ-induced neuronal cell death (Mattson et al. 1998), we further looked at the levels of the apoptotic protease cleaved caspase-3 in primary cortical neurons subjected to Aβ for 24 h. The elevated level of cleaved caspase-3 observed in neurons treated with Aβ was significantly reduced following IVIg treatment (Fig. 2c, d). Immunocytochemistry was also performed to visualize the expression levels of cleaved caspase-3. Consistent with the immunoblot results, the level of cleaved caspase-3 was significantly reduced in IVIg-treated (10 mg/mL) neurons exposed to Aβ compared to neurons exposed to Aβ alone (Supplementary Fig. 1).

Figure 2. Treatment with IVIg reduces cell death in Aβ-treated neuronal cultures in a concentration-dependent manner.

Figure 2

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a. Treatment with Aβ42 in primary neuronal cultures for 24 h increased the level cell death significantly in a concentration-dependent manner. ***, P < 0.05 compared to vehicle. b. Treatment with 10 μM Aβ42 significantly increased neuronal cell death whereas the co-treatment with IVIg (1-10 mg/mL) significantly decreased Aβ42-induced neuronal cell death in a concentration-dependent manner. *, P < 0.05 compared to vehicle; ***, P < 0.01 compared to Aβ42 treated group. c and d. Similarly the treatment of primary neuronal cells with Aβ42 significantly increased cleaved caspase-3 levels, and co-treatment with IVIg significantly decreased cleaved caspase-3 levels in a concentration-manner. *, P < 0.05 compared to vehicle; +, P < 0.05 compared to Aβ42 treated group; +++, P < 0.01 compared to Aβ42 treated group. n = 3 or more biologically different cultures in each group.

Effect of IVIg on GD- or Aβ-induced stress-sensitive kinases and a marker of oxidative stress

Because the activation of p38-MAPK, JNK and NFκB pathways are implicated in ischemia-induced cellular stress responses and neuronal death, (Thundyil et al. 2010; Arumugam et al. 2011), we next measured levels of p38-MAPK, JNK and NFκB-p65 following GD in IVIg-treated neurons compared with vehicle-treated neurons. IVIg treatment significantly reduced p-JNK and NFκB-p65 (Fig. 3a, c, d) following GD condition as compared to vehicle-treated neurons. A similar trend was also observed in p38-MAPK levels in the IVIg-treated neuron groups as compared to the vehicle groups (Fig. 3a, b). JNK and p38MAPK has also been shown to be activated by Aβ (Yang et al. 2010; Ramin et al. 2011) and their activation leads to the phosphorylation and activation of several transcription factors, involved in Aβ-induced cell death (Vukic et al. 2009). Similar to these findings, we found that exposure of primary neurons to Aβ significantly increased levels of p38MAPK (Fig. 4a, b) and p-JNK (Fig. 4a, c) and IVIg treatment significantly reduced these levels (Fig. 4a, b, c). To further explore the mechanism underlying the protective effect of IVIg against Aβ-induced cell death, the production of 4-hydroxynonenal (4-HNE) was examined. It has previously been shown that the lipid peroxidation product 4-HNE, generated in response to membrane-associated stress, is involved in Aβ-induced neuronal damage (Mark et al. 1997; Tang et al. 2008). Aβ-induced protein modification by 4-HNE was significantly reduced by IVIg treatment (Fig. 4a, d).

Figure 3. Treatment with high concentrations of IVIg reduces the level of pro-apoptotic proteins during GD-induced neuronal cell death.

Figure 3

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a-c. Low concentration of IVIg (0.1 mg/mL) increased the expression level of P-p38, P-JNK and P-p65 whereas high concentrations (2.5 mg/mL, 5 mg/mL) of IVIg decreased the expression level of P-p38 (b), P-JNK (C) and NFκB P-p65 (c) significantly after 12 h in GD-exposed primary neuronal cells. ns, non significant reduction compared to vehicle; **, P < 0.05 compared to vehicle; n = 3 or more biologically different cultures in each group.

Figure 4. IVIg treatment reduces the level of pro-apoptotic proteins and the generation of lipid peroxidation product 4-HNE in Aβ42-induced neuronal cell death.

