In this study, we have performed a series of experiments demonstrating that the PPAR-γ agonist, pioglitazone is capable of promoting the survival of cortical neurons in the presence of inflammatory mediators in vitro, including microglia-induced injury in neuron-microglia transwell cultures. In addition, we have demonstrated that pioglitazone may have axonoprotective effects in vitro. Furthermore, this compound produced a two-fold increase in the specific activity of the peroxisomal enzyme catalase, together with a significant induction of PPAR-γ gene expression, in the presence of a NO donor, suggesting that modulation of PPAR-gamma activity and peroxisomal function by pioglitazone may contribute to improved neuronal survival.
Inflammation plays a pivotal role in the pathogenesis of several neurological disorders [39
]. Activated microglia are capable of generating vast amounts of oxidizing radicals such as superoxide, hydrogen peroxide and nitric oxide [40
] as well as proinflammatory mediators such as proteases and arachidonic acid derivatives which are all capable of eliciting tissue damage in the central nervous system [41
]. It has been previously demonstrated that, at certain concentrations, the NO donor, DETANONOate is directly neurotoxic [5
] and that microglial-derived NO significantly reduces the number of SMI312-positive axons per surviving neuron [6
The role of PPAR-γ in regulating immune function has been extensively investigated, with a previous study demonstrating that pioglitazone can alleviate inflammation in experimental autoimmune encephalomyelitis (EAE) and reduce clinical severity [27
]. Furthermore, previous reports have suggested that PPAR-γ agonists may reduce NO production by cultured microglial cells in vitro
]. In the current study, however, pioglitazone did not have any inhibitory effect on NO production in IFN-γ/LPS activated microglia cultured alone or when neurons and microglia were separated with the use of transwell co-cultures. Despite the failure of pioglitazone to induce downregulation of NO production under these experimental conditions, pioglitazone (10 μM) significantly protected cortical neurons from injury by IFN-γ/LPS activated microglia in transwell co-culture. The neuroprotective effect of pioglitazone in transwell co-cultures cannot, therefore, be explained by the reduction in microglial activation commonly observed when PPAR-γ agonists are used [42
], and suggests that the major effects of pioglitazone may occur through a primary protective effect specifically on neurons. However, the possibility should be considered that pioglitazone may exert an inhibitory effect on the microglial secretion of inflammatory mediators which may contribute to the observed neuroprotective effects [43
Activated microglia are capable of causing widespread tissue damage in neuroinflammatory disorders [45
] through their generation of vast amounts of oxidizing radicals, such as superoxide, hydrogen peroxide and nitric oxide [40
]. In addition to demonstrating a protective effect of pioglitazone on neurons, we examined whether this drug could influence axonal morphology in the presence of activated microglia. Neurofilament phosphorylation is vital in the maintenance of axon stability and dephosphorylation of neurofilaments occurs within axons of inflammatory disorders such as multiple sclerosis leading to subsequent axon degeneration [47
]. Our results demonstrate that microglial derived NO significantly reduces the total number of SMI-312-positive axons per field and that pioglitazone attenuates this severe microglia derived NO-mediated axon destruction.
Interestingly, when NO is secreted, it can intereact with O2-
to form peroxynitrite (ONOO-
), an anion with strong oxidant properties [48
]. Anti-oxidant enzymes such as Cu/Zn superoxide dismutase (SOD), Mn SOD and catalase (the latter being mainly localized to peroxisomes), work in concert to detoxify superoxide radicals into water and oxygen [49
].We found that activation of PPAR-γ with neuroprotective doses of pioglitazone upregulated the expression of the PPAR-γ receptor and the activity of catalase, thus providing cortical neurons with a higher anti-oxidant capability. These results are consistent with the ability of PPAR-γ agonists to upregulate the levels of anti-oxidant enzymes in other experimental paradigms [49
]. Furthermore, it is known that catalase contains functional PPAR-γ response elements in its promoter region [36
], thus explaining the upregulation of this enzyme by pioglitazone.
In addition, our study confirmed a previous report [15
] that cortical neurons express PPAR-γ, with its immunoreactivity being clearly localized to the nucleus. Neuroprotective doses of pioglitazone elicited a five-fold increase in the transcription of PPAR-γ, suggesting the existence of a positive feedback mechanism by which pioglitazone could sustain the responsiveness of cortical neurons by increasing the expression of its receptor. Interestingly, this early rise in PPAR-γ gene transcription was seen in neurons also exposed to the NO donor, suggesting that the effect of PPAR-γ agonism on PPAR-γ gene expression can occur independently of co-signalling with inflammatory mediators.
Anti-oxidant enzymes such as catalase and glutathione peroxidase work in concert to detoxify hydrogen peroxide into water and oxygen. We have demonstrated that neuroprotective doses of pioglitazone elicited a significant increase in activity of catalase. Therefore, in addition, we examined whether pioglitazone could also protect cortical neurons against the neurotoxic effects of hydrogen peroxide. As expected, pioglitazone (1 μM), protected cortical neurons from injury by hydrogen peroxide. Furthermore, our results demonstrate for the first time, that hydrogen peroxide reduces the total number of SMI312-positive axons, and that pioglitazone attenuates this severe H2
-mediated axon destruction. Neuroprotection afforded by pioglitazone against both NO and H2
was abrogated by the presence of the specific PPAR-γ antagonist, GW9662. This is in accordance with a previous study whereby pioglitazone was found to exert its effects via a receptor-dependent mechanism [49