Microglia-Derived Cytokines/Chemokines Are Involved in the Enhancement of LPS-Induced Loss of Nigrostriatal Dopaminergic Neurons in DJ-1 Knockout Mice.

Mutation of DJ-1 (PARK7) has been linked to the development of early-onset Parkinson's disease (PD). However, the underlying molecular mechanism is still unclear. This study is aimed to compare the sensitivity of nigrostriatal dopaminergic neurons to lipopolysaccharide (LPS) challenge between DJ-1 knockout (KO) and wild-type (WT) mice, and explore the underlying cellular and molecular mechanisms. Our results found that the basal levels of interferon (IFN)-γ (the hub cytokine) and interferon-inducible T-cell alpha chemoattractant (I-TAC) (a downstream mediator) were elevated in the substantia nigra of DJ-1 KO mice and in microglia cells with DJ-1 deficiency, and the release of cytokine/chemokine was greatly enhanced following LPS administration in the DJ-1 deficient conditions. In addition, direct intranigral LPS challenge caused a greater loss of nigrostriatal dopaminergic neurons and striatal dopamine content in DJ-1 KO mice than in WT mice. Furthermore, the sensitization of microglia cells to LPS challenge to release IFN-γ and I-TAC was via the enhancement of NF-κB signaling, which was antagonized by NF-κB inhibitors. LPS-induced increase in neuronal death in the neuron-glia co-culture was enhanced by DJ-1 deficiency in microglia, which was antagonized by the neutralizing antibodies against IFN-γ or I-TAC. These results indicate that DJ-1 deficiency sensitizes microglia cells to release IFN-γ and I-TAC and causes inflammatory damage to dopaminergic neurons. The interaction between the genetic defect (i.e. DJ-1) and inflammatory factors (e.g. LPS) may contribute to the development of PD

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

<div><p>DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.</p></div

Effects of IL-1β on cell viability, proliferation, adhesion and colony formation.

<p>(A) Cell viability was evaluated using MTT assay. Note that treatment of IL-1β (20 ng/ml) increased cell viability of melanoma cells. (B) Cell proliferation was measured using Brdu incorporation method. Note that treatment of IL-1β (20 ng/ml) enhanced cell proliferation. (C) Effects of IL-1β on cell adhesive ability. Note that IL-1β (2 and 20 ng/ml) can increase adhesion of melanoma cells on type I collagen, but not on albumin and fibronectin. (D) Colony formation of melanoma cells in soft agar with or without addition of IL-1β. Upper panel: microscopic photos showed the colony of large sizes (> 0.02mm, arrow) and small sizes (< 0.02mm, arrow head). Lower panel: bar chart showed that IL-1β increased the number of colony with large sizes. Data are presented as mean ± S.E.M. n = 3 for each group, * p < 0.05 compared with control groups.</p

LPS-induced death of dopaminergic neurons is enhanced by local LPS administration in DJ-1 KO mice.

<p>LPS (1 μg /1 μl) or saline (1 μl) was locally injected into substantia nigra. One day and five days later, mice were sacrificed respectively. (A) Immunohistochemical images of substantia nigra showed the differential loss of TH-positive neurons in WT (upper panel) and DJ-1 KO (lower panel) mice Day 1 (left 4 images) or Day 5 (right 4 images) after local injection of LPS. Note that LPS-induced death of dopaminergic neurons was enhanced on Day 5 in DJ-1 KO mice. The statistical results were shown in (B). The mean of the summated neuron number was shown in each group and the data are presented as mean ± S.E.M. (n = 7 for each group) * p<0.05, Scale bar: 0.25 mm.</p

LPS-induced up-regulation of IFN-γ and I-TAC is enhanced in DJ-1 knockout mice.