Figure 4

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a-c. Treatments with different concentrations of IVIg (0.1 mg/mL, 2.5 mg/mL, 5 mg/mL) against Aβ42-induced neuronal cell significantly decreased the expression level of P-p38 (b), P-JNK (c) and the production of 4-HNE (D). *, P < 0.05 compared to vehicle; **, P < 0.01 compared to vehicle; n = 3 or more biologically different cultures in each group.

IVIg treatment elevates Bcl-2 levels in neurons subjected to GD or exposed to Aβ

A well-established anti-death protein, Bcl-2, has been shown to be downregulated in neurons under stress conditions, such as in ischemic stroke and Aβ toxicity (Prehn et al. 1994; Alves da Costa et al. 2003). We therefore evaluated Bcl-2 protein levels in primary neurons treated with IVIg or vehicle and then subjected to GD or exposure to Aβ. IVIg treatment significantly increased the level of Bcl-2 in GD conditions (Fig. 5a, b). Furthermore, neurons treated with IVIg and then exposed to Aβ exhibited Bcl-2 levels that were significantly greater than vehicle-treated neurons exposed to Aβ (Fig. 5 c, d).

Figure 5. IVIg treatment increases the level of the anti-apoptotic protein Bcl2 in cultured neurons.

Figure 5

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Increasing concentrations of IVIg significantly increased the level of Bcl2 during GD-induced neuronal cell death (a and b). Aβ42 treatment decreased the expression level of Bcl2, however treatment with increasing concentrations of IVIg increased Bcl2 expression level in Aβ42-treated cultures (c and d). **, P < 0.05 compared to vehicle; ***, P < 0.001 compared to vehicle; +, P < 0.05 compared to Aβ42 treated group; +++, P < 0.001 compared to Aβ42-treated group; n = 3 or more biologically different cultures in each group.

IVIg treatment improves neuronal survival, reduces infarct size and improves functional outcome following ischemic stroke in vivo

We previously reported that IVIg treatment can reduce brain damage and neurological deficits measured 3 days after cerebral I/R in mice (Arumugam et al. 2007). Cerebral blood flow measurements obtained immediately before and after middle cerebral artery occlusion showed ~90% reduction in blood flow to the middle cerebral artery occlusion infarct region, which did not differ between groups. To test whether IVIg was able to enter brain tissue, we first performed immunohistochemistry to locate human IgG (hIgG) in control and IVIg-treated mice. Immunohistochemistry analysis showed more intense and extensive staining for human IgG at the site of injury (Fig. 6a). In order to investigate whether IVIg was able to protect neurons in core and/or penumbra regions of ischemic cerebral cortex, we evaluated levels of the neuronal marker microtubule-associated protein 2 (MAP2). We found that the extent of MAP2 positive cell loss was significantly reduced by both pre- and post-stroke IVIg treatment as compared to vehicle-treated mice (Fig. 6b, d, e). We next investigated the amount of TUNEL positive cells in the penumbra regions of ischemic stroke. Both pre- and post-treatment of IVIg significantly reduced I/R induced apoptosis compared to vehicle treated group (Fig. 6c) and most of TUNEL positive cells in the penumbra regions were found to be primarily neurons (Supplementary Fig. 2). To further confirm the protective effect of IVIg against stroke-induced infarct development, we used in vivo magnetic resonance imaging methods. In vivo results obtained using T2 (Fig. 7a) and diffusion-based (Fig. 7b) analysis showed that both pre- and post-stroke IVIg treated mice had a significantly lower brain damage compared to the vehicle treated group (Fig. 7a, b, c). We further evaluated the functional aspect of our stroke model in vivo by scoring cerebral I/R induced neurological deficit in a blinded manner. The data show that the IVIg-treated mice had significantly reduced neurological deficit score compared to vehicle-treated mice (Fig. 7d).

Figure 6. IVIg treatment reduces neuronal cell death in vivo following ischemic stroke.

