In the current studies, we demonstrated that microglial-mediated neuroinflammation induces MAPT phosphorylation in several different models: a single LPS injection model of modest systemic inflammation, the hTau mouse model of tauopathy mated to Cx3cr1−/− mice and an in vitro microglial/neuronal culture system.
While other studies have linked microglial activation to MAPT phosphorylation, a majority of these studies were performed in vitro
or were correlational in nature in postmortem tissue. The only two previous in vivo
studies to address the relationship between neuroinflammation and MAPT phosphorylation and aggregation was a study examining the effects of LPS administration in the 3xTg mouse model of AD and a separate study on the effects of the immunosuppressant drug FK506 in P301S transgenic mice. However, the 3xTg mice contains mutant APP
as well as MAPT
transgenes, making it difficult to assess the direct contribution of LPS to MAPT pathology, as microglial activation is also observed in response to the aggressive beta-amyloid (Aβ) deposition observed in this model. In addition the effects of LPS can act through numerous cell types within the brain. By contrast, the P301S study provided the first evidence that microglial neuroinflammation precedes MAPT pathology (in the absence of Aβ pathology) and that intervention with FK506 could ameliorate MAPT pathology and increase lifespan (Yoshiyama et al., 2007
). While these studies suggested a links between microglial neuroinflammation and MAPT pathology, the current study documents the effects of a specific microglial receptor in modulating MAPT pathology and provides mechanistic insights into the downstream signaling molecules.
The present study demonstrates that a single dose of LPS, administered peripherally, is sufficient to induce MAPT hyperphosphorylation. LPS is an endotoxin that is part of the outer wall of Gram negative bacteria and can induce systemic inflammation via engagement of TLR4 (Lien et al., 2000
). LPS has also been utilized as model to examine the role neuroinflammation via either peripheral administration in systemic inflammation or direct administration into the brain (Hauss-Wegrzyniak et al., 1998
). Depending upon the dose of LPS, the route of administration, the time frame and the model examined, LPS can induce either neurodegeneration or neuroprotection with both short term (hours to days) and long term (months to years) effects. The LPS paradigm we have employed to examine the effects of induction of systemic inflammation has been widely utilized in numerous animal models of neurodegeneration (Cunningham et al., 2009
; Cunningham et al., 2005
; Masocha, 2009
; Qin et al., 2007
). Notably, several recent studies have suggested that the acute effects of LPS can have long lasting effects (months to years) within the CNS (Masocha, 2009
; Qin et al., 2007
). Our studies demonstrate a graded response to LPS with increased MAPT phosphorylation that is dependent upon the dose of LPS and deficiency of CX3CR1. Furthermore, either TLR4 or IL1R1 deficiency blocked the enhanced MAPT phosphorylation observed upon LPS administration, demonstrating that LPS-induced MAPT phosphorylation is acting through a canonical inflammatory pathway mediated in part by IL1. In addition, in vitro
studies demonstrated that blocking IL1 signaling with an IL1 receptor antagonist reduced both p38 MAPK activation as well as MAPT phosphorylation within neurons. Future studies will be required to examine the relative contribution of IL1α versus IL1β and to determine whether the enhanced MAPT phosphorylation and aggregation observed in CX3CR1 deficient hTau mice is also dependent upon IL1-p38 MAPK signaling.
Previous studies demonstrated that hTau mice develop age-related MAPT pathology (Andorfer et al., 2003
) and neurodegeneration (Andorfer et al., 2005
). In the current studies, we document that hTau mice develop age-related alterations in microglia activation at 12 to 18 months of age. This is consistent with several other recent observations in the hTau mice (Kelleher et al., 2007
; Noble et al., 2009
) as well as other mouse model of tauopathies (Kitazawa et al., 2005
; Yoshiyama et al., 2007
), suggesting that robust microglia activation occurs upon development of extensive MAPT pathology. In addition, our studies suggest that at six months of age, an age at which there is minimal development of MAPT aggregates, there are already alterations in expression of several cytokines and chemokines, including CCL2, NOS2 and most notably CX3CL1. CX3CL1 is produced by neurons in the CNS and signals directly to the CX3CR1 receptor, which is exclusively produced by microglia. Finally, consistent with the findings in the 3xTg mouse model, LPS administration to hTau mice induced enhanced MAPT phosphorylation (Kitazawa et al., 2005
CX3CL1 and its cognate receptor, CX3CR1, play an important role in neuroinflammation via paracrine signaling between neurons and microglia (). Previous studies from our laboratory (Cardona et al., 2006
) as well as others (Jung et al., 2000
) demonstrated that CX3CR1 deficiency alone does not lead to acute microglial activation or neurodegeneration. However, in mouse models of systemic inflammation, PD and ALS, CX3CR1 deficiency leads to enhanced neurodegeneration (Cardona et al., 2006
), while conversely in mouse models of stroke, CX3CR1 deficiency lead to neuroprotection (Denes et al., 2008
Model of fractalkine (CX3CL1/CX3CR1) signaling in neurodegenerative tauopathies
Notably, a recent study by Fuhrmann and colleagues suggested that CX3CR1 deficiency could prevent neuronal loss in the 3xTg mouse model of AD (Fuhrmann et al., 2010
). A 1.8% loss of neurons in cortical layer II per month observed in the 3xTg mouse model detected by two photon microscopy was prevented in the CX3CR1 deficient 3xTg animals. While this study suggested that CX3CR1 deficiency could modulate phenotypes in an AD mouse model, there are a number of important differences between the two studies, their endpoints and their conclusions.
