Silencing of Abcd1 and Abcd2 genes sensitizes astrocytes for inflammation: implication for X-adrenoleukodystrophy

Abstract

X-linked adrenoleukodystrophy is a metabolic disorder arising from a mutation/deletion in the ABCD1 gene, leading to a defect in the peroxisomal adrenoleukodystrophy protein (ALDP), which inhibits the oxidation of very long chain fatty acids (VLCFAs). Thus, these VLCFAs accumulate. In a cerebral form of ALD (cALD), VLCFA accumulation induces neuroinflammation that leads to loss of oligodendrocytes and myelin, which ultimately shortens the lifespan. To establish a relationship between the metabolic disease and inflammatory disease induction, we document that small interfering RNA (siRNA)-mediated silencing of Abcd1 (ALDP) and Abcd2 [adrenoleukodystrophy-related protein (ALDRP)] genes in mice primary astrocyte cultures resulted in accumulation of VLCFA and induction of an inflammatory response characteristic of human cALD. Correction of the metabolic defect using monoenoic FAs in Abcd1/Abcd2-silenced cultured astrocytes decreased inducible nitric oxide synthase and inflammatory cytokine expression, suggesting a link between VLCFA accumulation and inflammation. The inflammatory response was found to be mediated by transcription factors NF-kappaB, AP-1, and C/EBP in Abcd1/Abcd2-silenced mouse primary astrocytes. Although mechanisms of VLCFA-mediated induction of the inflammatory response have been investigated here in vitro, the in vivo mediators remain elusive. Our data represent the first study to suggest a direct link between the accumulation of VLCFA and the induction of inflammatory mediators.

Fig. 1.

Transient Abcd1 and Abcd2 gene silencing. A: Both of the siRNAs induced gene silencing in mice primary astrocytes, indicated by a significant decrease in Abcd1 and Abcd2 expression analyzed by RT-PCR and Western blot immunoassay detected with monoclonal antibodies against Abcd1 and Abcd2 proteins. The same membranes were stripped and reprobed with anti-actin antibodies as a loading control. B: Transfection efficiency was more than 90% as seen by Cy3-labeled negative control-siRNA transfection. C: siRNA transfection or oleic acid/erucic acid treatment was not cytotoxic. Cell viability of Abcd1- and/or Abcd2-silenced and oleic acid and erucic acid mixture-treated astrocytes are reported as absorbance (A570–A690 nm) obtained with an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. D: Mouse primary astrocytes were silenced for Abcd1 and/or Abcd2. After 6 days, β-oxidation was measured in the cell suspension as described in Materials and Methods. Lignoceric acid oxidation was significantly decreased upon silencing of Abcd1 or Abcd2 genes in primary astrocytes. Silencing of both Abcd1 and Abcd2 simultaneously further significantly (P < 0.001) inhibited β-oxidation. There was no significant change in palmitic acid oxidation upon silencing with Abcd1/Abcd2. E: Fatty acid methyl ester was prepared directly from cells as described in Material and Methods. FAs were analyzed by GC after adding C_27:0 as an internal standard. C_26.0, C_24.0, and C_22:0 were measured as a percent of total FAs and expressed as percent C_26:0 of total FAs (i), C_26:0/C_22:0 (ii). Results represent the means ± SE of duplicates from three different experiments. * P < 0.01, Abcd1 or Abcd2 silencing compared with control; ** P < 0.05, Abcd2 silencing compared with control; *** P < 0.001, Abcd1+Abcd2 silencing compared with control; **** P < 0.001, Abcd1 or Abcd2 silencing compared with control; ^@@ P < 0.001, Abcd1+Abcd2 silencing compared with Abcd1 or Abcd2 silencing; ^$ P < 0.001, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; ^$$ P < 0.001, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; ^$$$ P < 0.001, oleic acid/erucic acid (40 μM) treatment compared with Abcd1+Abcd2 silencing; NS, nonsignificant, control compared with scrambled RNA-treated.

Fig. 2.

Reactive oxygen species (ROS) generation and expression of 5-lipoxygenase (5-LOX) and cycloxygenase-2 (COX-2) is induced in Abcd1/Abcd2-silenced astrocytes. A: ROS generation in primary astrocytes after 6 days of silencing or oleic acid/erucic acid-treated astrocytes. B: The immunoblot (i, ii) and expression of 5-LOX and COX-2 (iii) examined using the iCycler iQ real-time PCR detection system, as discussed in Materials and Methods. The target gene expression was normalized to GAPDH expression, and the result is presented as mean normalized expression. Data are means ± SD of three experiments. * P < 0.001, Abcd1 silencing compared with control; ** P < 0.001, Abcd2 silencing compared with control; *** P < 0.001, Abcd1+Abcd2 silencing compared with Abcd1 or Abcd2 single silencing; ^@ P < 0.05, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; ^@@ P < 0.01 oleic acid/erucic acid (40 μM) treatment compared with Abcd1+Abcd2 silencing; NS, nonsignificant, oleic acid/erucic acid (10 μM) treatment compared with Abcd1+Abcd2 silencing.

