Figure 4. Effects of CBD on reactive gliosis, neuronal survival, and neurogenesis in rat hippocampi. A: representative photomi- crographs of the CA1 area of rat hippocampus showing the results of immunohistochemical evaluation of GFAP. B: representative photomi- crographs showing the results of Nissl staining of the whole rat hippocampus (2X magnification) and the corresponding CA1 region (10X magnification). C: immunofluorescence photomicrographs show- ing a particular (10X magnification) of DCX-labeled cells in the dentate gyrus (DG) of rat hippocampi. doi:10.1371/journal.pone.0028668.g004 reducing pro-inflammatory molecules and cytokine release that strongly occurs in Ab neurotoxicity. This activity was linked to its ability to act as a potent inhibitor of NFkB activation induced by Ab challenge [21]. The present findings, confirming the formerly obtained results and extending our knowledge about CBD pharmacology, indicate that a selective PPARc activation occurs upstream to CBD-mediated NFkB inhibition. Such activation appears to be responsible for a large plethora of CBD effects. Indeed, the interaction of CBD at the PPARc site results in a profound inhibition of reactive gliosis as showed by the reduction of both GFAP and S100B protein expression together with a marked decline of pro-inflammatory molecules and cytokine release observed in Ab challenged astrocytes. The in vitro findings were replicated in in vivo experiments, since once again CBD treatment resulted in a profound inhibition of astrocytic activation surrounding CA1 area Ab-injected and induced a rescue of CA1 neuronal viability in comparison to control. Along this line, results from hippocampal homogenates fully matched with in vitro data previously obtained from primary cultured astrocytes. Also in this case, CBD through the activation of PPARc provoked a marked reduction of NO, TNFa, and IL-1b release in association with a parallel decline of GFAP, S100B, and iNOS protein expression. The observation that in the hippocam- pus the selective activation of PPARc caused a decrease in p50 and p65 protein expression, further reinforces the importance of CBD Promotes Neuronal Survival and Neurogenesis the sequence of events PPARc activation/NFkB inhibition as responsible for the CBD anti-inflammatory effect. The PPARc mediated inhibition of S100B induced by CBD represents a crucial step in interrupting self-perpetuation of the reactive gliosis cycle. Indeed, the over-release of this astroglial-derived neurotrophin actively exacerbates the pro-inflammatory cytokine loop fuelled by Ab stimulation, massively accelerates amyloidogenicity by pro- moting cleavage of APP to Ab and induces tau protein hyperphosphorylation, by disrupting the Wnt pathway [22–24]. Notably, both in vivo and in vitro, a selective involvement of PPARc at the base of the neuroprotective and anti-gliotic effect of CBD appears peremptory, since a complete loss of any beneficial pharmacological activity of this phytocannabinoid occurs when it was co-administered with the PPARc antagonist GW9662. Neuroprotective effects exerted by PPARc agonists in neuro- pathological conditions, including Ab induced neuroinflammation and neurodegeneration have been largely described in the past years [25]. Besides microglial cells, emerging data indicate astrocytes and neurons as fundamental cell type targets for the beneficial role of PPARc ligands [26]. Astrocytes represent the most abundant glial cells type in the CNS. Once these cells undergo reactive activation, they produce cytokines and other molecules involved in inflammatory response, which are thought to significantly contribute to expand brain damage. Interestingly astrocytes express the highest level of PPARc in the CNS [27,28], and accumulating evidence over the last decade indicate that PPARc agonists may finely regulate their detrimental functions during a protracted activation exerting a profound anti-inflam- matory and neuroprotective effects [29]. Such well-defined and highly effective beneficial activity due to PPARc activation has recently demonstrated to functionally inactivate NFkB promoters inducing a massive down regulation of the activation of this transcription factor. Traditional PPARc agonists like thiazolidinediones were reported to inhibit the overproduction of NO, IL-6, and TNF-a as well as the increased expression of the inducible enzymes iNOS and COX2 induced in LPS-stimulated astrocytic and microglial cultures [30–32]. Taken together all these findings fully match those concerning the anti- inflammatory and neuroprotective activities exhibited by CBD in this study. AD neuroinflammation is a highly important feature that has been considered responsible for the progressive worsening of the disease. Given the tremendous complexity of AD, however, it appears to be oversimplified reasoning to consider only reactive astrogliosis and neuronal degeneration as the unique factors at the basis of the functional decline provoked by the course of the illness. Neuropathological investigation has provided evidence that in AD brains progressive neuronal loss is not accompanied by a new neuronal replacement while alterations in neurogenesis have been reported to occur [33]. Both aspects are considered to be important in contributing to the long-term development of disease. Therefore the disease symptoms could partly be due to the impaired replacement of new hippocampal neurons from endogenous neuronal stem cells [34,35], which is believed to promote learning and memory. Although there were some controversies over whether neurogenesis is increased or reduced in the pathogenesis of AD, later studies have confirmed deficient maturation of new neurons in AD patients [36]. As a result, an approach to enhance neurogenesis and/or maturation should be considered potential therapies for AD. Moreover, activation of PPARc has been reported to promote neurogenesis and agonists at these receptors regulate neuronal stem cell proliferation and differentiation as well [37]. In addition PPARc activation promotes neurite outgrowth in mature neurons, PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28668