Journal: Frontiers in Molecular Neuroscience

Article Title: Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

doi: 10.3389/fnmol.2017.00062

Figure Lengend Snippet: Interleukin 4 (IL4) protects midbrain dopaminergic (mDA) neurons from MPP + -induced neurodegeneration in mixed neuron-glia cultures. E14 ventral midbrain neuron-enriched cultures (A) and E14 ventral midbrain mixed neuron-glia cultures (C) were treated with 0.2 μM MPP + in the presence or absence of IL4 (10 ng/ml) for 2 days and numbers of TH + neurons were counted. Scale bars indicate 50 μm. Quantifications and statistical evaluations revealed that IL4 failed to protect mDA neurons in E14 neuron-enriched cultures (B) but was sufficient to significantly protect mDA neurons in E14 mixed neuron-glia cultures (D) . Data are given as mean ± SEM from four (neuron-enriched cultures) and five (neuron-glia cultures) independent experiments performed in duplicates. P -values derived from one-way ANOVA followed by Bonferroni’s multiple comparison post-test are ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: IL4 (10 ng/ml) and IGF-1 (50 ng/ml) were obtained from Peprotech (Germany) and prepared and dissolved according to the manufacturer’s instructions.

Techniques: Derivative Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

doi: 10.3389/fnmol.2017.00062

Figure Lengend Snippet: Neutralization of endogenous IL4 increases MPP + -induced neurodegeneration in vitro . (A) Cellular expression of IL4 in E14 mixed neuron-glia cultures was determined after co-staining with Gfap as an astrocyte marker and FITC-coupled tomato lection as a microglia marker. White arrows mark IL4 + cells. Scale bar indicates 30 μm. (B) Secretion of IL4 in E14 mixed neuron-glia cultures under control conditions and 2 days after MPP + treatment was determined using an IL4 ELISA. The levels of secreted IL4 in supernatants of primary microglia cultures 24 h after MPP + treatment (0.2 μM) was analyzed using an IL4 ELISA. (C) Validation of IL4 neutralization in vitro . Primary microglia cultures were treated with recombinant IL4 (10 ng/ml) in the presence (1:50 and 1:100) or absence of an IL4 neutralizing antibody and IL4-mediated increase of Arg1 expression was assessed by PCR. Gapdh was used as housekeeping gene. (D) E14 ventral midbrain mixed neuron-glia cultures were treated with 0.2 μM MPP + in the presence or absence of an IL4 neutralizing antibody (IL4nAB) at a dilution of 1:50 for 2 days and numbers of TH + neurons were counted. Scale bars indicate 50 μm. (E) Quantifications and statistical evaluation demonstrates that neutralization of IL4 increases MPP + -induced degeneration of mDA neurons in vitro . Data are given as mean ± SEM from six (B) and three (D,E) independent experiments performed in duplicates. P -values derived from one-way ANOVA followed by Bonferroni’s multiple comparison post-test are ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: IL4 (10 ng/ml) and IGF-1 (50 ng/ml) were obtained from Peprotech (Germany) and prepared and dissolved according to the manufacturer’s instructions.

Techniques: Neutralization, In Vitro, Expressing, Staining, Marker, Control, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Recombinant, Derivative Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