Figure 6

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a. Intravenously-injected IVIg reached the brain as a result of blood brain barrier breakdown. (Scale bars: 100 μM.) b-d. MAP2-positive neuronal cells were significantly decreased in number in infarcted brain area whereas the treatment with IVIg in both pre- and post-treatment significantly reduced the amount of neuronal loss in infarcted area. ***, P < 0.05 compared to MCAO/R group; n = 3-6 in each group. e and f. TUNEL staining showed that both pre- and post-treatment of IVIg decreased the amount of cell apoptosis in the infarcted region of the brain. ***, P < 0.05 compared to MCAO/R group; n = 3-6 in each group.

Figure 7. IVIg treatment reduces brain damage and improves functional outcome in a mouse stroke model.

Figure 7

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a and b. T2-based magnetic resonance imaging analysis and diffusion-based analysis show that both pre- and post-IVIg treatment significantly reduced cerebral I/R induced infarct volume compared to the vehicle-treated group. *, P < 0.05 compared to MCAO/R group; n = 6 in each group. c. Corresponding representative images show rows one to four MRI slices. Provided are normalized T2-weighted images obtained using TE=14ms (left panel), calculated T2-map (ms) (middle panel), and apparent diffusion coefficient (ADC) (right panel). The images show increased T2-weighted image signal intensity and reduced ADC value used to estimate stroke volume. d. A five-point neurological score was applied to the sham (n=7), vehicle MCAO/R (n=10), IVIg (2 g/kg) pre-treated MCAO/R (n=10) and IVIg (2 g/kg) post-treated MCAO/R mice; *, P < 0.05 compared with vehicle MCAO/R group.

DISCUSSION

IVIg therapy has been shown to be effective in the treatment of various autoimmune and inflammatory disorders, and several mechanisms have been proposed to explain these beneficial effects. Some of these mechanisms include inhibiting the activation of complement pathways and release of inflammatory cytokines, neutralization of T and B cell activation, and their differentiation and effector functions (Samuelsson et al. 2001; Kaneko et al. 2006; Basta, 2008). IgG can exert both pro- and anti-inflammatory activities, depending upon its concentration (Durandy et al. 2009). The proinflammatory activity of low dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific Fc receptors. This results in an increase in intracellular calcium levels and activation of proinflammatory or death signaling pathways. Our data also showed that low dose of IVIg significantly increased GD- and Aβ-induced neuronal cell death. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. We previously provided pre-clinical evidence supporting the possibility of using high-dose IVIg as a stroke therapy targeting the complement cascade and inflammatory mechanisms (Arumugam et al. 2007). Since naturally occurring autoantibodies against Aβ (nAbs-Aβ), which can block the toxic effects of Aβ, are found in intravenous immunoglobulin (IVIg), such preparations have been suggested to be efficacious and safe in the treatment of AD (Dodel et al. 2004). Recent studies have suggested the possibility that IVIg treatment may reduce the risk of developing AD and related disorders (Fillit et al. 2009). Furthermore, two open-label pilot studies have reported that IVIg-treated AD patients show improved cognition (Dodel et al. 2004; Relkin et al. 2009). However, little is known about how IVIg protects neurons in such conditions. Numerous studies have shown that MAPK signaling pathways such as p38, JNK, and NFκB-p-p65 are two major signaling cascades involved in neuronal cell death (Legos et al. 2001; Tamatani et al. 2000; Tang et al. 2007; Arumugam et al. 2011). Furthermore, the role of Bcl-2 in stroke outcome has been studied extensively. Increased Bcl-2 expression promotes cell survival and protects against neuronal apoptosis and cellular necrosis. In addition, treatments that increase Bcl-2 expression have proven to be neuroprotective (Linnik et al., 1995; Paradis et al. 1996; Mattson et al. 2000). In this study we found that IVIg directly protects cortical neurons against ischemic and Aβ-induced cell death by preventing the activation of caspase-3, p38MAPK, JNK and NFκB signaling pathways, by reducing the production of 4-HNE, and by elevating Bcl2 protein levels.

