The idea that brain accumulation of β-amyloid (Aβ) is the primary influence that triggers the cascade of pathogenic events leading to neuronal damage and dysfunction as the final common pathway in AD is the current leading hypothesis in the field (Hardy and Allsop, 1991
; Hardy and Selkoe, 2002
). Favoring this idea is the long time standing observation that amyloid plaques are associated with dendritic and axonal changes with local spine loss, axonal swellings, and dysmorphic processes surrounding the plaques. However, the exact molecular mechanisms by which accrual of brain Aβ leads to neuronal toxicity and morphological disruption of dendrites and axons remain unknown. Here we present data supporting the idea that aberrant activation of GSK-3β and subsequent inhibition of a transcriptional program mediated by CREB are likely key mechanisms that link Aβ to the structural damage of neural networks. Exposure to Aβ results in aberrant activation of GSK-3β that leads in turn to phosphorylation of targets critically involved in synaptic stability and neuronal survival. In particular, GSK-3β activation appears to inhibit a transcriptional program mediated by CREB that may compromise synaptic anatomical integrity and transmission by promoting dendritic spine loss and neuritic dystrophies, and distortion of the normal straight patterns of dendrites and axons. Importantly, we show reversal of Aβ induced morphological neurodegenerative phenotypes in vitro and in vivo by pharmacological GSK-3β inhibition, providing an important proof that this therapeutic intervention protects neuronal structure in cultured neurons and in a transgenic mouse model.
GSK-3β is a multi-tasking serine/threonine kinase with crucial roles in many major signaling processes in the brain and has recently proposed to promote most of the key steps involved in the neuronal dysfunction associated with AD including tau hyperphosphorylation and increased β-amyloid production (Hooper et al., 2008
). In vitro and in vivo overexpression of GSK-3β has been shown to result in apoptotic neuronal cell death (reviewed in Beurel and Jope, 2006
; Bhat et al., 2000b
; Hetman et al., 2000
; Lucas et al., 2001
). GSK-3β has also been involved in memory formation through mechanisms controlling synaptic function and synaptic plasticity and transgenic mice overexpressing GSK-3β exhibited pronounced impairment in spatial memory and long-term potentiation (LTP) in CA1 and dentate gyrus (Hernandez et al., 2002
). CREB-mediated gene expression plays a critical role in the best established form of synaptic plasticity related to learning, long-term potentiation (LTP), (reviewed in Barco et al., 2002
; Josselyn and Nguyen, 2005
; Lonze and Ginty, 2002
). Importantly, CREB DNA binding activity is inhibited by phosphorylation by GSK-3β and facilitated by lithium, the first known GSK-3 inhibitor (Grimes and Jope, 2001
). We had previously demonstrated an age-dependent aberrant increase in GSK-3β activity in the hippocampi of transgenic mice overexpressing human mutant APP and tau (APPsw
line) accompanying learning and memory deficits exhibited by these mice in the Morris water maze and contextual fear conditioning paradigms (Sereno et al., 2009
). Moreover, we have recently demonstrated that memory impairment can be completely prevented in these mice by sustained pharmacological inhibition of GSK-3β (Sereno et al., 2009
). These results suggest that the synaptic dysfunction and memory retention deficits that occur in AD patients and animal AD models might result, at least in part, from aberrantly increased GSK-3β activity and the resulting inhibition of the transcriptional program mediated by CREB.
In the present study, we have shown that exposure of neurons in culture to Tg2576 CM, which contains high levels of naturally secreted Aβ, and to oligomeric Aβ isolated from human brain, trigger aberrant activation of GSK-3β and this can be blocked by immunodepletion of the Tg2576 CM with an Aβ-specific antibody and by treatment with TDZD-8 which inhibits GSK-3β (). In this model, GSK-3β activation results in turn in decreased expression of one of the CREB targeted genes involved in synaptic plasticity, BDNF (). Importantly, aberrant activation of GSK-3β and decreased levels of two of the CREB transcriptional targets extensively implicated in synaptic plasticity and neuronal survival, ARC and BDNF, were also found in AD postmortem temporal cortex, indicating that the increase in the activity of this enzyme and the compromise of CREB signaling pathway also occurs in AD. Moreover, in cultures, GSK-3β inhibition provided very significant neuroprotection from Tg2576 CM and restored BDNF levels ( and ).