doi: 10.3389/fnmol.2017.00062

Figure Lengend Snippet: Microglia-conditioned medium after IL4 treatment is neuroprotective in vitro . (A) Treatment of primary microglia with recombinant IL4 (10 ng/ml) for 24 h results in modest changes of secreted chemokines and cytokines. As expected, IL4 was detected in IL4-treated samples and thus was not included in the densitometric spot analysis. Levels of MCP-5 and MIP-2 were increased after IL4 treatment. Data are given as mean ± SEM from two independent experiments. (B) E14 ventral midbrain neuron-enriched cultures were treated for 2 days with serum-free medium (control) and IL4 (10 ng/ml) or with microglia conditioned medium obtained after treatment of primary microglia for 24 h with serum-free medium (MCM) and IL4 at 10 ng/ml (MCM IL4). Scale bar indicates 50 μm. (C) Quantifications of TH + neurons indicate that microglia conditioned medium after treatment with IL4 (MCM IL4) significantly increased neuron survival. IL4 alone was not able to promote neuroprotection. (D) IL4 treatment increases IGF-1 expression in primary microglia. After treatment for 3, 6, 12, and 24 h IGF-1 expression was determined using qPCR and is presented as fold change compared to untreated control cultures. Significant increases in IGF-1 expression was observed after 12 h. (E) IGF-1 secretion under control conditions and 24 h after treatment with IL4 (10 ng/ml) was detected using an IGF-1 ELISA. (F) Recombinant IGF-1 (50 ng/ml) increased neuron survival in E14 ventral midbrain neuron-enriched cultures without reaching significance. Data are given as mean ± SEM from four (B–D) , three ( C , ELISA) and five (E) independent experiments performed in duplicates. P -values derived from student’s t -test (D) are ∗ p < 0.05 and ∗∗ p < 0.01. P -values derived from one-way ANOVA followed by Bonferroni’s multiple comparison post-test are ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: IL4 (10 ng/ml) and IGF-1 (50 ng/ml) were obtained from Peprotech (Germany) and prepared and dissolved according to the manufacturer’s instructions.

Techniques: In Vitro, Recombinant, Control, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

doi: 10.3389/fnmol.2017.00062

Figure Lengend Snippet: Knockout of IL4 has no effect on the cellular composition of the nigrostriatal system. Changes in numbers of TH + neurons (A,B) , Iba1 + microglia (C,D,E,I,J) and Gfap + astrocytes (F,G,H,K,L) in the nigrostriatal system were analyzed in 4 months old male WT and IL4 KO mice using unbiased stereology. A significant change in cell numbers was only detected for Iba1 + microglia in the SNpc, which were reduced in IL4 KO mice. Scale bar indicates 200 μm. Data are given as mean ± SEM from at least three animals per genotype. P -values derived from student’s t -test are ∗ p < 0.05. VTA, ventral tegmental area; SNpc, substantia nigra, pars compacta; SNpr, substantia nigra, pars reticularis; CC, corpus callosum; CPu, caudate putamen.

Article Snippet: IL4 (10 ng/ml) and IGF-1 (50 ng/ml) were obtained from Peprotech (Germany) and prepared and dissolved according to the manufacturer’s instructions.

Techniques: Knock-Out, Derivative Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Interleukin-4 Protects Dopaminergic Neurons In vitro but Is Dispensable for MPTP-Induced Neurodegeneration In vivo

doi: 10.3389/fnmol.2017.00062

Figure Lengend Snippet: Lack of IL4 has no impact on the susceptibility toward 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration. (A) Scheme for MPTP injections and time points used for qPCR, HPLC and immunohistochemistry. (B) Expression of IL4 in total tissue samples from substantia nigra (SN) and caudate putamen (CPu) of wild type (WT) mice 1 and 2 days after MPTP injections. qPCR results were normalized to Gapdh and are given as fold changes ( n = 3 PBS, n = 3 MPTP). (C) Immunohistochemical detection of TH + neurons in the SN 7 days after injections with PBS and MPTP. Scale bar indicates 300 μm. (D) Striatal dopamine levels in PBS- and MPTP-injected mice after 7 days. Equal reductions in dopamine levels were observed in both genotypes. (E) Quantification of TH + neuron numbers in the SN of PBS- and MPTP-injected mice. No significant changes in neurodegeneration were detected between WT and mutant (IL4 KO) mice. (F) Immunohistochemical detection of TH + neurons in the SN 90 days after injections with PBS and MPTP. (G) Striatal dopamine levels in PBS- and MPTP-injected mice after 90 days. Similar recoveries of dopamine levels were observed in WT and IL4 KO mice. (H) TH + neuron numbers in the SN of PBS- and MPTP-injected mice after 90 days were not significantly different between WT and IL4 KO mice, indicating normal regeneration of mDA neurons in IL4-deficient mice. Scale bars indicate 300 μm. Data are given as mean ± SEM from at least three mice per genotype and time point. P -values derived from one-way ANOVA followed by Bonferroni’s multiple comparison post-test are ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: IL4 (10 ng/ml) and IGF-1 (50 ng/ml) were obtained from Peprotech (Germany) and prepared and dissolved according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Injection, Mutagenesis, Derivative Assay, Comparison