A series of studies have demonstrated that

A series of studies have demonstrated that GABAergic neurons were found to be affected in AD patients (Inaguma et al., 1992; Mikkonen et al., 1999; Takahashi et al., 2010), as well as cholinergic neurons and glutamatergic neurons (Danysz et al., 2000; Muir et al., 1994; Selkoe, 2002). However, some researchers believe that GABAergic neurons were relatively resistant in AD due to their high expression of calcium-binding proteins (CBPs) (Mattson and Magnus, 2006). In our cellular AD model, we employed a mixed neuronal culture expressing GABAergic, glutamatergic, and cholinergic neuron markers and we did not find that specific types of neurons demonstrated significantly selective sensitivity to Aβ1–42 (Supplemental Fig. 5). It is possible that the neurons used in these experiments are not mature enough to express high levels of CBPs to afford the protection, compared with the ones in AD patients. Moreover, different neuron culture protocols, Aβ species, and treatment time may also lead to the different neuronal susceptibility to toxic exposure (Krantic et al., 2011; Pakaski et al., 1998). Further investigations may be necessary to clarify the association between neuronal mature extent and their susceptibility to Aβ toxic effects. Cdk inhibitors have been widely tested in animal models of central nervous system diseases including AD, PD, stroke and traumatic order DYKDDDDK injury (Hilton et al., 2008; Jorda et al., 2003; Osuga et al., 2000). Though they have been demonstrated to improve behavioral outcomes and increase neuronal survival in those animal models, the lack of specificity of those Cdk inhibitors may cause side effects and other issues. In previous reports, Park et al. demonstrated that dominant negative forms of Cdk4 and Cdk6, but not Cdk2 and Cdk3, prevented Aβ induced neuronal death (Giovanni et al., 1999; Park et al., 1997). While Copani et al. found that a dominant negative mutant of Cdk2 also protected neurons against Aβ toxicity (Copani et al., 1999, 2001). In our study, we found that both shCdk2 and shCdk4 could decrease neuronal death caused by Aβ, but shCdk2 were more effective than shCdk4. This discrepancy might be caused by the different experimental paradigms including different forms of Aβ aggregates, different gene silencing methods and different neuronal cell types used in those studies. It is still not clear from the current study which factors from the aberrant CCEs trigger the apoptosis pathways. The E2F1 related signaling pathways are generally accepted to be involved in neuronal apoptosis. For instance, E2F1 could induce caspase activation by directly increasing intracellular Apaf-1 levels (Furukawa et al., 2002). It also inhibits the NF-KB related survival signals and favors the accumulation of ROS, which induce the occurrence of apoptosis (Phillips et al., 1999). Furthermore, E2F1 also might induce the expression of other apoptosis related genes, such as Bcl2 (Eischen et al., 2001), Cdc2 (Konishi and Bonni, 2003) and Bim (Biswas et al., 2005). In this study, we found that Aβ1–42 treatment triggered DNA synthesis and induced the increased expression of Cyclin D1, Cdk2 and Cyclin A, though it should be noted that the cell cycle re-entry is only one of the potential risk factors from the multitude of activated signaling pathways in AD. The evidence suggests that other activated mitogenic pathways also exist in a similar manner. For example, MAPK/ERK1/2 signaling and PLC/IP3/PKC/JNK signaling have been reported to be involved in the cell cycle-reentry in AD researches (Frasca et al., 2004; Grant et al., 2001; Lopez-Bergami and Ronai, 2008). The complexity in AD mechanisms may account for our results demonstrating that targeting the single molecule (Cdk2 inhibition) only partly rescues and delays the neuronal apoptosis rather than completely blocks neuronal death. Therefore, it will likely be necessary to find additional signaling components in addition of targeting Cdk2 for therapeutic interventions for AD.