Inflammation-induced degeneration of DA neurons in mesencephalic neuron-glia cultures is a useful in vitro
model for studying the mechanism and identifying the potential therapeutic application in PD (2
). Using the well-characterized models of LPS and MPP+
-induced neurodegeneration, we sought to identify the mechanism by which TGFβ1, a major anti-inflammatory cytokine, mediates the neuroprotection of inflammation-induced neurotoxicity in vitro.
Our results showed that TGFβ1 exerted potent effects in inhibiting LPS and MPP+
-induced inflammation and neuronal destruction through the inhibition of oxidative stress responses in microglial cells. Three salient features of this protective role of TGFβ1 were observed in this study: 1) TGFβ1 exerts potent anti-inflammatory and neuroprotective effects through the inhibition of both direct microglial activation by LPS, and reactive microgliosis elicited by MPP+
; 2) TGFβ1 decreases NADPH oxidase-mediated superoxide production mainly through the inhibition of p47phox
translocation to the cellular membrane, a novel site of action for the neuroprotective effect of TGF β1; and 3) The inhibition of p47phox
translocation is mediated through the inhibition of PHOX subunit p47phox
phosphorylation at Ser345 via suppression of the ERK signaling pathway.
Our results showed that TGFβ1 has protective effects in both the LPS and the MPP+
model of PD even though the target of these two agents is different. LPS leads to the direct activation of microglia, which results in death of DA neurons through the production of inflammatory mediators. Reports from our laboratory and others have shown that MPP+
can cause reactive microgliosis, and that oxidative stress is involved in MPP/MPP+
-induced neurotoxicity (19
). Even though MPP+
directly damages DA neurons, we show that TGFβ1 still provides significant neuroprotective effects through the inhibition of reactive microgliosis in neuron-glia cultures. However, when microglial cells are removed, TGFβ1 can no longer show any protective effects on DA neurons, suggesting that TGFβ1 does not work directly on DA-neurons, but rather indirectly by inhibiting activated microglia which contribute to additional neurotoxicity by producing toxic inflammatory mediators. Based on our current evidence, we propose that the anti-inflammatory effect of TGFβl is capable of inhibiting both LPS-induced microglial activation and MPP+
-induced reactive microgliosis, and its ultimate result is to suppress the inflammation that mediates chronic neurodegeneration in PD.
Our studies indicated that TGFβl functions in neuroprotection by inhibiting the initial events in the inflammatory response, the activation of oxidative stress response, and the subsequent production of inflammatory mediators TNFα and NO. It has been shown that DA neurons in the substantia nigra are uniquely vulnerable to oxidative stress due to lower antioxidant capacity, increased accumulation of iron, high content of dopamine auto-oxidative metabolites and high density of microglia in the substantia nigra (7
). The fact that TGFβl significantly inhibits the production of superoxide induced by LPS within a few minutes after stimulation, led us to examine this factor in greater details by using PHOX-deficient mice. The findings that TGFβl could significantly lessen the LPS-induced DA uptake reduction in cells from wild-type mice, but has no significant protective effect on cells from PHOX−/−
mice () strongly support the contention that the protective effect of TGFβl is most likely mediated through the inhibition of PHOX activity.
While our rat primary midbrain cultures consist of a variety of different types of cells, it is clear that the primary source of LPS-induced ROS in these cultures is the microglial cell. This notion was supported by our previous reports, which indicated that LPS fails to produce extracellular superoxide in neuron-glia cultures devoid of microglial cells, or in cultures of enriched microglia prepared from PHOX−/−
). Thus, these findings indicate that PHOX is the key enzyme involved in superoxide production in these cultures. Activation of PHOX in microglia not only increases the production of superoxide, but indirectly increases the intracellular ROS concentration, possibly through the conversion of superoxide to H2
, which is membrane permeable. Increase of intracellular ROS can intensify the activation of NF-κB, which leads to higher TNFα production (2
). In addition, it was reported that PHOX inhibitors prevented LPS/IFNγ-induced degradation of IκBα, and thus, inhibited the activation of NF-κB (47
). However, the ability to activate NF-κB-dependent genes such as TNFα in PHOX−/−
cells suggests that PHOX plays an important but not exclusive role in regulating inflammation in microglial cells. These data are consistent with the notion that inhibition of NF-κB activity by TGFβ1 may be mediated at least in part through its inhibition of PHOX, and that PHOX is the major target of the anti-inflammatory activity and neuroprotective effects of TGFβ1.
It is known that translocation of the cytosolic components p47phox
, p40 phox
, and rac2 to the plasma membrane is required for the activation of PHOX (25
). The phosphorylation of Ser345 of p47phox
by pro-inflammatory agents enhances this translocation event (41
). While investigating the mechanism by which TGFβ1 inhibits PHOX activity, we found that TGFβ1 significantly inhibits this LPS-induced p47phox
phosphorylation at Ser345, resulting in the inhibition of p47phox
translocation. As Ser345 is located in the MAPK consensus sequence, we investigated whether TGFβ1 inhibited components of the MAPK signaling pathway, and our results indicate that TGFβ1 shows significant inhibitory effect on LPS-induced ERK phosphorylation, but not p38 or JNK (data not shown).Furthermore, a specific Erk inhibitor, U0126, showed strong inhibitory effects against LPS-induced neurodegeneration, superoxide production and p47phox
translocation, suggesting a central role for ERK in these effects (). These findings, coupled with our previous findings on the role of ERK in PHOX activation (22
), strongly suggests it is ERK that regulates p47phox
phosphorylation and is the crucial target for TGFβ1-mediated inhibition of PHOX activation. Previously, we have shown that ERK is a crucial mediator of GM-CSF-induced activation of Ser345 on p47phox
in neutrophils (41
), and it appears that ERK also plays a central in the LPS-induced phosphorylation of Ser345 on p47phox
in microglial cells. Taken together, we suggest that TGFβ1 inhibits LPS-induced ROS production in microglia by inhibiting p47phox
phosphorylation and translocation, which is regulated by ERK signaling pathway.
These results suggest a central role for microglia in the pathogenesis of PD, and that by limiting their pro-inflammatory response with anti-inflammatory mediators TGFβ1 we can significantly inhibit the neurotoxicity associated with this disease. In addition, we have previously shown that IL-10 also has similarly potent neuroprotective properties (48
), and the combination of both TGFβ1 and IL-10 might even act in concert to regulate chronic inflammation in the brain. It has recently been shown that regulatory T cells (Treg cells), a significant source of both IL-10 and TGFβ1, show strong therapeutic efficacy in the treatment of neuroinflammation (49
). It is yet to be determined the exact role TGFβ1 plays in the physiological regulation of chronic CNS inflammation in PD, and how TGFβ1 may synergize with other anti-inflammatory mediators to regulate microglia-mediated neurotoxicity. In addition, the level of the immune response in the brain necessary to effectively control infections without resulting in neuropathology is yet to be understood. Consequently, much work remains to determine if TGFβ1, either therapeutically delivered or produced in the CNS, can play an important anti-inflammatory role by limiting the activation of microglia and promoting a well-regulated immune response that is lacking during the pathophysiology of CNS disorders.