Activation of the p38MAPK pathway and its downstream signaling has been shown to play a role in the post-ischemic brain damage in animal models of stroke (Legos et al. 2001). The stress-activated p38MAPK plays important roles in transducing stress-related signals by phosphorylating intracellular enzymes, transcription factors and cytosolic proteins involved in apoptosis and inflammatory cytokine production. Sustained activation of p38MAPK is associated with neuronal death/apoptosis following ischemic stroke and its inhibition is neuroprotective (Legos et al. 2001). There is accumulating evidence that p38MAPK plays a role in AD pathophysiology (Munoz and Ammit, 2010). Furthermore, recent findings suggest that p38MAPK operates in other events related to AD, such as excitotoxicity, synaptic plasticity and tau phosphorylation. Our findings show that IVIg treatment significantly reduces ischemic or Aβ-induced neuronal p38MAPK levels.

The c-Jun NH2-terminal kinase (JNK) signaling pathway is frequently induced by cellular stress and is correlated with neuronal death. Activation of the JNK pathway may trigger cell death by phosphorylating transcription factors regulating cell death (Tang et al. 2007). Several studies with JNK inhibitors such as SP600125 decreased apoptotic neuronal cell death following ischemic stroke (Guan et al. 2006). Recent observations have also linked the JNK pathway to AD, including the ability of JNK to phosphorylate Tau and APP, promoting the accumulation of two neurotoxic species, hyperphosphorylated Tau and Aβ42 (Sahara et al. 2008; Tang et al. 2008). Our findings suggest that IVIg protects neurons by down-regulating the JNK pathway during both ischemic and Aβ42-induced neuronal cell death.

There is ample evidence that NFκB is activated in ischemic stroke (Tamatani et al. 2000). Most investigators have found NFκB to be activated in neurons following stroke (Arumugam et al. 2011). NFκB activation involves nuclear translocation of the subunits p65 and p50 where it initiates transcription by binding to regulatory DNA sequences. Studies have shown that expression of the pro-apoptotic Bcl-2-homology domain 3 (BH3)-only genes, Bim and Noxa, depend on p65 (Inta et al. 2006). NFκB stimulates Bim and Noxa gene transcription in primary cortical neurones and binds to the promoter of both genes. As the ratio of pro- and anti-apoptotic Bcl-2 family members determines cell fate, Bim and Noxa could be candidates to mediate the detrimental effects of NFκB activation in ischemia-induced neuronal cell death. Our data demonstrate that IVIg treatment significantly reduces levels of NFκB-p65.

There is a growing body of evidence indicating that Aβ damages neurons by inducing lipid peroxidation of brain cell membranes and this has been shown to be inhibited by antioxidants. Peroxidation of membrane unsaturated fatty acids leads to the production of several different aldehydes, and one of the major products is 4HNE (Mattson, 2009). 4HNE is lipid-soluble and may damage proteins by rapidly conjugating to lysine, histidine and cysteine, and it can be toxic to neurons via this mechanism (Mark et al. 1997; Keller and Mattson, 1998). Furthermore, Aβ is able to induce the production of 4-HNE in hippocampal neurons and can inhibit DNA, RNA and protein synthesis as well as alter the activity of degradative, glycolytic and transport proteins (Mark et al. 1997). This will then result in the disruption of calcium homeostasis, reduction in the activity of Na+/K+-ATPase and impairment of glucose transport, thus increasing neuronal vulnerability to excitotoxicity, leading to neuronal cell death (Mark et al. 1997). Consistent with the latter scenario, our findings demonstrate that Aβ led to a significant increase in the level of 4-HNE in primary cortical neurons. IVIg treatment ameliorated Aβ-induced accumulation of 4-HNE in cultured neurons. Interestingly, 4-HNE has been shown to activate both the p38MAPK and JNK signaling pathways, which can mediate neuronal apoptosis (Tamagno et al. 2003). Therefore, it is postulated that the ability of IVIg to reduce the production of 4-HNE induced by Aβ leads to inhibition of both p38MAPK and JNK signaling pathways, thereby protecting neurons against Aβ-induced oxidative injury.

