Background
Extensive evidence, both clinical and preclinical, implicates neuroinflammation and overproduction of proinflammatory cytokines as a contributor to pathophysiology of chronic neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease, and multiple sclerosis [for review, see: [
1]]. Proinflammatory cytokine overproduction has also been documented as detrimental to recovery in acute brain injuries such as trauma or stroke [
2‐
5]. In the brain, activated microglia are a major mediator of neuroinflammation and can release a number of potentially neurotoxic substances, such as reactive oxygen species, nitric oxide, and various proinflammatory cytokines, of which two main proinflammatory cytokines TNFα and IL-1β are generally considered primary mediators leading to neurotoxicity [for detailed reviews on microglia, see: [
6,
7]].
There are many critical roles for innate immunity, and thereby the primary effector cells, microglia, in the classically immune privileged CNS. For example, microglia are rapid responders to local tissue stressors [
8,
9], can efficiently clear apoptotic cells during neurodevelopment [
10], and can promote neuro-repair through the production of growth factors [
7]. The spectrum of activated microglia phenotypes is diverse and generally beneficial. It is only when the activation becomes exaggerated or dysregulated does the response become neurotoxic. Therefore, it is of critical importance to elucidate the mechanisms that are specifically involved in the dysregulated response of microglia which contribute to neuronal damage.
Intracellular signal transduction cascades regulate the production of proinflammatory cytokines. By targeting a specific signal transduction pathway it is possible to determine if a pathway is involved in the dysregulated response that is neurotoxic and if the dysregulated response is amenable to intervention. One of the most well established signal transduction cascades that regulate the production of proinflammatory cytokines in peripheral tissue inflammatory diseases, such as rheumatoid arthritis, is the p38 mitogen activated protein kinase (MAPK) family [
11,
12]. The p38 MAPK family consists of at least four isoforms (p38α, β, δ, γ), which are encoded by separate genes, expressed in different tissues and have distinct functions [
13]. Activation of p38 MAPK signaling has been shown to regulate gene expression and lead to increased production of proinflammatory cytokines by a number of different mechanisms [for review, see: [
14]]. The p38 MAPK pathway has been suggested to play a central role in various pathological CNS conditions including cerebral ischemia [
15,
16] and Parkinson's disease [
17‐
19], as well as in AD [
20,
21], where postmortem studies find p38 MAPK activation occurs at the very early stage of the disease [
20,
22].
Previously we have shown using both a pharmacological approach with a selective small molecule p38α MAPK inhibitor and a genetic approach with primary microglia that are deficient in p38α that the α isoform of p38 MAPK is critical for the production of IL-1β and TNFα from activated microglia [
23]. Moreover, suppression of p38α MAPK with the small molecule inhibitor in an AD-relevant mouse model was also found to decrease brain proinflammatory cytokine production, and attenuate synaptic protein loss [
24]. These data suggested that microglia p38α MAPK is critical to inflammation-induced neurotoxicity. In the current study, we explored whether there is a causative link between microglia p38α MAPK signaling and neuronal damage, as well as a potential mechanism for microglia-dependent neurotoxicity. We used primary microglia from either wild-type (WT) mice or from p38α MAPK conditional knockout (KO) mice in co-culture with cortical neurons from WT mice. In WT microglia/neuron co-cultures, LPS treatment led to a significant increase in TNFα production, loss of synaptic proteins, and neuronal death. Neurons in co-culture with p38α-deficient microglia showed reduced LPS-induced TNFα production and were protected against synaptic loss and neuronal death. The mechanism of neurotoxicity was explored by showing that addition of a neutralizing TNFα antibody prevented neuronal degeneration in WT microglia-neuron co-cultures, and addition of recombinant TNFα to KO microglia-neuron co-cultures led to enhanced neuronal degeneration. Our data support the conclusion that activation of p38α MAPK and the downstream overproduction of the proinflammatory cytokine TNFα play a major role in the dysregulated microglial response to LPS that leads to neuron degeneration.
Discussion
In the current study, we used microglia/neuron co-cultures to document several important findings about the mechanisms by which activated microglia can produce neurodegenerative responses. First, the importance of microglia p38α MAPK signaling was demonstrated by the observations that neurons in co-culture with p38α-deficient microglia were protected against LPS-induced neurotoxicity, synaptic protein loss, and neurite degeneration. Second, p38α-dependent microglia TNFα production was shown to be involved in the mechanism of the LPS-induced neuron damage by the findings that p38α KO microglia produce much less TNFα in response to LPS compared to WT microglia, that adding back TNFα to p38α KO microglia increases the LPS-induced neurotoxicity, and that neutralization of TNFα in WT microglia decreases the LPS-induced neuron damage. Altogether, our results demonstrate the critical importance of the p38α MAPK signaling pathway and overproduction of the proinflammatory cytokine TNFα in the dysregulated microglia inflammatory responses to an LPS stressor, leading to microglia-induced neuronal dysfunction.
