Adult neurogenesis is the process by which the brain continuously generates and adds new neurons into specific regions, and the discovery and isolation of the progenitor cells responsible for this process has created exciting new possibilities for the treatment of multiple neurological diseases and injuries. Additionally, adult neurogenesis may play roles in learning, memory, stress, depression, and aging. Adult neural progenitor cells (NPCs) from the sub-granular zone (SGZ) of the mammalian hippocampus have the ability to differentiate into the three major cell types of the brain: neurons, astrocytes, and oligodendrocytes. They also have a fourth option, which is to simply divide and remain as stem cells, a process known as self-renewal. It is this mechanism by which NPCs replenish themselves, and it appears to be a significant regulatory point of adult neurogenesis. Therefore, the goal of this work is to investigate the intracellular molecular regulatory mechanisms of NPC proliferation and self-renewal.
There are multiple signaling mechanisms/pathways that could potentially promote NPC proliferation and self-renewal. To determine which of these pathways may be most critical, we used a series of established pharmacological inhibitors. The inhibitors of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway dramatically reduced NPC proliferation; and multiple NPC mitogens, including Sonic hedgehog (Shh) and basic fibroblast growth factor (FGF-2, the strongest NPC mitogen), activated Akt signaling. Additionally, retroviral vector-mediated overexpression of wild type Akt increased proliferation, while a dominant negative mutant decreased proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (β-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation.
Downstream of the Akt signal, we show that activation of the cAMP response element binding protein (CREB) occurs in cells stimulated by FGF-2, and it is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Overexpression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. Most importantly, Akt overexpression promotes expression of the transcription factor SRY-related HMG-box 2 (Sox2), which is important for the self-renewal of multiple stem cell types, including NPCs. It is also widely used as a multipotent NPC marker. Akt drives Sox2 expression by increasing its mRNA concentration. Additionally, Akt inhibition decreases Sox2 expression. Interestingly, however, Sox2 overexpression did not promote NPC proliferation. Taken together, these results indicate that Akt is a master promoter of proliferation and self-renewal, and that self-renewal is mediated by increased Sox2 expression.
Despite its demonstrated importance, the Akt signaling cascade is unlikely to be the only cascade responsible for NPC maintenance. Growth factor stimulation similar to that of FGF-2 is known to promote the activity of other key pathways, including Ras/mitogen activated protein kinase (MAPK) and calcium ion (Ca2+)-mediated pathways, in addition to Akt. There is also a great deal of cross-talk observed in multiple cell types between all three signaling modules. The precise importance of Akt signaling was investigated using tamoxifen-inducible, conditionally active Akt and PI3K mutants (Akt-ER and PI3K-ER). While these results show that Akt is sufficient for NPC proliferation, PI3K is not. Since Akt activation is mediated by two phosphorylation events, but only one of these events is driven by PI3K, these data indicate that Akt is acting as an AND gate and that the second phosphorylation of Akt is PI3K independent. Using pharmacological and genetic manipulation of Ras, calmodulin (CaM), CaM kinase II (CaMKII), CaM kinase kinase (CaMKK), CaM kinase IV (CaMKIV), and calcineurin, I investigated the importance of these pathways for NPC proliferation and self-renewal. The results of these studies provide no clear indication that any of these molecules are important for NPC proliferation and self-renewal. However, this is a critical step toward ruling out several major pathways.
Intracellular signaling and gene expression pathways can be very complex. In addition to the cross-talk mentioned above, they may involve intricate feed-forward loops, feedback loops, and parallel processing. Systems biology applies computational and experimental approaches to investigate the emergent behavior of collections of molecules and strives to explain how these numerous components interact to regulate molecular, cellular, and organismal behavior. Here, I also review systems biology, and in particular computational, efforts to understand the intracellular mechanisms of stem cell fate choice. These tools could potentially improve our understanding of NPC proliferation and self-renewal. I discuss deterministic and stochastic models that synthesize molecular knowledge into mathematical formalism, enable simulation of important system behaviors, and stimulate further experimentation. In addition, statistical analyses such as Bayesian networks and principal components analysis (PCA)/partial least squares (PLS) regression can distill large datasets into more readily-managed networks and principal components that provide insights into the critical aspects and components of regulatory networks. These computational tools, coupled with traditional experimentation may further our understanding of the critical networks governing NPC proliferation and self-renewal.