Members of the Bcl-2 gene family, such as B-cell-leukemia/lymphoma-2-associated X protein (Bax), Bcl-2 and B-cell lymphoma-extra large (Bcl-xL), play pivotal roles in determining whether cells survive or die under stressful conditions. Bcl-2 is able to prevent ischemia-induced neuronal death (Mattson et al. 2000). The pro-apoptotic Bax and anti-apoptotic Bcl-2 have been shown to be involved in the Aβ-induced neurotoxic mechanism (Paradis et al. 1996). In this study, ischemic conditions and exposure to Aβ decreased Bcl-2 protein levels, consistent with several studies suggesting that both ischemia and Aβ lead to downregulation of Bcl-2 protein (Paradis et al. 1996). The results obtained in this study demonstrated that IVIg is able to support Bcl-2 protein levels in primary neurons subjected to ischemic conditions or exposure to Aβ. Upregulation of Bcl-2 expression suggested that IVIg is able to prevent apoptosis.

We have shown previously that after 3 days, the degree of brain damage and neurological deficit resulting from cerebral I/R is reduced in mice treated with IVIg compared to vehicle-treated controls (Arumugam et al. 2007). Here, we report similar results obtained using in vivo magnetic resonance imaging method. Our in vivo data also confirm the neuroprotective mechanisms of IVIg observed in vitro as IVIg preserved the numbers of MAP2-positive neuronal cells in both core and infarct areas compared to vehicle-treatment. Furthermore, stroke-induced neurological deficit was also improved with IVIg treatment. Our findings suggest that IVIg targets multiple cell death pathways in ischemic stroke and Aβ toxicity to protect neurons. These studies further suggest that IVIg may prove effective in reducing neuronal cell death and brain injury in ischemic stroke and AD.

Supplementary Material

Supp Fig S1 -S3

ACKNOWLEDGEMENT

This work was supported by the National Heart Foundation of Australia for a Grant-In-Aid (G 09B 4272), the Australian National Health and Medical Research Council grant (NHMRC APP1008048), ARC Future Fellowship (FT100100427) awarded to TVA, and the National Institute on Aging Intramural Research Program.

Abbreviations

4HNE

4-Hydroxynonenal

A β

amyloid beta

AD

Alzheimer’s disease

ADC

apparent diffusion coefficient

APP

amyloid precursor protein

Bax

B-cell-leukemia/lymphoma-2-associated X protein

Bcl-XL

B-cell lymphoma-extra large

Bcl-2

B-cell lymphoma

BSA

bovine serum albumin

CCA

common carotid artery

DMSO

dimethyl sulfoxide

ECA

external carotid artery

GD

glucose deprivation

HFIP

1,1,1,3,3,3-Hexafluro-2-propanol

ICA

internal carotid artery

IVIg

intravenous immunoglobulin

I/R

ischemia/reperfusion

JNK

c-Jun NH2-terminal kinase

MAP2

microtubule associated protein

MCA

middle cerebral artery

MCAO/R

middle cerebral artery occlusion/ reperfusion

NFκB

nuclear factor kappa B

OGD

oxygen and glucose deprivation

MAPK

mitogen activated protein kinase.

Footnotes

AUTHORSHIP CONTRIBUTIONS

Participated in research design: Arumugam, Mattson, Basta, Tang, Magnus, Jo, Chan, Sobey, Tang and Reutens.

Conducted experiments: Widiapradja, Vegh, Lok, Manzanero, Thundyil, Cheng and Gelderblom.

Performed data analysis: Widiapradja, Vegh, Lok, Manzanero and Arumugam

Wrote or contributed to the writing of the manuscript: Arumugam, Mattson, Reutens, Sobey, Jo, Chan, Magnus and Basta

The authors declare no conflict of interest.

A.W., V.V., K.Z.L. contributed equally to this work.