Our demonstration that microglia p38α MAPK signaling is important in the mechanism of LPS-induced neuron damage is consistent with numerous findings that have implicated p38 MAPK activation in the process of neuronal death in a variety of neurodegenerative disorders. In addition, our studies here using cell-selective, isoform-specific KO mice extend previous findings by showing that the p38α MAPK isoform in microglia is a key mediator of LPS-induced neuronal and synaptic dysfunction. We also provide evidence that one mechanism by which LPS activation of microglia p38α MAPK signaling leads to neuron death is through up-regulation of the proinflammatory cytokine TNFα.
The p38 MAPK family consists of four major isoforms (p38α, β, δ, γ) that have different cell and tissue expression patterns, substrate specificities, and functions [for reviews, see: [
14,
28]]. The patterns of expression and activation of the p38α isoform in peripheral immune cells [
29,
30] suggested that this isoform might play a major role in the inflammatory response. Early attempts using genetic KO approaches to explore the role of p38α in inflammatory responses were hampered because of embryonic lethality seen with global KO of p38α. However, a number of more recent studies have used conditional ablation of p38α in specific cell types to provide direct evidence that the p38α isoform is of central importance for many peripheral inflammatory responses, such as inflammation-induced arthritic bone loss [
31], inflammatory skin injuries [
13], inflammatory responses of myeloid cells in an experimental colitis model [
32], immune cell recruitment and pathogen clearance in intestinal epithelial cells [
33], and LPS-induced cytokine production in macrophages [
25]. These and other studies using selective p38α inhibitors and drug-resistant forms of the kinase have demonstrated the importance of p38α signaling in mediating peripheral inflammatory responses [
34‐
37].
Although there is broad agreement that p38α plays a key role in cytokine production and other inflammatory responses in peripheral immune cells, the contribution of p38α to pathological microglial activation and detrimental inflammation in CNS disorders is less well understood. Increasing evidence suggests that p38 signaling cascades contribute to CNS cytokine overproduction and neurodegenerative sequelae [for reviews, see: [
14,
38,
39]], but few studies have tested the specific role of microglia p38α. Expression of the p38α isoform in microglia was reported to increase early after transient global ischemia [
40], and administration of p38 inhibitors reduced infarct volume [
15,
41] and suppressed proinflammatory cytokine production [
41]. We recently demonstrated [
23] a direct linkage between microglia p38α and proinflammatory cytokine production in response to different stressors by showing that inhibition of p38α in microglia with either a pharmacologic or genetic approach suppresses proinflammatory cytokine up-regulation induced by toll-like receptor ligands or beta-amyloid.
In the present study, we explored the consequences of the microglial p38α-dependent proinflammatory cytokine response on neuronal endpoints. By using microglia deficient in p38α, we showed definitively that microglial p38α is critical for LPS-induced neuron dysfunction and we implicated p38α-dependent production of the proinflammatory cytokine TNFα in the mechanism of neuron damage. The potential involvement of TNFα was not unexpected, as this proinflammatory cytokine has been shown to induce neurotoxicity in models of CNS neurodegenerative disorders [
42‐
44], and blocking TNFα signaling can be neuroprotective [
45,
46]. However, TNFα is pleiotropic and can also have neuroprotective functions [for review, see: [
47]]. Multiple factors influence whether TNFα will exert neurotoxic or neuroprotective actions, including the level and duration of expression in a particular cell type or brain region, the microglia activation state, the particular disease or disease stage, the levels of different TNF receptors and adapter proteins, and the upstream activators and downstream effectors in the signaling pathways. Thus, it was somewhat surprising that microglia p38α-dependent production of TNFα in response to an LPS insult appeared to be sufficient to induce neuron death, as evidenced by the observations that anti-TNFα antibody treatment resulted in increased neuronal survival back to control values, and addition of TNFα to KO microglia reduced neuronal survival to the same levels as WT. Altogether, our data demonstrate that microglia p38α activation in response to an LPS stressor stimulus and the consequent dysregulated TNFα signaling can lead to neuron damage.
