Effects of mitochondrial dysfunction on the immunological properties of microglia

<p>Abstract</p> <p>Background</p> <p>Neurodegenerative diseases are characterized by both mitochondrial dysfunction and activation of microglia, the macrophages of the brain. Here, we investigate the effects of mitochondrial dysfunction on the activation profile of microglial cells.</p> <p>Methods</p> <p>We incubated primary mouse microglia with the mitochondrial toxins 3-nitropropionic acid (3-NP) or rotenone. These mitochondrial toxins are known to induce neurodegeneration in humans and in experimental animals. We characterized lipopolysaccharide- (LPS-) induced microglial activation and the alternative, interleukin-4- (IL-4-) induced microglial activation in these mitochondrial toxin-treated microglial cells.</p> <p>Results</p> <p>We found that, while mitochondrial toxins did not affect LPS-induced activation, as measured by release of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β), they did inhibit part of the IL-4-induced alternative activation, as measured by arginase activity and expression, induction of insulin-like growth factor 1 (IGF-1) and the counteraction of the LPS induced cytokine release.</p> <p>Conclusions</p> <p>Mitochondrial dysfunction in microglial cells inhibits part of the IL-4-induced alternative response. Because this alternative activation is considered to be associated with wound healing and an attenuation of inflammation, mitochondrial dysfunction in microglial cells might contribute to the detrimental effects of neuroinflammation seen in neurodegenerative diseases.</p

Fumaric Acid Esters Stimulate Astrocytic VEGF Expression through HIF-1α and Nrf2

<div><p>Fumaric acid esters (FAE) are oral analogs of fumarate that have recently been shown to decrease relapse rate and disease progression in multiple sclerosis (MS), prompting to investigate their protective potential in other neurological diseases such as amyotrophic lateral sclerosis (ALS). Despite efficacy in MS, mechanisms of action of FAEs are still largely unknown. FAEs are known to activate the transcription factor Nrf2 and downstream anti-oxidant responses through the succination of Nrf2 inhibitor KEAP1. However, fumarate is also a known inhibitor of prolyl-hydroxylases domain enzymes (PhD), and PhD inhibition might lead to stabilization of the HIF-1α transcription factor under normoxic conditions and subsequent activation of a pseudo hypoxic response. Whether Nrf2 activation is associated with HIF-1α stabilization in response to FAEs in cell types relevant to MS or ALS remains unknown. Here, we show that FAEs elicit HIF-1α accumulation, and VEGF release as its expected consequence, in astrocytes but not in other cell types of the central nervous system. Reporter assays demonstrated that increased astrocytic VEGF release in response to FAEs was dependent upon both HIF-1α and Nrf2 activation. Last, astrocytes of transgenic mice expressing SOD1(G93A), an animal model of ALS, displayed reduced VEGF release in response to FAEs. These studies show that FAEs elicit different signaling pathways in cell types from the central nervous system, in particular a pseudo-hypoxic response in astrocytes. Disease relevant mutations might affect this response.</p> </div

Reporter assay evidence of HIF-1α involvement in VEGF release.

<p>Wild type astrocytes were transfected with a HRE-Luciferase reporter plasmid (A), with a VEGF-Luciferase reporter plasmid (B), or were cotransfected with both VEGF-Luciferase plasmid and either an empty vector or a vector encoding a shRNA targeting HIF-1α (two different used, shRNA1 or 2), or an expression vector encoding a dominant negative Nrf2 isoform (C). After 24h cells were treated with 30µM DEF, 30µm DMF or 0,05% DMSO for 6h (A), for 8h (B) or 18h (A-C). Luciferase activity was then measured in cell extract by luminescence. *p<0,05 - ****p<0,0001; significantly different from corresponding control. Values are mean +/- SEM of n=3 independent experiments.</p

Transgenic (mSOD-G93A) astrocytes show abnormal response to FAEs.

<p>Transgenic (mSOD-G93A) astrocytes were treated with 30µM DEF (A,C,D,F) or 30µM DMF (B,C,E,F) for indicated times and the following read-outs were measured; HIF-1α level by Western-Blot (A,B); HIF-1α (VEGF, GLUT1) target genes using qPCR (C). VEGF release as determined by ELISA (D,E); and Nrf2 target genes (NQO-1, HO-1) (F) *p<0,05; **p<0,01; ***p<0,001; significantly different from corresponding control. Values are mean +/- SEM of n=3 independent experiments.</p

FAEs induce HIF-1α accumulation and VEGF release in astrocytes.

<p>Wild type astrocytes were treated with 30µM DEF (A, C, E) or 30µM DMF (B, D, E) for the indicated times. HIF-1α level was measured by Western-Blot (A, B, C, D). VEGF in the supernatant was quantified by ELISA (E, F); *p<0,05; ***p<0,001; significantly different from corresponding control. Values are mean+/-SEM of n=3 independent experiments.</p

Transcriptional effects of FAEs.

<div><p>(<b>A</b>) mRNA levels of NAD(P)H dehydrogenase quinone 1 (NQO-1) and Heme Oxygenase 1 (HO-1), two Nrf2 target genes in astrocytes, microglia, neurons and oligodendrocytes of wild type mice after vehicle (empty columns, Ctrl), or after 6 hours or 18 hours of either 30µM DEF (grey columns, DEF) or DMF (black columns, DMF). Note the robust upregulation of these two genes in all cell types except microglia.</p> <p>(<b>B</b>) mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1), two HIF1-α target genes in astrocytes, microglia, neurons and oligodendrocytes of wild type mice after vehicle (empty columns, Ctrl), or after 6 hours or 18 hours of either 30µM DEF (grey columns, DEF) or DMF (black columns, DMF). FAEs induced the expression of HIF1-α target genes in astrocytes and microglia but not in oligodendrocytes or neurons. *p<0,05; **p<0,01; ***p<0,001; significantly different from corresponding control. Values are mean+/- SEM of n=3 independent experiments.</p></div

Pharmacological evidence of HIF-1α involvement in VEGF release.

<p>Wild type astrocytes were treated with 30µM DMF for indicated times. HIF1-α level was measured by Western-Blot (A) 6h after DMF treatment (B) or 16h after DMF treatment (C). The HIF1-α inhibitor YC-1 was incubated 30min before treatment with DMF. VEGF in the supernatant was quantified by ELISA (C) *p<0,05; ***p<0,001; significantly different from corresponding control. Values are mean+/-SEM of n=3 independent experiments.</p