GSK-3β activation has profound effects on spine density and morphology. We show in cultures that overexpression of GSK-3β is sufficient to mimic the Aβ induced neurodegenerative phenotype in the absence of Aβ (dendritic spine loss and reduction in the proportion of thin and mushroom spines) ( and ), and transfection of neurons with a constitutively active form of GSK-3β results in a complete loss of dendritic spines () further favoring the idea that aberrant activation of this enzyme has a dramatic detrimental effect on neuron sculpture. GSK-3β inhibition in cultured neurons was associated with increases in spine density and the proportion of stubby spines and these effects may be mediated by BDNF signaling, as it has been shown in CA1 pyramidal neurons of hippocampal slice cultures (Chapleau et al., 2008
), reinforcing the notion that CREB-dependent transcription plays a key role in spine remodeling. GSK-3β inhibition in the mouse brain completely rescued the Aβ-related neurite morphological changes that occur both far from and near plaques, decreased the level of the inhibitory phosphorylation of CREB at Serine 129 and restored the level of expression of two of CREB target genes, c-fos and ARC ( and ) confirming in vivo relevance of our in vitro observations. All together these data point to a pivotal role of GSK-3β activation and CREB signaling pathway in Aβ-related neurite alterations, providing a mechanistic link between aberrant activation of GSK-3β, inhibition of CREB transcriptional targets, and Aβ-induced morphological disruption of neural systems both in vitro and in vivo. GSK3β is reportedly the predominant NFAT kinase, and inhibits NFAT activity (Beurel et al., 2010
). Our prior work (Wu et al., 2010
) suggested that increased (rather than decreased) NFAT activity mediated by calcineurin activation disrupts neurite structure around plaques. We have confirmed that oligomeric Aβ exposure increases the levels of nuclear NFAT in cultured neurons (data not shown), despite concomitant increase in GSK-3β activity, and this is consistent with the idea that calcineurin/NFAT is one of the signaling pathway involved in Aβ toxicity. The finding reported here that GSK-3β activation alone, even in the absence of Aβ, is sufficient to produce a phenocopy of Aβ-induced dendritic spine loss in neurons in culture provides evidence for the involvement of multiple mechanisms (e.g. calcineurin/NFAT activation, GSK-3β activation/CREB inhibition) in Aβ mediated neurotoxicity. Interestingly, nuclear NFAT levels did not increase after treatment with TDZD-8, as it could be predicted a priori. We argue that this is most likely mediated by the reported agonistic effect of TDZD compounds on the peroxisome proliferator-activated receptor gamma (PPAR-gamma) (Luna-Medina et al., 2007
). It has been previously shown that PPAR-gamma agonists and overexpression of PPAR-gamma inhibit the nuclear translocation of NFAT in different human cell types, pointing to a cross-talk between PPAR-gamma and calcineurin/NFAT (Yang et al., 2000
.; Bao et al., 2008
). Thus, some of the effects of TDZDs on neurite changes may be mediated in part by PPAR-gamma agonism in addition to GSK-3β inhibition, and this reinforces the potential usefulness of these compounds in AD.
Interestingly, sustained pharmacological GSK-3β inhibition robustly decreases the amount of total Aβ plaques and oligomeric Aβ deposits in the brain of APPsw
mice, () favoring a model where, on the one hand, activation of GSK-3β lies downstream of soluble Aβ neurotoxic effects and, on the other hand, inhibition of this enzyme impacts oligomeric brain Aβ accrual in vivo. These observations are in agreement with recent data showing reduction of Aβ42 levels upon GSK-3 inhibition in a Drosophila model of AD (Sofola et al., 2010
), and point to a direct role of GSK-3 in the regulation of Aβ levels. The mechanism/s responsible for the reduction of brain Aβ deposition observed after pharmacological inhibition of GSK-3β in the APPsw
mouse model need now to be elucidated.
One important implication of our results is that they confirm that Aβ induces aberrant activation of GSK-3β (Akiyama et al., 2005
; Kim et al., 2003
; Ryan and Pimplikar, 2005
) and we now show that aberrantly increased activity of GSK-3β in neurons causes loss of dendritic spines and spine morphological alterations. Although activation of GSK-3 has multiple effects, including tau phosphorylation, regulation of APP processing/Aβ production, activation of NF-kB and its proinflammatory targeted genes, and inhibition of multiple transcription factors involved in cell fate and survival (reviewed in Jope et al., 2007
), and thus it is possible that Aβ exposure induces neurotoxicity through many different mechanisms, our in vitro and in vivo data are consistent with the possibility that GSK-3β inhibition of the transcriptional program mediated by CREB plays a prominent role in the Aβ triggered neurodegenerative process. Moreover, our results implicate a soluble form of Aβ, likely oligomeric, as the responsible bioactive molecule that mediates the neuronal alterations that occur near plaques (Koffie et al., 2009
). A second relevant implication of our results is that increased activity of GSK-3β produces a phenocopy of these Aβ effects. Importantly, either immunodepletion of Aβ or pharmacological inhibition of GSK-3β can inhibit these changes and lead to recovery of spine density and neurite structure. Moreover, our current data show that pharmacological reduction of aberrantly increased GSK-3β activity not only blocks the morphological changes induced by Aβ on neuronal processes but also decreases brain oligomeric Aβ accrual in vivo, reinforcing the importance of this therapeutic strategy and the disease-relevance of these observations. However, our in vitro data also alert about potential adverse effects of excessive GKS-3β inhibition on dendritic spines. Concordant with these data a recent study showed that administration of a novel ATP competitive GSK3 inhibitor (SB216763) to wt mice induced inflammation and behavioral deficits further indicating that the window to safely achieved beneficial GSK-3 inhibition needs to be carefully defined.
Finally, our results suggest that GSK-3β and CREB mediated transcriptional program play a major role in neural sculpting. It has been established that GSK-3β has a prominent role in memory and CREB-mediated gene expression plays a critical role in the best established form of synaptic plasticity related to learning, long-term potentiation (LTP), (Barco et al., 2002
; Josselyn and Nguyen, 2005
; Lonze and Ginty, 2002
). However, a major role for GSK-3β-CREB mediated transcriptional events in neurite remodeling in the adult brain or in disease conditions as we observed here has not been previously reported. Our data favor a model where the neurotoxic and structural damage effects of Aβ accrual may be mediated, at least in part, by aberrant activation of GSK-3β and its consequent inhibition of CREB mediated transcriptional program providing a molecular mechanism of neurodegeneration in AD and further evidence that inhibition of GSK-3β has the potential to be a disease-modifying strategy in this devastating disorder.