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PMP70 knock-down generates oxidative stress and pro-inflammatory cytokine production in C6 glial cells
Rita Di Benedetto1, Michela Alessandra Denti, Serafina Salvati
1Department of Food Science, Nutrition and Health, Istituto Superiore di Sanità, Rome, Italy.
Abstract:
By using RNA interference (RNAi) in rat C6 glial cells, we previously generated the cell line abcd3kd in which the peroxisomal half-transporter PMP70 was stably knocked-down. The observations that abcd3kd cells had peroxisomal beta-oxidation impairment and an increase of hexacosenoic acid in cholesterol ester fraction, indicated an overlapping function of PMP70 with adrenoleukodystrophy protein (ALDP), the peroxisomal half-transporters involved in X-linked adrenoleukodystrophy (X-ALD). The objective of the present study was to investigate whether PMP70 could affect some oxidative and inflammatory parameters, since many findings indicate oxidative damage in the brain of ALD patients and inflammation is a hallmark of the cerebral forms of X-ALD. We thus measured parameters indicative of oxidative stress, the expression or activity of antioxidant enzymes, and the production of some pro-inflammatory cytokines. Our results show that, due to inducible nitric oxide synthase up-regulation, abcd3kd cell line produces higher levels of nitrites than native C6 cells. The enhanced production of superoxide and thiobarbituric acid-reactive substances, the increased expression of mitochondrial superoxide dismutase, and the reduction of catalase and glutathione peroxidase activities confirm the presence of an oxidative process. We then measured the concentrations of TNFalpha, IFNgamma, and IL-12 and we observed that abcd3kd cells produce higher amounts of pro-inflammatory cytokines compared to native C6 cells. By using neutralizing antibodies against IL-12, not only inflammatory parameters significantly decrease, but nitrite and superoxide production is also affected. This demonstrates that oxidative status of abcd3kd cells is not a direct PMP70 knock-down consequence, but depends on IL-12 release. The scenery induced by the knock-down of PMP70 in C6 cells recall the oxidative and inflammatory status observed in human X-ALD and thus reinforce the idea that PMP70 could affect the clinical course of the disease.
Insights
Knocking down peroxisomal transporter PMP70 in glial cells increased oxidative stress and inflammation, linked to Interleukin-12 (IL-12). This suggests PMP70 influences X-linked adrenoleukodystrophy (X-ALD) progression.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- X-linked adrenoleukodystrophy (X-ALD) is characterized by oxidative damage and neuroinflammation.
- Peroxisomal transporter adrenoleukodystrophy protein (ALDP) is implicated in X-ALD pathogenesis.
- PMP70, a peroxisomal half-transporter, shares functional similarities with ALDP.
Purpose of the Study:
- To investigate the role of PMP70 in oxidative and inflammatory processes relevant to X-ALD.
- To determine if PMP70 knockdown affects oxidative stress markers and pro-inflammatory cytokine production in glial cells.
Main Methods:
- Utilized RNA interference (RNAi) to generate a PMP70-knockdown rat C6 glial cell line (abcd3kd).
- Assessed oxidative stress parameters including nitrite, superoxide, thiobarbituric acid-reactive substances, and antioxidant enzyme activities (catalase, glutathione peroxidase, mitochondrial superoxide dismutase).
- Measured pro-inflammatory cytokine levels (TNF-alpha, IFN-gamma, IL-12) and evaluated the effect of IL-12 neutralization.
Main Results:
- abcd3kd cells exhibited increased nitrite and superoxide production, indicative of oxidative stress.
- Enhanced expression of mitochondrial superoxide dismutase and reduced activities of catalase and glutathione peroxidase were observed.
- Pro-inflammatory cytokine production, particularly IL-12, was significantly elevated in abcd3kd cells.
- Neutralization of IL-12 reduced inflammatory markers and also affected nitrite and superoxide levels, indicating IL-12's role in the oxidative status.
Conclusions:
- PMP70 knockdown in C6 glial cells induces an oxidative and inflammatory state resembling X-ALD.
- The oxidative stress observed is dependent on IL-12 release, suggesting a complex interplay between inflammation and oxidative pathways.
- These findings highlight PMP70 as a potential modulator of the clinical course in X-ALD.
