To unravel the mechanisms underlying the effects of CNF1 on cognition in apoE4 mice, we performed molecular studies on the hippocampus and frontal cortex, focusing on different markers involved in memory, energy and neuroinflammation processes. We found, interestingly, that there is a genotype specificity in hippocampus, apoE4 mice displaying an hyper-activation state of Rho proteins. In this context, it has been shown that an excessive Rho activity, negatively affects synaptic and cognitive Alprostadil functions and errors in cellular modulators of APP processing induced by polymorphisms predisposes an individual to early or late-onset AD induced by an hyperactivation of the Rho family GTPases. Our results demonstrate that CNF1 is able to switch off the hyper-activation of Rho proteins in the hippocampus and the mechanism by which CNF1 counteracts this phenomenon most probably involves the ubiquitin-mediated proteasomal degradation of activated Rho GTPases. The involvement of the ubiquitin-proteasome pathway in CNF1 activity was first reported by Doye and coworkers in 2002, and subsequently confirmed by several other authors. All studies so far conducted on this matter have been performed in cell cultures but, obviously, the in vivo situation is much more complex. In fact, there is not only a genotypedependent difference in terms of Rho/Rac activation but also there is a difference between the two brain areas. This is a well known phenomenon, hippocampus and cortex differing in term of neurotransmitter dynamics, structures and plasticity and our results highlight a different Rho GTPases activation state by CNF1 in the two brain areas, CNF1 decreasing Rho proteins’activation in the hippocampus while activating them in the frontal cortex. This is probably due to the fact that CNF1 most certainly stimulates the activation/degradation process of Rho GTPases in both areas, but with a different outcome depending on initial activation status of Rho proteins. It is also relevant that CNF1 increases ATP availability in both hippocampus and cortex of apoE4 mice, although at different extent. How RhoA and Rac1 signaling can increase ATP is still uncharted and under investigation by our group, but we can hypothesize that the increase in ATP content observed in both brain regions could probably be linked to the CNF1-induced activation/degradation process. Furthermore, we have previously reported that CNF1 influences the mitochondrial homeostasis, induces a remarkable modification in the mitochondrial network architecture, with the appearance of elongated and interconnected mitochondria, and promotes an increment of proteins such as creatine and phosphocreatine, which are involved in ATP regeneration in the brain of pathological murine models. All these effects persist for long periods of time in mouse brains, suggesting the persistence of the CNF1 molecular effects Sipeimine rather the persistence of the toxin in the CNS. On this basis, mitochondria may be regarded as one of the target cell organelle of CNF1, this toxin playing an important role for the restoration of apoE4 brain energy balance. Furthermore, such an increase in ATP content could promote the amelioration of cognitive functions observed in apoE4 mice, where the hippocampal synaptic plasticity is altered by Ab accumulation. In this context, it is interesting to note that in apoE4 hippocampus the CNF1-dependent decrease of Rho and Rac, which occurs with a very significative increment of ATP content, is accompanied by counteraction of important neuroinflammatory markers of AD, such as Ab deposition and IL-1b overexpression.