Inhibition of p38α MAPK restores neuronal p38γ MAPK and ameliorates synaptic degeneration in a mouse model of DLB/PD

Abstract

Alterations in the p38 mitogen-activated protein kinases (MAPKs) play an important role in the pathogenesis of dementia with Lewy bodies (DLB) and Parkinson's disease (PD). Activation of the p38α MAPK isoform and mislocalization of the p38γ MAPK isoform are associated with neuroinflammation and synaptic degeneration in DLB and PD. Therefore, we hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. To test this hypothesis, we treated in vitro cellular and in vivo mouse models of DLB/PD with SKF-86002, a compound that attenuates inflammation by inhibiting p38α/β, and then investigated the effects of this compound on p38γ and neurodegenerative pathology. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-synuclein. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-synuclein in mice overexpressing human α-synuclein. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-synuclein mutation) and healthy controls. Treatment with SKF-86002 ameliorated α-synuclein-induced neurodegeneration in these neurons only when microglia were pretreated with this compound. However, direct treatment of neurons with SKF-86002 did not affect α-synuclein-induced neurotoxicity, suggesting that SKF-86002 treatment inhibits α-synuclein-induced neurotoxicity mediated by microglia. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD.

Fig. 1.. SKF-86002 pharmacokinetics and target engagement in the CNS of mice.

(A) Concentration of SKF in the blood and brain of the non-tg mouse was determined by pharmacokinetic analysis (n = 3 per group). (B to D) Immunoblot analysis of non-tg mice brain homogenates administrated with indicated amounts of SKF is shown. Cytosolic (B) and particulate (C) fractions were probed with phosphor-Atf2 (p-Atf2), total Atf2, and β-actin. Whole brain lysates (D) were probed with phosphor-p38 (p-p38), total p38, and β-actin. Phosphor-Atf2 and phosphor-p38 band intensities were determined by densitometric quantification and normalized to total Atf2 and p38, respectively. Data are means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 (one-way ANOVA with Tukey’s multiple comparison post hoc test). n = 3 per group. (E) Immunoblot analysis of p38 kinase assay is shown. Human recombinant ATF2 was incubated with non-tg mouse brain homogenate in the presence and absence of ATP and SKF. The blot was probed with phosphor-ATF2 and total ATF2. (F to J) Immunoblot analysis of non-tg and α-syn tg mice administrated with either vehicle (Veh) or SKF. Whole brain lysates (F) and cytosolic/particulate fractions (G) were probed with phosphor-Atf2, total Atf2, phosphor-p38, and β-actin. Phosphor-Atf2 (H) and phosphor-p38 (I and J) band intensities were determined by densitometric quantification. Phosphor-Atf2 and phosphor-p38 were normalized to total Atf2 and β-actin, respectively. Data are means ± SEM. *P < 0.05 (two-way ANOVAwith Tukey’s multiple comparison post hoc test). n = 4 per group. (K) Representative images from immunohistochemical analysis for phosphor-Atf2 in the neocortices of injected mice are shown. Scale bar, 10 μm.

Fig. 2.. The p38 inhibitor SKF rectifies elevated p38α and neuroinflammation in α-syn tg mice.

Non-tg and α-syn tg mice were injected with vehicle or SKF (20 mg/kg) five times weekly for 12 weeks. (A) Representative images from immunohistochemical stainings for p38α in the neocortices, hippocampi, and striata of injected mice are shown. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (B to D) Percentage of p38α-positive cell area was analyzed in the neocortices (Ctx) (B), hippocampi (HP) (C), and striata (ST) (D) of the mice. Data are means ± SEM. ***P < 0.001 and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF. (E) Representative images from immunohistochemical analysis for Gfap in the neocortices, hippocampi, and striata of injected mice are shown. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (F to H) Optical density of Gfap immunoreactivity was analyzed in the neocortices (F), hippocampi (G), and striata (H) of the mice. Data are means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF. (I) Representative images from immunohistochemical stainings for Iba-1 in the neocortices, hippocampi, and striata of injected mice are shown. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (J to L) Number of Iba-1–positive cells was analyzed in the neocortices (J), hippocampi (K), and striata (L) of the mice. Data are means ± SEM. *P < 0.05 and **P < 0.01 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg–SKF. (M to Q) Quantitative gene expression analysis from the brains of mice injected with vehicle or SKF. Expressions of Tnf (M), Il-6 (N), Il-13 (O), Cxcl1 (P), and Ccl2 (Q) were analyzed by quantitative PCR. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 7 for non-tg + Veh, n = 6 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF.

Fig. 3.. The effects of p38 inhibitors in α-syn–induced microglial cytokine gene expression.

Primary microglia from WT mice were pretreated with control, SKF, p38 MAPK inhibitor IV (IV), VX-745 (VX), SB 706504 (SB), or DBM 1285 (DBM) for 30 min, followed by challenge with LZCM (control) or αSCM for 2 hours. Microglial cytokine gene expression was determined by quantitative PCR. (A) Schematic illustration of experiment procedure. (B to D) Expressions of Il-1β (B), Il-6 (C), and Il-10 (D) were analyzed by quantitative PCR. Data are means ± SEM. ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 6 per group.

Fig. 4.. SKF rescues the aberrant expression and localization of p38γ in brains of α-syn tg mice.