Of note is our finding that p38α MAPK deficiency in microglia attenuates LPS-induced loss of specific synaptic proteins in the co-cultures. Previous studies have shown a correlation between p38 MAPK activation and a decline in synaptophysin levels in AD transgenic mouse models and in primary microglia and cortical neuron co-cultures stimulated with LPS [
48,
49], and pharmacological inhibition of p38α MAPK significantly reduced TNFα and IL-1β production and prevented synaptophysin loss in an AD mouse model [
24]. Our results here demonstrate for the first time a linkage of p38α MAPK and TNFα to LPS-induced decreases in SNAP25 and drebrin. Because drebrin, a postsynaptic protein found within dendritic spines, is important for spine morphogenesis and maintenance [
50,
51], future studies should examine in more detail the mechanisms by which p38α MAPK influences dendritic pathology and synaptic deterioration such as seen in many neurodegenerative disorders. Future studies should also explore whether microglia p38α MAPK is involved in beneficial responses of activated microglia, as the current study focused only on detrimental consequences of microglia p38α activation.
Methods
Animals
All experiments were conducted in accordance with the principles of animal care and experimentation in the Guide For the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of the University of Kentucky approved the use of animals in this study. C57BL/6 mice were obtained from Harlan Laboratories. The p38α MAPK conditional knockout mice were generated as previously described [
23,
25], following a standard breeding scheme for conditional gene inactivation [
52]. The first exon of the p38α gene (MAPK14) was flanked by two loxP sites. The mice were backcrossed to homozygosity so that both alleles of the p38α gene contained loxP sites (p38α
fl/fl) and maintained on a C57BL/6 background. LysM-Cre mice expressing the Cre recombinase transgene under control of the lysozyme M promoter (B6.129-Lyzs
tm1(cre)Ifo/J) were then crossed with the p38α
fl/fl mice. The LysMCre
+ p38α
fl/fl offspring were then crossed with the p38α
fl/fl mice to generate experimental and control animals. This generates litters where ~50% mice are p38α
fl/fl(+cre) (KO) and ~50% are p38α
fl/fl(-cre) (used as WT controls). The restricted cell-type expression of the lysozyme promoter [
53,
54] results in cell-specific deletion of p38α MAPK in myeloid cells including microglia. Genotyping was performed by Transnetyx, Inc (Cordova, TN).
Primary neuronal culture
Primary neuronal cultures were derived from embryonic day 18, C57BL/6 mice, as previously described [
26]. Briefly, cerebral cortices were dissected and the meninges were removed. Cells were dissociated by trypsinization (0.25% trypsin, 2.21 mM EDTA) for 15 min at 37°C and triturated, followed by passing through a 70 μm nylon mesh cell strainer. The neurons were plated onto poly-D-lysine-coated 12-mm glass coverslips at a density of 5 × 10
4/well in 24 well plates. Neurons were grown in neurobasal medium (Invitrogen) containing 2% B27 supplement (Invitrogen), 0.5 mM L-glutamine, (Mediatech), and 100 IU/ml penicillin, 100 μg/ml streptomycin (Mediatech); no serum or mitosis inhibitors were used. Every 3 days, 50% of the media was replenished with fresh medium. The purity of the primary neuronal cultures was verified as 93% by immunocytochemistry for the neuronal marker NeuN, astrocyte marker GFAP, and microglia marker Iba-1 (data not shown).
Microglia culture
Microglia cultures were prepared as previously described [
23]. Briefly, mixed glial cultures (~95% astrocytes, ~5% microglia) were prepared from the cerebral cortices of 1-3 day old mice. The tissue was trypsinized as above, and the cells were resuspended in glia complete medium [α-minimum essential medium (α-MEM; Mediatech) supplemented with 10% fetal bovine serum (FBS) (US Characterized FBS; Hyclone; Cat no. SH30071.03), 100 IU/ml penicillin, 100 μg/ml streptomycin (Mediatech) and 2 mM L-Glutamine (Mediatech)]. After 10-14 days in culture, microglia were isolated from the mixed glial cultures by the shake-off procedure [
55]. Specifically, loosely attached microglia were shaken off in an incubator shaker at 250 rpm for 2 h at 37°C, the cell-containing medium was centrifuged at 1100 rpm for 3 min, and the cells were seeded onto 12-mm glass coverslip at the density of 2 × 10
4 in 24 well plates, unless otherwise specified. Prior to plating the microglia on the coverslip, three equally spaced 1 mm glass beads (Borosilicate; Sigma) were attached to the coverslip with paraffin wax. The microglia cultures were verified to be > 99% microglia by immunocytochemistry. Microglia were incubated for one day before placing into co-culture with neurons.
