Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease

Fyn kinase is rapidly activated in microglial cells and in the ventral midbrain following inflammogen stimulation. A, Fyn kinase assay shows that Fyn activity was highly induced in BV2 microglia treated with 1 μg/ml LPS for 10, 15, and 30 min. *p < 0.05. **p < 0.01. B, Immunoblots showing a concomitant rise in p-Y416 SFK levels in BV2 cell lysates after LPS treatment. C, D, Immunoprecipitation studies revealed that WT Fyn, but not activation loop tyrosine-mutant Fyn (Y417A Fyn), when overexpressed in BV2 microglia, was activated following LPS stimulation. E, F, Treatment of (E) primary microglia with LPS and TNFα (F) for 15 and 30 min increased p-Y416 SFK levels in primary microglia obtained from Fyn^+/+, but not Fyn^−/− mice, identifying Fyn as the primary Src family kinase that was activated by inflammogen stimulation. G, Pretreatment of primary microglia with the TLR-signaling antagonist IAXO-101 or the TNFα receptor decoy etanercept abolished Fyn activation by LPS or TNFα stimulation (p44/42 phosphorylation used as marker for early microglial activation). H, Immunocytochemistry of LPS-treated WT primary microglia showing that activated Fyn expression greatly increased and was localized preferentially to the membrane periphery of the microglial cell. Scale bar, 20 μm. I, Immunoblots of ventral midbrain lysates showed that peripheral administration of the inflammogen LPS (5 mg/kg) increased p-Y416 SFK levels in Fyn^+/+, but not in Fyn^−/− ventral midbrain tissues.

Fyn contributes to LPS- and TNFα-induced tyrosine phosphorylation and activation of PKCδ in primary microglia. Western blot analysis revealed that stimulation of microglia with LPS (A, B) and TNFα (C, D) induced a time-dependent increase in p-Y311 PKCδ levels in wild-type but not Fyn^−/− microglia. *p < 0.05. **p < 0.01. E, LPS-induced PKCδ kinase activity was reduced in Fyn^−/− microglial lysates in contrast to wild-type lysates, as measured by PKCδ kinase assay. F, G, Coimmunoprecipitation studies showed that LPS stimulation elicited a physical interaction between Fyn and PKCδ in WT Fyn-transfected BV2 microglial cells.

The Fyn-PKCδ signaling axis mediates MAP kinase activation in microglial cells. A, B, Immunoblot analysis demonstrated diminished LPS-induced p38 and p44/42 (ERK) phosphorylation in Fyn^−/− and PKCδ^−/− microglia. *p < 0.05. ***p < 0.001. C, D, Diminished TNFα-induced p38 and p44/42 (ERK) phosphorylation in Fyn^−/− and PKCδ^−/− microglia. *p < 0.05.

Fyn contributes to inflammogen-mediated NFκB pathway activation in microglial cells. A, B, Immunoblot analyses of whole-cell lysates of wild-type and Fyn^−/− microglia treated with LPS for 15–45 min revealed reduced IκBα degradation in Fyn^−/− microglia at 15 min, and attenuated IκBα resynthesis at 30 and 45 min. *p < 0.05. **p < 0.01. C, D, Cytosolic and nuclear fractionation of LPS- and TNFα-treated wild-type and Fyn^−/− microglia revealed diminished nuclear translocation of the p65 subunit of the NFκB complex in the Fyn^−/− microglia. **p < 0.01. ***p < 0.001. E, Immunocytochemistry also showed reduced nuclear p65 in LPS-treated Fyn^−/− microglia. Scale bar, 50 μm.

LPS- or TNFα-induced proinflammatory cytokine production is suppressed in Fyn/PKCδ-deficient microglia. A, Luminex analyses of supernatants from LPS-treated wild-type, PKCδ^−/−, and Fyn^−/− microglia revealed reduced secretion of the proinflammatory cytokines IL-6, IL-12, and TNFα. **p < 0.01. ***p < 0.001. B, Wild-type primary microglia were transfected with nontargeting and Fyn-specific siRNA for 72 h. Knockdown of Fyn was evaluated by Western blot. C, Fyn-depleted microglia demonstrated diminished IL-6 and TNFα secretion in response to LPS stimulation. **p < 0.01. ***p < 0.001. D, TNFα stimulation of Fyn^−/− microglia reduced IL-6 and TNFα production in contrast to wild-type microglia. **p < 0.01. ***p < 0.001. E, Immunoblots showing reduced TNFα levels in Fyn-deficient microglia after TNFα stimulation in contrast to wild-type microglia. F, G, Overexpressing the FLAG-tagged activation loop tyrosine mutant of Fyn in BV2 microglia attenuated IL-6 and IL-12 production when the cells were treated with LPS, as shown by Luminex cytokine analysis. *p < 0.05. **p < 0.01. ***p < 0.001.

