12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation

Abstract

Background:Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system.Methodology:We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain.Results:We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo.Conclusions:This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical agents to facilitate neuronal renewal.

ELAC (3,12‐di‐O‐acetyl‐8‐O‐tigloilingol), a plant‐derived lathyrane diterpene, induces subventricular zone neural progenitor cell proliferation through PKCβ activation

“…To test the effects of our compounds on NPC proliferation, single cells from mechanically disaggregated neurospheres were plated in anti‐adherent 96‐well plates (Corning, NY, USA) at a density of 20 000 cells mL −1 (4000 cells in 200 μL per well), making triplicates for each condition in each experiment. Lathyranes and other pharmacological agents were added at the time of seeding, and neurosphere size and number were measured 72 h later, as previously described (Geribaldi‐Doldán et al, ). Experiments were repeated a minimum of five independent times, and each of these five independent experiments was run in triplicate.…”

“…Some of these primary neurospheres were mechanically disaggregated again after completion of the 72 h treatments with the different compounds, seeded at an identical density (4000 cells in 200 μL per well) in triplicates and cultured for an extra 72 h in the absence of any added drugs; then, the number of secondary neurospheres formed from pretreated NPC was determined by direct counting (Geribaldi‐Doldán et al, ). Quantification of the size and number of neurospheres were blinded (samples were coded and experiments were performed and quantified by different individuals).…”

“…These phorboids included the commercially available PMA and the non‐tumour‐promoting phorboid prostratin, in addition to six natural products isolated by us from the plant Euphorbia resinifera . Two of these compounds, which did not show tumour‐promoting activities, prostratin and ER272 (13‐ O ‐isobutyroyl‐12‐deoxyphorbol), also augment endogenous neurogenesis in response to a traumatic brain injury in vivo (Geribaldi‐Doldán et al, ). Interestingly, Sung et al () have reported that cerebral hypoxia induces NPC proliferation through a PKC‐dependent mechanism, suggesting that the neurogenic response to brain insults may be mediated by PKC activation.…”

“…Certain diterpenes with a lathyrane skeleton can mimic prostratin and other PKC activators in their ability to antagonize HIV‐1 latency in Jurkat cells, by a PKC‐dependent mechanism (Avila et al, ). These have been reported, based on its structures, as non‐tumour‐promoting agents; thus, our first aim was to test the capacity of four different lathyranes to promote NPC proliferation, in an attempt to identify new molecules useful to improve neuronal replacement through PKC modulation (Blumberg et al, ; Geribaldi‐Doldán et al, ). We show that one of these lathyranes, which we denominated ELAC, stimulates NPC proliferation in vivo and in vitro by a mechanism involving classical PKC activation and without negatively affecting stemness.…”

Microglia induce neurogenesis by stimulating PI3K/AKT intracellular signaling in vitro

“…Application of the PI3K/AKT inhibitor, LY294002, blocks microglial-enhanced neuronal survival and proliferation following injury (Figure 8) and the expression of neurogenic markers ( Figure 9). Inhibition of other intracellular signaling pathways associated with viability, proliferation, and neurogenesis such as MEK [84], p38MAPK [62], PKCα/βI/βII/γ [85][86][87], and Janus Kinase 2 protein [88,89] did not block the increased metabolic activity and viability of cortical cells co-cultured with microglia (see supplemental Figure 2S).…”