Abstract
Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.
Highlights
The PPARγ agonist rosiglitazone up-regulates Bcl-2 and protects dorsal root ganglion (DRG) neurons from nerve growth factor (NGF) withdrawal-induced apoptosis− To determine whether the protective effect of rosiglitazone (RGZ), previously described in neuronal cells, might be attributed to PPARγ-induced upregulation of Bcl-2, we first determined the effect of RGZ in Bcl-2 protein content and mRNA expression in DRG neurons
Since DRG cultures are contaminated by non-neuronal cells (30-40% under our conditions), quantitative immunofluorescence of Bcl-2 was assessed in NFH positive cells
To assess whether RGZ-induced up-regulation of Bcl-2 was reflected in decreased sensitivity to apoptosis we used NGF withdrawal, a widely used model to induce apoptosis in embryonic DRG neurons [35,36,37]
Summary
Materials- Chemicals and drugs were from Sigma (St. Louis, MO), and culture media and sera were from Gibco BRL (Paisley, UK). Cells were maintained for 48 hr in Ham-F12 containing 10% horse serum (HS, Invitrogen, San Diego, CA), 100 U/ml penicillin, 100μg/ml streptomycin, 50 ng/ml NGF, 5 μM cytosine arabinoside (Sigma Aldrich, St. Louis, MO USA), and 20 μM fluorodeoxyuridine (Sigma Aldrich-Fluka, St. Louis, MO, USA ) at 37oC, 5% CO2, either in 3.5 cm dishes (real-time PCR) or polylysine-coated covers (immunofluorescence studies). MO, USA ) at 37oC, 5% CO2, either in 3.5 cm dishes (real-time PCR) or polylysine-coated covers (immunofluorescence studies) After this step over 65% of cells were neurons, as assessed by counting cells with round, phase-bright bodies and intact neurites. Bcl-2 mean fluorescence intensity, under non-saturated conditions, was determined in at least 50 NFH positive control or RGZ-treated cells in each experiment, in digital images from random fields using the Image-Pro express software (Media Cybernetics Inc., Silver Spring, MD, USA). Fluorescence signal of treated cells was obtained using a 488-nm argon laser to excite 2,7-DCF fluorescence and signals were collected at 505–530 nm
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