The accumulation of soluble Aβ from the sequential proteolytic processing of APP and the hyperphosphorylation of tau is a primary hallmark of AD pathogenesis [4, 50]. In this study, we discovered that RAPGEF2 acts as a novel signaling component involved in Aβ oligomer-mediated synaptic and cognitive impairments in AD. Using multiple AD mouse models and postmortem human AD hippocampal tissue, our results indicate that the Aβ oligomer triggers the upregulation of RAPGEF2 levels in AD brains. Increased RAPGEF2 expression induces the loss of synapses via the activation of the Rap2-JNK pathway. Furthermore, the in vivo knockdown of endogenous RAPGEF2 prevents oligomeric Aβ-induced synaptic and cognitive dysfunction. This scenario showing the involvement of RAPGEF2 in the early synaptopathy of AD is summarized in Fig. 8.
One of the best-characterized familial AD models is the 3xTg-AD mouse model, which develops Aβ deposits prior to tangle formation, consistent with the amyloid cascade hypothesis [6]. Interestingly, we observed heightened levels of RAPGEF2 in 3xTg-AD mice at 3 months of age before the onset of synaptic and cognitive deficits (Fig. 2). An analogous result was found in 1.5-month-old 5xFAD mice expressing APP and PSEN1 mutations (Fig. 2) but not in rTg4510 tau mutants at 2.5 and 5.5 months of age (Additional file 2). These results indicate that either full-length APP or its proteolytic product Aβ augments RAPGEF2 expression in early AD brains, suggesting the potentially causative role of RAPGEF2 in subsequent synaptic and cognitive disturbances. Our results revealed that the Aβ oligomer, rather than full-length APP, induces the upregulation of RAPGEF2 levels (Fig. 3, Additional file 2, and Additional file 3). Furthermore, we provided direct evidence that oligomeric Aβ treatment stimulated RAPGEF2 expression in cultured hippocampal neurons (Fig. 3).
Importantly, augmented RAPGEF2 levels were also identified in the postmortem human AD hippocampus (Fig. 1). Because most cases of AD are sporadic (late-onset) and ~ 5% of cases are genetic (early-onset) in origin [51], we assume that the increased RAPGEF2 levels in the human AD hippocampus may reflect the relatively slow and gradual increase in Aβ oligomers in sporadic AD compared to familiar AD. Although it is unclear when RAPGEF2 expression begins to be stimulated in human AD patients, our data show that RAPGEF2 levels are commonly induced in the brains of early- and late-onset AD brains.
Rap1 and Rap2, the downstream effectors of RAPGEF2, are members of the Ras family of small GTPases [22]. Rap1 and Rap2 share close to 60% sequence homology [52], but they seem to exert distinct functions by activating different signaling pathways. For instance, Rap1 and Rap2 regulate the synaptic removal of AMPA-type glutamate receptors via the activation of p38 MAPK during long-term depression and JNK in synaptic depotentiation, respectively [20, 26, 28]. Furthermore, the activation of Rap2 causes the loss of excitatory synapses, while Rap1 has no effects on axonal or dendritic morphology in spiny neurons [21]. In line with these findings, our data show that the Aβ oligomer-mediated stimulation of RAPGEF2 expression selectively activates Rap2-JNK, rather than Rap1-p38 MAPK (Fig. 4), suggesting the distinct roles of Rap proteins in the pathogenetic mechanisms of AD.
The activation of JNK, a downstream target of Rap2, has been reported in human AD brains [53]. Our results also revealed that phosphorylation levels of JNK are significantly higher in cortical lysates from 3xTg-AD and 5xFAD mice during the same period of RAPGEF2 upregulation (Fig. 4 and Additional file 4), which is consistent with the Aβ-mediated activation of the JNK pathway in cultured cortical neurons [46, 54]. Conversely, JNK phosphorylates APP at the Thr688 residue and facilitates Aβ aggregation [55, 56], and the inhibition of JNK reduces soluble Aβ oligomers [57]. Thus, it is likely that Aβ and JNK could create a reciprocal activation circuit that further exacerbates Aβ pathology.
What are the upstream molecules that trigger the upregulation of RAPGEF2? Polo-like kinase 2 (Plk2) could be one of the candidates involved in Aβ-mediated synaptopathy, as Plk2 has been shown to promote RAPGEF2 activity in the hippocampus [25]. In support of this idea, increased levels of Plk2 were also observed in the APP-SwD1 AD mouse brain and in the human AD brain [58]. Furthermore, the pharmacological inhibition of Plk2 function ameliorates synapse loss and memory decline in an AD mouse model [58]. Thus, these findings suggest that Plk2 may act as an upstream regulator in RAPGEF2-mediated synaptic dysfunction and cognitive deficits. Further studies are warranted to identify additional upstream signaling molecules that trigger the upregulation of RAPGEF2.
Previous studies have reported that constitutively active Rap2 reduces the number and length of dendritic spines in CA1 hippocampal neurons [27], and JNK inhibition rescues Aβ oligomer-mediated spine loss [46]. Thus, it has been speculated that the Aβ oligomer-mediated upregulation of RAPGEF2 levels may induce changes in the number and morphology of dendritic spines by activating the Rap2-JNK signaling cascade. We found that the knockdown of RAPGEF2 restored the Aβ oligomer-mediated loss of dendritic spines but not the length and head size of spines (Fig. 5). These findings indicate that Aβ oligomers may induce multiple signaling pathways that act in parallel to affect distinct morphological aspects of dendritic spines. Indeed, a recent study showed that the Pyk2-Graf1-RhoA pathway plays a role in Aβ-mediated dendritic spinopathy [59].
To our surprise, in vivo silencing of RAPGEF2 in the hippocampal CA1 area was sufficient to preserve contextual fear memory retention (Fig. 6), which requires the intact function of the dorsal hippocampus [60]. Notably, electron microscopy analysis revealed that RAPGEF2 knockdown was sufficient to block the reduction in excitatory synapses in the hippocampal CA1 stratum radiatum of 3xTG-AD mice (Fig. 7).
In summary, our results demonstrate that RAPGEF2 mediates Aβ oligomer-induced synaptic and cognitive deficits in the AD hippocampus. Therefore, early intervention regarding RAPGEF2 expression might be a potential therapeutic option to help mitigate Aβ oligomer-induced synaptic and behavioral impairments in AD.