Fig. 3.

Abcd1/Abcd2 silencing induces the expression of proinflammatory mediators in primary astrocytes. Tumor necrosis factor-α (TNF-α) (A) and interleukin-1β (IL-1β) (B) were measured by ELISA in the supernatant of primary astrocytes after 6 days of silencing. TNF-α (C) and IL-1β (D) expression was examined using the iCycler iQ real-time PCR detection system as discussed in Materials and Methods. The target gene expression was normalized to GAPDH expression, and the result is presented as mean normalized expression. Data are means ± SD of three experiments. *P < 0.001, Abcd1 silencing compared with control; ** P < 0.001, Abcd2 silencing compared with control; *** P < 0.001 Abcd1/Abcd2 silencing compared with Abcd1 or Abcd2 single silencing; ^@ P < 0.01, Abcd1 silencing compared with control; ^@@ P < 0.01, Abcd2 silencing compared with control; NS, nonsignificant, scramble-treated compared with control.

Fig. 4.

Abcd1/Abcd2 silencing induces nitric oxide (NO) and inducible nitric oxide synthase (iNOS) expression in primary astrocytes. A: For detection of iNOS protein expression by immunoblot in response to Abcd1/Abcd2 silencing, cell lysate from astrocytes was prepared. B: NO was measured by ELISA in the supernatant of primary astrocytes after 6 days of silencing or oleic acid/erucic acid-treated astrocytes. C: iNOS expression was examined using the iCycler iQ real-time PCR detection system as discussed in Materials and Methods. The target gene expression was normalized to 18S rRNA expression, and the result is presented as mean normalized expression. Data are means ± SD of three experiments. ^@ P < 0.001, Abcd1 or ABCD2 silencing compared with control; ^@ P < 0.05, Abcd1 or ABCD2 silencing compared with Abcd1+Abcd2 silencing; * P < 0.001, Abcd1 silencing compared with control; ** P < 0.001, Abcd2 silencing compared with control; *** P < 0.001, Abcd1+Abcd2 silencing compared with Abcd1 or Abcd2 single silencing; NS, nonsignificant, scramble-treated compared with control.

Fig. 5.

Lorenzo's Oil treatment downregulates expression of proinflammatory mediators and iNOS in Abcd1/Abcd2-silenced astrocytes. TNF-α (A) and IL-1β (B) were measured by ELISA in the supernatant of primary astrocytes after 6 days of silencing. TNF-α (C) and IL-1β (D) expression was examined using the iCycler iQ real-time PCR detection system as discussed in Materials and Methods. The target gene expression was normalized to GAPDH expression, and the result is presented as mean normalized expression. E: For the detection of iNOS protein expression by immunoblot in response to Abcd1/Abcd2 silencing, cell lysate from astrocytes was prepared (i); NO was measured by ELISA in the supernatant of primary astrocytes after 6 days of silencing or oleic acid/erucic acid-treated astrocytes (ii); iNOS expression was examined using the iCycler iQ real-time PCR detection system as discussed in Materials and Methods (iii). The target gene expression was normalized to 18S rRNA expression, and the result is presented as mean normalized expression. *P < 0.001, Abcd1+Abcd2 silencing compared with control; ** P < 0.001, oleic acid/erucic acid (10 μM) treatment compared with Abcd1+Abcd2 silencing; *** P < 0.001, oleic acid/erucic acid (40 μM) treatment compared with Abcd1+Abcd2 silencing; ^@ P < 0.01, oleic acid/erucic acid (10 μM) treatment compared with Abcd1+Abcd2 silencing; ^@@ P < 0.05, oleic acid/erucic acid (10 μM) treatment compared with Abcd1+Abcd2 silencing; ^$ P < 0.05, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; ^$$ P < 0.01, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; ^$$$ P < 0.001, oleic acid/erucic acid (20 μM) treatment compared with Abcd1+Abcd2 silencing; NS, nonsignificant, scramble-treated compared with control.

Fig. 6.

Abcd1/Abcd2 silencing induces expression and DNA binding of NF-κB, AP1, and C/EBP in primary astrocytes. Nuclear extracts were prepared from control, scrambled, and Abcd1/Abcd2-silenced astrocytes (A). Nuclear extracts from control (i), scrambled (ii), and Abcd1/Abcd2-silenced (iii) astrocytes were incubated with a set of biotin-labeled DNA binding oligonucleotides (TranSignal probe mix) to allow the formation of protein/DNA (or transcription factor/DNA) complexes. The protein/DNA complexes were then separated from the free probes. The probes in the complexes were extracted and hybridized to the TranSignal array. Signals were detected using a chemiluminescent imaging system. B: Nuclear extracts were used for electrophoretic mobility shift assay to check the DNA binding activity of NF-κB, AP-1, and C/EBP.