<p>Levels of IFN-γ and I-TAC in substantia nigra (SN) were up-regulated by local injection with LPS (1 μg /1 μl) in DJ-1 KO mice. (A) Immunohistochemical staining of IFN-γ in substantia nigra of WT and DJ-1 KO mice Day-5 after injection of LPS. (B) Quantitative results (ELISA) show the increase of IFN-γ expression in substantia nigra of DJ-1 KO mice. (C) Immunohistochemical staining of I-TAC in substantia nigra of WT and DJ-1 KO mice Day-5 after injection of LPS. (D) Quantitative results (ELISA) show the up-regulation of I-TAC expression in substantia nigra of DJ-1 KO mice. The typical stained-cells were indicated by arrows. Data were normalized as percentage of the mean of basal expressional levels in WT mice (con) and presented as mean ± S.E.M. (n = 4–5 for each group) * p<0.05. SNpc: substantia nigra pars compacta; SNr: substantia nigra pars reticulate. Scale bar: 0.2 mm.</p

IFN-γ and I-TAC reduce the survival of dopaminergic neurons in primary midbrain neuron-glia mixed cultures but not in neuron-enriched cultures.

<p>(A) Midbrain neuron-glia mixed cultures were derived from E14 rat embryos and treated with recombinant IFN-γ protein (30 and 300 ng/ml) or I-TAC protein (1 and 10 ng/ml) on Day-7 cultures for 48 hours. LPS (500 ng/ml) was used as positive control. Note that IFN-γ and I-TAC reduced the survival of TH-positive neurons in neuron-glia mixed cultures (B) The neuron-enriched cultures were obtained from E14 rat embryos and treated with IFN-γ and I-TAC on Day-7 cultures for 48 hours. Note that IFN-γ and I-TAC did not affect survival of TH-positive neurons in neuron-enriched cultures. The dopaminergic neurons (TH-positive) were counted and normalized as percentage of TH-positive neurons in control cells. Data were presented as mean ± S.E.M. (n = 4–5 for each group) * p<0.05 as compared with control.</p

Up-regulation of IFN-γ and I-TAC in DJ-1 KO mice.

<p>(A) IFN-γ-regulated biological network predicted from the bioinformatics tool STRING. IFNG (i.e. IFN-γ) was predicted to serve as a hub protein to control CXCL11 (i.e. I-TAC), IL-17, IL-1β, IL1RN, and ICAM1. (B) The basal mRNA levels of IFN-γ (left panel) and I-TAC (right panel) in substantia nigra were up-regulated in DJ-1 KO mice as compared with those in WT mice. (C) Increase of basal IFN-γ expression in microglia in substantia nigra of DJ-1 KO mice. The immunofluorescent labeling images of IFN-γ (green), CD11b (red) and DAPI (blue) in substantia nigra of WT (upper images) and DJ-1 KO (lower images) mice were shown. (D) Increase of basal I-TAC expression in microglia in substantia nigra of DJ-1 KO mice. The immunofluorescent labeling images of I-TAC (green), CD11b (red) and DAPI (blue) in substantia nigra of WT (upper images) and DJ-1 KO (lower images) mice were shown. The arrow indicates the brain area that is magnified and shown in the right panel. The merged image shows the co-localization of two proteins in microglia. Data were normalized as percentage of mean mRNA level in WT mice and presented as mean ± S.E.M. (n = 5 for each group) * p<0.05. SNpc: substantia nigra pars compacta; SNr: substantia nigra pars reticulate.</p

Schematic diagram of molecular and cellular mechanisms involved in DJ-1 deficiency-associated loss of dopaminergic neurons.

<p>(A) DJ-1 deficiency and (B) PAMPs may have a synergistic effect to up-regulate (C) IFN-γ and I-TAC through the activation of (D) NF-κB. The released IFN-γ then serves as a paracrine and/or an autocrine to impact on the (E) cytokine network, which in turn amplifies the inflammatory activity of microglia to cause the (F) death of dopaminergic neurons in substantia nigra and (G) depletion of striatal dopamine and its metabolites.</p

Increase of serum levels of IL-1β and other cytokines in DJ-1 KO mice.

<p>(A) Representative protein array images displayed the serum cytokine profiles in WT (left) and DJ-1 KO (right) mice. Each protein was measured in duplicate on an array. Positions of IL-1β spots are indicated by blue arrows. (B) Bar charts showed the fold differences of proteins in cytokine arrays between WT and DJ-1 KO mice. The data were presented as mean ± S.E.M., and IL-1β bars are indicated by the blue arrow. All data were normalized to respective protein spots in WT groups. n = 4 for each group, * p < 0.05 compared with WT mice.</p