REFERENCE

  1. Alves da Costa C, Mattson MP, Ancolio K, Checler F. The C-terminal fragment of presenilin 2 triggers p53-mediated staurosporine-induced apoptosis, a function independent of the presenilinase-derived N-terminal counterpart. J Biol Chem. 2003;278:12064–12069. doi: 10.1074/jbc.M212379200. [DOI] [PubMed] [Google Scholar]
  2. Anthony RM, Kobayashi T, Wermeling F, Ravetch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature. 2011;475:110–113. doi: 10.1038/nature10134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arumugam TV, Cheng YL, Choi Y, et al. Evidence that gamma-secretase-mediated Notch signaling induces neuronal cell death via the nuclear factor-kappaB-Bcl-2-interacting mediator of cell death pathway in ischemic stroke. Mol Pharmacol. 2011;80:23–31. doi: 10.1124/mol.111.071076. [DOI] [PubMed] [Google Scholar]
  4. Arumugam TV, Salter JW, Chidlow JH, Ballantyne CM, Kevil CG, Granger DN. Contributions of LFA-1 and Mac-1 to brain injury and microvascular dysfunction induced by transient middle cerebral artery occlusion. Am J Physiol Heart Circ Physiol. 2004;287:H2555–2560. doi: 10.1152/ajpheart.00588.2004. [DOI] [PubMed] [Google Scholar]
  5. Arumugam TV, Selvaraj PK, Woodruff TM, Mattson MP. Targeting ischemic brain injury with intravenous immunoglobulin. Expert Opin Ther Targets. 2009;12:19–29. doi: 10.1517/14728222.12.1.19. [DOI] [PubMed] [Google Scholar]
  6. Arumugam TV, Tang SC, Lathia JD, et al. Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death. Proc Natl Acad Sci U S A. 2007;104:14104–14109. doi: 10.1073/pnas.0700506104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Arumugam TV, Woodruff TM, Lathia JD, Selvaraj PK, Mattson MP, Taylor SM. Neuroprotection in stroke by complement inhibition and immunoglobulin therapy. Neuroscience. 2009;158:1074–1089. doi: 10.1016/j.neuroscience.2008.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Aschermann S, Lux A, Baerenwaldt A, Biburger M, Nimmerjahn F. The other side of immunoglobulin G: suppressor of inflammation. Clin Exp Immunol. 2010;160:161–167. doi: 10.1111/j.1365-2249.2009.04081.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Baerenwaldt A, Biburger M, Nimmerjahn F. Mechanisms of action of intravenous immunoglobulins. Expert Rev Clin Immunol. 2010;6:425–434. doi: 10.1586/eci.10.9. [DOI] [PubMed] [Google Scholar]
  10. Basta M, Langlois PF, Marques M, Frank MM, Fries LF. High-dose intravenous immunoglobulin modifies complement-mediated in-vivo clearance. Blood. 1989;74:326–333. [PubMed] [Google Scholar]
  11. Basta M, Kirshbom P, Frank MM, Fries LF. Mechanism of therapeutic effect of high-dose intravenous immunoglobulin. Attenuation of acute, complement-dependent immune damage in a guinea pig model. J Clin Investig. 1989;84:1974–1978. doi: 10.1172/JCI114387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Basta M, Van Goor F, Luccioli S, et al. F(ab)’2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins. Nat Med. 2003;9:431–438. doi: 10.1038/nm836. [DOI] [PubMed] [Google Scholar]
  13. Basta M. Ambivalent effect of immunoglobulins on the complement system: activation versus inhibition. Mol Immunol. 2008;45:4073–4079. doi: 10.1016/j.molimm.2008.07.012. [DOI] [PubMed] [Google Scholar]
  14. Bederson JB, Pitts LH, Tsuiji M. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986;17:472–476. doi: 10.1161/01.str.17.3.472. [DOI] [PubMed] [Google Scholar]
  15. Dodel R, Hampel H, Depboylu C, et al. Human antibodies against amyloid beta peptide: a potential treatment for Alzheimer’s disease. Ann Neurol. 2002;52:253–256. doi: 10.1002/ana.10253. [DOI] [PubMed] [Google Scholar]