Non-tg and α-syn tg mice were injected with vehicle or SKF (20 mg/kg) five times weekly for 12 weeks. (A) Representative images from immunohistochemical stainings for p38γ in the neocortices, hippocampi, and striata of injected mice. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (B to D) Percentage of p38γ-positive neuropil area analyzed in the neocortices (B), hippocampi (C), and striata (D) of injected mice. (E to G) Percentage of p38γ-positive cell area analyzed in the neocortices (E), hippocampi (F), and striata (G) of injected mice. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (two-way ANOVAwith Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF. (H to J) Immunoblot analysis of injected mice. Cytosolic and particulate fractions were probed with total p38γ and β-actin (H). Cytosolic (I) and particulate (J) total p38γ band intensity was determined by densitometric quantification and normalized to β-actin. Data are means ± SEM. *P < 0.05 and **P < 0.01 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 4 per group.

Fig. 5.. The p38 inhibitor SKF rectifies abnormal accumulation of α-syn and mislocalization of p38 in brains of α-syn tg mice.

Non-tg and α-syn tg mice were injected with vehicle or SKF (20 mg/kg) five times weekly for 12 weeks. (A) Representative images from immunohistochemical stainings for α-syn in the neocortices, hippocampi, and striata of injected mice are shown. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (B to D) Percentage of α-syn–positive neuropil area analyzed in the neocortices (B), hippocampi (C), and striata (D) of injected mice. (E to G) Number of α-syn–positive cells analyzed in the neocortices (E), hippocampi (F), and striata (G) of injected mice. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF. (H to J) Immunoblot analysis of injected mice. (H) Whole brain lysates were probed with α-syn and β-actin. Band intensities of monomeric (I) and oligomeric (J) α-syn were determined by densitometric quantification and normalized to β-actin. Data are means ± SEM. **P < 0.01 and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 4 per group. (K and L) Amounts of human α-syn in cytosolic (K) and particulate (L) fractions were determined by ELISA. Data are means ± SEM. **P < 0.01 (unpaired two-tailed Student’s t test). n = 6 per group. (M and N) Representative images from double immunostaining analysis for p38γ with synaptophysin (M) and p38γ with α-syn (N) in the neocortices of injected mice. Scale bars, 10 μm (low magnification) and 5 μm (high magnification). (O and P) Percentages of p38γ/synaptophysin– (O) and p38γ/α-syn (P)–positive cells were analyzed in the neocortices of the injected mice. Data are means ± SEM. *P < 0.05, ***P < 0.001, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 4 per group.

Fig. 6.. The p38 inhibitor SKF reduces neurodegeneration in α-syn tg mice.

Non-tg and α-syn tg mice were injected with vehicle or SKF (20 mg/kg) five times weekly for 12 weeks. (A) Representative images from immunohistochemical stainings for NeuN in the neocortices, hippocampi, and striata of injected mice are shown. Scale bars, 250 μm (low magnification) and 25 μm (high magnification). (B to D) Number of NeuN-positive cells was analyzed in the neocortices (B), hippocampi (C), and striata (D) of injected mice. (E) Representative images from immunohistochemical stainings for TH in the striata and substantia nigrae of injected mice are shown. Scale bars, 25 μm (high magnification). (F and G) Optical density of TH in striatum (F) and number of TH-positive cells in substantia nigra (G) were analyzed in the injected mice. Data are means ± SEM. *P < 0.05 and **P < 0.01 (two-way ANOVAwith Tukey’s multiple comparison post hoc test). n = 6 for non-tg + Veh, n = 7 for non-tg + SKF, n = 5 for α-syn tg + Veh, and n = 6 for α-syn tg + SKF.

Fig. 7.. The p38 inhibitor SKF restores p38γ and α-syn mislocalization and neurodegeneration.

Healthy control and A53T-mutant patient–derived human iPSC neurons and glia mixed cultures were treated with low (1 μM) or high (50 μM) concentrations of SKF. After 1 or 18 hours of incubation, the cells were analyzed by double immunostaining analysis. (A and B) Representative images from double immunostaining analysis of p38γ and α-syn in SKF-treated cells after 1 hour (A) and 18 hours (B) of incubations. Arrows indicate neurites. Scale bars, 50 μm (low magnification) and 25 μm (high magnification). (C and D) Pixel intensity of p38γ in the cell body (C) and percentage of α-syn–positive neurite area (D) were analyzed. ****P < 0.0001 (unpaired two-tailed Student’s t test). n = 8 per group. (E and F) Representative images from double immunostaining of synaptophysin and Map2 in SKF-treated cells after 1 hour (E) and 18 hours (F) of incubations. Scale bars, 50 μm (low magnification) and 25 μm (high magnification). (G and H) Percentages of synaptophysin-positive (G) and Map2-positive (H) area are analyzed. *P < 0.05 and **P < 0.01 (unpaired two-tailed Student’s t test). n = 8 per group.

Fig. 8.. The p38 inhibitor SKF reduces α-syn–mediated microglial neurotoxicity.

(A) Illustrated is the experimental design to obtain MgCM and to test microglial neurotoxicity. Non-tg mouse primary microglia were pretreated with either control or SKF for 30 min, followed by challenge with LZCM (control) or αSCM. After 1-hour incubation, cells were incubated with fresh neurobasal media for additional 6 hours, and cultured media were collected. (B) Non-tg mouse primary neurons were treated with MgCM-control, MgCM-SKF, MgCM-αSCM, or MgCM-SKF-αSCM for 18 hours. The neuronal viability was determined by CyQUANT assay. Data are means ± SEM. **P < 0.01 and ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 4 per group. (C) Non-tg mouse primary neurons were incubated with either MgCM-control or MgCM-αSCM in the presence and absence of SKF. (D) Neuronal viability was determined by cyquant assay. Data are means ± SEM. ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparison post hoc test). n = 4 per group.