First, the 3xTg mice exhibits both Aβ and MAPT pathologies due to the inclusion of three different mutant human transgenes (APP, PSEN1
) that causes AD and/or FTD. Because of this transgenic strategy, it remains difficult to attribute the effects of CX3CR1 deficiency to specific alterations in Aβ or MAPT pathologies. Notably, we have recently completed a separate study examining the effects of CX3CR1 deficiency on Aβ pathologies in two different mouse models of AD and demonstrate that blocking CX3CL1-CX3CR1 signaling actually reduces
Aβ pathologies via a mechanism involving alterations in microglial phagocytosis of extracellular Aβ aggregates (Lee et al., 2010
). Thus, taken together, our results suggest that CX3CR1 deficiency has opposing effects on the two primary pathologies of AD, extracellular Aβ plaques and intracellular MAPT aggregates, information that could not be discerned in the Fuhrmann et al. studies.
Second, Fuhrman et al. examined CX3CR1 deficient 3xTg animals at 4–6 months of age, which is just prior to substantial extracellular Aβ deposits exhibited in this model and at least 6–8 months prior to the first appearance of alterations in MAPT phosphorylation and aggregation (Oddo et al., 2003
). Notably, the authors also report that there are no observable alterations in MAPT phosphorylation in 4–6 month-old CX3CR1 deficient 3xTg mice. In addition, due to restrictions in live imaging within the brain by two photon microscopy, the neuronal loss detected by Fuhrman et al., was observed in the outer layers of the cortex and not within the hippocampus, where the initial alterations in MAPT phosphorylation and aggregation were observed in the hTau mouse model and have also been reported in the 3xTg mouse model. Because of this, the Fuhrman et al. study focuses on the abundant intracellular Aβ present at this age within this cortical neuronal population in the 3xTg mice as a potential neurotoxic trigger for the neuronal loss that can be blocked by CX3CR1 deficiency. How these results translate to effects on extracellular Aβ deposition, similar to that published by Lee et al., and intracellular MAPT aggregates, as presented here, remain to be determined.
Third, most of the transgenic mouse models of AD, including the 3xTg mice do not exhibit robust age-related neurodegeneration (reviewed in McGowan et al., 2006
), as assessed by other post-mortem methods utilized to detect neuronal cell loss. Thus, it remains to be determined how the neuronal loss observed by Fuhrmann et al. via two photon microscopy relates to the long-term Aβ toxicity observed in the AD mouse models.
Fourth, our studies suggest that CX3CR1 deficiency has opposing effects on Aβ and MAPT pathologies (Lee et al., 2010
). Given increasing evidence that Aβ pathologies precede MAPT pathologies by as much as 10 years in humans (Perrin et al., 2009
) our studies strongly suggest that blocking CX3CL1/CX3CR1 signaling has opposing effects at different stages of disease progression. Thus, while CX3CR1 deficiency at early stages of intracellular Aβ pathology (Fuhrmann et al., 2010
) and extracellular Aβ pathology (Lee et al., 2010
) may be protective, the effects of CX3CR1 deficiency are deleterious as the disease progresses towards development of enhanced microglia-mediated neuroinflammation and/or MAPT aggregation. Future studies will be required to assess the validity of this hypothesis in additional disease relevant animal models, as well as assess the effects on microglial phenotypes at different stages of disease progression.
Finally, the present study provides mechanistic links between CX3CR1 and specific AD pathologies and signaling pathways. CX3CR1 deficiency has specific effects on MAPT phosphorylation and aggregation, microglial activation and behavior in the hTau mouse model. In addition, these studies link CX3CR1 deficiency to the release of microglia factors (including IL1) that subsequently induces p38 MAPK and MAPT phosphorylation within neurons.
Interestingly, our previous studies demonstrated that administration of LPS to Cx3cr1−/−
mice induces neurodegeneration that is also dependent upon IL1 signaling (Cardona et al., 2006
), suggesting that IL1 may represent as an important signaling molecule between microglia and neurons. Notably, the relevance of CX3CL1-CX3CR1 signaling to human neurodegeneration has been highlighted by the identification of V249I and T280M polymorphisms in CX3CR1 that are associated with human age-related macular degeneration (Combadiere et al., 2007
). The current studies extends these observations and provides evidence that CX3CL1-CX3CR1 signaling plays a role in the development of tauopathies, as CX3CR1 deficient hTau mice exhibit enhanced microglial activation, pronounced MAPT hyperphosphorylation and pathology as well as memory disturbances (). These results also suggest that CX3CL1-CX3CR1 signaling may provide a novel therapeutic target for tauopathies ().
Several different studies have provided evidence that p38 MAPK is one of the MAPT kinases that links neuroinflammation and MAPT phosphorylation in tauopathies (Hartzler et al., 2002
; Hensley et al., 1999
; Sun et al., 2003
). In the present studies, we observed increased levels of activated p38 MAPK and ATF2 in the brains of CX3CR1 deficient hTau mice, but not other established MAPT kinases. Furthermore, our in vitro
studies demonstrated that soluble factors released by microglia (and enhanced in CX3CR1 deficient microglia) induces MAPT phosphorylation in cultured neurons that is dependent upon p38 MAPK activity and can be blocked by an IL1 receptor antagonist.
In conclusion, this study demonstrates that altered microglia activation plays a direct role in modulating the hyperphosphorylation and aggregation of MAPT within neurons and suggests potential strategies for therapeutic intervention in tauopathies.