  16. Dodel RC, Du Y, Depboylu C, et al. Intravenous immunoglobulins containing antibodies against beta-amyloid for the treatment of Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2004;75:1472–1474. doi: 10.1136/jnnp.2003.033399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Durandy A, Kaveri SV, Kuijpers TW, Basta M, Miescher S, Ravetch JV, Rieben R. Intravenous immunoglobulins--understanding properties and mechanisms. Clin Exp Immunol. 2009;158(Suppl 1):2–13. doi: 10.1111/j.1365-2249.2009.04022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fillit H, Hess G, Hill J, Bonnet P, Toso C. IV immunoglobulin is associated with a reduced risk of Alzheimer disease and related disorders. Neurology. 2009;73:180–185. doi: 10.1212/WNL.0b013e3181ae7aaf. [DOI] [PubMed] [Google Scholar]
  19. Guan QH, Pei DS, Liu XM, Wang XT, Xu TL, Zhang GY. Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis. Brain Res. 2006;1092:36–46. doi: 10.1016/j.brainres.2006.03.086. [DOI] [PubMed] [Google Scholar]
  20. Inta I, Paxian S, Maegele I, et al. Bim and Noxa are candidates to mediate the deleterious effect of the NF-kappa B subunit RelA in cerebral ischemia. J Neurosci. 2006;26:12896–12903. doi: 10.1523/JNEUROSCI.3670-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaneko Y, Nimmerjahn F, Ravetch JV. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science. 2006;313:670–673. doi: 10.1126/science.1129594. [DOI] [PubMed] [Google Scholar]
  22. Keller JN, Mattson MP. Roles of lipid peroxidation in modulation of cellular signaling pathways, cell dysfunction, and death in the nervous system. Rev Neurosci. 1998;9:105–116. doi: 10.1515/revneuro.1998.9.2.105. [DOI] [PubMed] [Google Scholar]
  23. Kotlan B, Stroncek DF, Marincola FM. Intravenous immunoglobulin-based immunotherapy: an arsenal of possibilities for patients and science. Immunotherapy. 2009;1:995–1015. doi: 10.2217/imt.09.67. [DOI] [PubMed] [Google Scholar]
  24. Linnik MD, Zahos P, Geschwind MD, Federoff HJ. Intravenous immunoglobulin-based immunotherapy: an arsenal of possibilities for patients and science. Immunotherapy. Stroke. 1995;26:1670–1674. [Google Scholar]
  25. Legos JJ, Erhardt JA, White RF, et al. SB 239063, a novel p38 inhibitor, attenuates early neuronal injury following ischemia. Brain Res. 2001;892:70–77. doi: 10.1016/s0006-8993(00)03228-5. [DOI] [PubMed] [Google Scholar]
  26. Magga J, Puli L, Pihlaja R, et al. Human intravenous immunoglobulin provides protection against Aβ toxicity by multiple mechanisms in a mouse model of Alzheimer’s disease. J Neuroinflammation. 2010;7:90. doi: 10.1186/1742-2094-7-90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mark RJ, Lovell MA, Markesbery WR, Uchida K, Mattson MP. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide. J Neurochem. 1997;68:255–264. doi: 10.1046/j.1471-4159.1997.68010255.x. [DOI] [PubMed] [Google Scholar]
  28. Mattson MP, Partin J, Begley JG. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 1998;807:167–176. doi: 10.1016/s0006-8993(98)00763-x. [DOI] [PubMed] [Google Scholar]
  29. Mattson MP, Culmsee C, Yu ZF. Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res. 2000;301:173–187. doi: 10.1007/s004419900154. [DOI] [PubMed] [Google Scholar]
  30. Mattson MP. Roles of the lipid peroxidation product 4-hydroxynonenal in obesity, the metabolic syndrome, and associated vascular and neurodegenerative disorders. Exp Gerontol. 2009;44:625–633. doi: 10.1016/j.exger.2009.07.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Munoz L, Ammit AJ. Targeting p38 MAPK pathway for the treatment of Alzheimer’s disease. Neuropharmacology. 2010;58:561–568. doi: 10.1016/j.neuropharm.2009.11.010. [DOI] [PubMed] [Google Scholar]
  32. Okun E, Arumugam TV, Tang SC, et al. The organotellurium compound ammonium trichloro(dioxoethylene-0,0′) tellurate enhances neuronal survival and improves functional outcome in an ischemic stroke model in mice. J Neurochem. 2007;102:1232–1241. doi: 10.1111/j.1471-4159.2007.04615.x. [DOI] [PubMed] [Google Scholar]