Primary neuron/microglia co-culture and cell treatments
Following previously described methods [
26], after 7-9 days in culture, neurons on coverslips were co-cultured with mouse microglia by placing the microglia-containing coverslips cell side down into the neuron-containing wells. In this co-culture system, the microglia and neurons are in close apposition and share the same neurobasal/B27 culture media, but are separated by the 1 mm glass beads and do not have direct cell-cell contact. Lipopolysaccharide (LPS) from
Salmonella typhimurium (Sigma) was resuspended in sterile saline at 100 mg/ml, and was used at a final concentration of 3 ng/ml for all experiments. A rat monoclonal IgG
1, anti-mouse TNFα neutralization antibody (clone # MP6-XT22) with a reported 50% neutralization dose in the range of 0.15-0.75 μg/ml, was reconstituted in sterile PBS according to manufacturer specifications (R&D Systems). A rat IgG
1 monoclonal antibody (clone # 43414) was used as a non-immune isotype control antibody (R&D Systems). Treatment with either antibody occurred 1 h prior to LPS treatment. Recombinant mouse TNFα (aa 80-235; R&D systems) was added at the same time as the LPS treatment.
Neuronal Viability Assay
Neuron viability was assayed by trypan blue exclusion [
26]. Neuron-containing coverslips were incubated with 0.2% trypan blue in Hanks' Balanced Salt Solution (HBSS) for 2 min in 37°C incubator and then rinsed 3 times with HBSS. Neurons were viewed under brightfield microscopy at 200× final magnification. Three to five fields were chosen per coverslip, and a total of 150 to 560 cells were counted per coverslip. Trypan blue-positive and negative neurons were counted per field and the ratio of negative cells to the total cells was taken as the index of neuronal survival rate.
Immunocytochemistry
Cells were fixed with 3.7% formalin containing 0.1% Triton X-100 in PBS for 10 min at room temperature. After washing three times with PBS, the coverslips were incubated with blocking buffer (PBS containing 5% goat serum, 3% bovine serum albumin (BSA; Fisher Scientific), 0.1% Triton X-100) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated with the cells at room temperature for 2 h. Primary antibodies used in this study were: chicken anti-MAP-2 antibody (1:100, Neuromics); mouse anti-NeuN (1:100, Millipore); rat anti-GFAP (1:1000, Invitrogen); rabbit anti-IBA1 (1:1000, Wako); rat anti-CD11b (1:100, Serotec); rat anti-F4/80 (1:100, Serotec); and p38α (1:100, R&D Systems). For detection of primary antibodies, species-appropriate Alexa Fluor® fluorescent conjugated secondary antibodies (1:1000, Invitrogen) were incubated in blocking buffer at room temperature for 2 h. Wide field fluorescent photomicrographs were obtained using a Zeiss Axioplan 2 microscope with an Axiocam MRc5 digital camera (Carl Zeiss).
Western blotting and ELISA assays
Western blotting was performed as previously described [
55]. Briefly, whole cell lysates were prepared in sodium dodecyl sulfate (SDS)- containing sample buffer, and equal volumes of lysates were separated by 10.5-14% SDS-PAGE Criterion precast gel (Bio-Rad Laboratories). Proteins were transferred to nitrocellulose membrane using a dry blotting system (iBlot
® Invitrogen). Blots were probed using reagents and manufacturer recommendations for Odyssey Infrared Imaging system (LI-COR Biosciences), with the following primary antibodies: mouse anti-drebrin (1:5000, Abcam); rabbit anti-PSD95 (1:2000, Cell Signaling); mouse anti-synaptophysin (1:1000, Millipore); rabbit anti-syntaxin 1 (1:10, 000, Millipore), mouse anti-SNAP 25 (1:4000, BD Biosciences); rabbit anti-p38α/β (1:1000, Cell Signaling), and mouse anti-β-Actin (1:10, 000, Cell Signaling). Blots were visualized and analyzed on the Odyssey Infrared imaging system (LI-COR Biosciences), and integrated intensity values were used in statistics.
After 24 h, 48 h, and 72 h in the co-cultures, 20 μl conditioned medium was harvested for TNFα ELISA assay using kits from Meso Scale Discovery (MSD) according to the manufacturer's instructions.
Sholl analysis
The Sholl method [
27] was used in the quantification of MAP-2 labeled neurites. A series of concentric circles were drawn at 10 μm intervals starting with a diameter of 20 μm to a final diameter of 200 μm. Intersections of smooth or blebbed neurites with the concentric circles were counted. The total number of intersections for each neuron was plotted as a measure of neurite arborization. Per experimental condition, 20-30 neurons were analyzed from two independent experiments by an observer blinded to treatment conditions.
Statistics
Statistical analysis was conducted using GraphPad prism software V.5 (GraphPad Software, La Jolla, CA). Unless otherwise indicated, values are expressed as mean ± SEM. Groups of two were compared by unpaired t-Test. One-way ANOVA followed by Bonferroni's multiple comparison test was used for comparisons among three or more groups. Statistical significance was defined as p < 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BX, ADB and LVE designed the studies. BX performed the experiments in cell culture. BX and ADB performed the data analysis. BX and ADB jointly drafted the manuscript together with LVE. All authors read and approved the final version. BX and ADB contributed equally to this study.