Fyn plays a role in LPS-induced iNOS expression, nitrite production, and neuroinflammatory marker expression. A, Griess nitrite measurement assay demonstrated that LPS-induced nitrite production was reduced in Fyn^−/− microglia. *p < 0.05. ***p < 0.001. B–D, Diminished iNOS expression in LPS-treated Fyn^−/− microglia. **p < 0.01. Scale bar, 100 μm. E, F, Reduced gp91^phox and Iba-1 expression in LPS-treated Fyn^−/− and PKCδ^−/− microglia, as shown by immunoblotting analysis. *p < 0.05. **p < 0.01.

Fyn^−/− and PKCδ^−/− mice are resistant to LPS- and MPTP-induced neuroinflammatory responses. A, Wild-type, PKCδ^−/−, and Fyn^−/− mice were injected intraperitoneally with 5 mg/kg LPS for 3 h. Striatal cytokine mRNA levels, assessed by qRT-PCR, showed significantly reduced induction of pro-IL-1β and TNFα mRNA levels in PKCδ^−/− and Fyn^−/− mice in contrast to wild-type mice. *p < 0.05. **p < 0.01. ***p < 0.001. B, The transitional stages of microglial activation, from ramified (inactivated, Type A) to amoeboid (activated, Types B-D), are shown by representative images. C, D, Iba-1-DAB immunohistochemistry in MPTP-injected Fyn^−/− and wild-type ventral midbrain sections demonstrated nigral microgliosis, assessed by quantification of microglial morphology, in the wild-type, but not the Fyn^−/− sections. Scale bar, 75 μm. *p < 0.05. **p < 0.01. ns, Not significant. E, F, Fyn^−/− mice showed diminished induction of the proinflammatory marker gp91^phox in ventral midbrain lysates following the acute MPTP regimen. *p < 0.05.

Fyn^−/− mice are protected against 6-OHDA-induced nigrostriatal dopaminergic neuronal deficits and microgliosis. A, TH-DAB immunohistochemistry in 6-OHDA-injected Fyn^−/− and wild-type mouse striatal sections. Scale bar, 1000 μm. B, Schematic diagram of a coronal section through the mouse striatum at the level of the injection. C, Significant preservation of 6-OHDA-induced degeneration of dopaminergic terminals is seen in the Fyn^−/− mice in contrast to wild-type mice. **p < 0.01. ***p < 0.001. D, Immunofluorescence staining of 6-OHDA-injected Fyn^−/− and wild-type ventral midbrain sections reveals diminished microgliosis and concomitant nigral neuroprotection in Fyn^−/− mice after 6-OHDA administration, in contrast to the massive microgliosis and nigral dopaminergic neuronal death observed in the wild-type mice. Scale bar, 200 μm.

PKCδ^−/− mice are resistant to 6-OHDA-induced nigrostriatal dopaminergic neuronal deficits and microgliosis. A, TH-DAB immunohistochemistry in 6-OHDA-injected PKCδ^−/− and wild-type mouse striatal sections. Scale bar, 1000 μm. B, Schematic diagram of a coronal section through the mouse striatum at the level of the injection. C, Significant preservation of dopaminergic terminals is seen in the 6-OHDA-treated PKCδ^−/− mice in contrast to wild-type mice. *p < 0.05. **p < 0.01. ***p < 0.001. D, Immunofluorescence staining of 6-OHDA-injected PKCδ^−/− and wild-type ventral midbrain sections reveals reduced nigral TH degeneration and microgliosis in PKCδ^−/− mice after 6-OHDA administration, in contrast to the wild-type mice. Scale bar, 200 μm. E, High-magnification image of 6-OHDA-injected PKCδ^−/− and wild-type ventral midbrain sections. Scale bar, 50 μm.

Diminished 6-OHDA-induced glial-neuronal contact (gliapse) formation in the Fyn^−/− SN. A, C, Confocal Z-stack maximum projection image analysis of ventral midbrain sections reveals a strongly increased number of microglial-neuronal contacts and appositions upon 6-OHDA treatment of Fyn^+/+ but not Fyn^−/− mice. Scale bar, 12 μm. B, D, Confocal Z-stack images were rotated and optically sectioned along the Z-plane using Imaris software, allowing easy visualization of gliapse formation. Scale bar, 10 μm. E, Diagrams of Process-Body (Pr-B) and Body-Body (B-B) gliapses formed between dopaminergic neurons and microglia. F, Fyn ventral midbrain sections revealed significantly fewer gliapses formed per dopaminergic neuron in the SN. ***p < 0.001. ns, Not significant.

Prolonged inflammogen stimulation induces Fyn upon microglial activation. A, B, Stimulation of primary microglia with LPS for 12 h and TNFα for 24 h increased Fyn expression, as evidenced by Western blotting. *p < 0.05. C, Immunohistochemistry analysis of Fyn expression. Scale bar, 20 μm. D, qRT-PCR analysis of Fyn mRNA levels in LPS-stimulated primary microglia and BV2 microglia revealed induction of Fyn at the message level. *p < 0.05. **p < 0.01. E, Induction of Fyn promoter activity in primary microglia following LPS activation of wild-type primary microglia. *p < 0.05. F, Increased striatal Fyn mRNA levels were seen in the Fyn^+/+ mice injected intraperitoneally with LPS (5 mg/kg) for 12 h, as assessed by qRT-PCR. **p < 0.01.