  33. Paradis E, Douillard H, Koutroumanis M, Goodyer C, LeBlanc A. Amyloid beta peptide of Alzheimer’s disease downregulates Bcl-2 and upregulates bax expression in human neurons. J Neurosci. 1996;16:7533–7539. doi: 10.1523/JNEUROSCI.16-23-07533.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prehn JH, Bindokas VP, Marcuccilli CJ, Krajewski S, Reed JC, Miller RJ. Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons. Proc Natl Acad Sci U S A. 1994;91:12599–12603. doi: 10.1073/pnas.91.26.12599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ramin M, Azizi P, Motamedi F, Haghparast A, Khodagholi F. Inhibition of JNK phosphorylation reverses memory deficit induced by β-amyloid (1-42) associated with decrease of apoptotic factors. Behav Brain Res. 2011;217:424–431. doi: 10.1016/j.bbr.2010.11.017. [DOI] [PubMed] [Google Scholar]
  36. Relkin NR, Szabo P, Adamiak B, et al. 18-Month study of intravenous immunoglobulin for treatment of mild Alzheimer disease. Neurobiol Aging. 2009;30:1728–1736. doi: 10.1016/j.neurobiolaging.2007.12.021. [DOI] [PubMed] [Google Scholar]
  37. Sahara N, Murayama M, Lee B, et al. Active c-jun N-terminal kinase induces caspase cleavage of tau and additional phosphorylation by GSK-3beta is required for tau aggregation. Eur J Neurosci. 2008;27:2897–2906. doi: 10.1111/j.1460-9568.2008.06258.x. [DOI] [PubMed] [Google Scholar]
  38. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor. Science. 2001;291:484–486. doi: 10.1126/science.291.5503.484. [DOI] [PubMed] [Google Scholar]
  39. Stine WB, Jr, Dahlgren KN, Krafft GA, LaDu MJ. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem. 2003;278:11612–11622. doi: 10.1074/jbc.M210207200. [DOI] [PubMed] [Google Scholar]
  40. Tamagno E, Robino G, Obbili A, et al. H2O2 and 4-hydroxynonenal mediate amyloid beta-induced neuronal apoptosis by activating JNKs and p38MAPK. Exp Neurol. 2003;180:144–155. doi: 10.1016/s0014-4886(02)00059-6. [DOI] [PubMed] [Google Scholar]
  41. Tamatani M, Mitsuda N, Matsuzaki H, et al. A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-kappaB and Bcl-2. J Neurochem. 2000;75:683–693. doi: 10.1046/j.1471-4159.2000.0750683.x. [DOI] [PubMed] [Google Scholar]
  42. Tang SC, Arumugam TV, Xu X, et al. Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A. 2007;104:13798–13803. doi: 10.1073/pnas.0702553104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tang SC, Lathia JD, Selvaraj PK, et al. Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid beta-peptide and the membrane lipid peroxidation product 4-hydroxynonenal. Exp Neurol. 2008;213:114–121. doi: 10.1016/j.expneurol.2008.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thundyil J, Tang SC, Okun E, et al. Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death. Exp Transl Stroke Med. 2010;2:e15. doi: 10.1186/2040-7378-2-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vukic V, Callaghan D, Walker D, et al. Expression of inflammatory genes induced by beta-amyloid peptides in human brain endothelial cells and in Alzheimer’s brain is mediated by the JNK-AP1 signaling pathway. Neurobiol Dis. 2009;34:95–106. doi: 10.1016/j.nbd.2008.12.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Woodruff TM, Thundyil J, Tang SC, Sobey CG, Taylor SM, Arumugam TV. Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener. 2011;6:e11. doi: 10.1186/1750-1326-6-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yang ZH, Sun K, Suo WH, et al. N-stearoyltyrosine protects primary neurons from Aβ-induced apoptosis through modulating mitogen-activated protein kinase activity. Neuroscience. 2010;169:1840–1847. doi: 10.1016/j.neuroscience.2010.05.069. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supp Fig S1 -S3
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