**2. NFκB initiates and maintains neuronal fate decision from neural stem cells**

NFκB is activated through a series of signaling cascades (Figure 1). The NFκB family con‐ tains 5 members including RelA(p65), RelB, c-Rel, p50/p105 (NFκB1) and p52/p100 (NFκB2),

© 2013 Zhang and Hu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Zhang and Hu; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

which form various combination of homodimers or heterodimers [8, 16]. In non-stimulated cells, the NFκB dimer is sequestered in the cytoplasm by the Inhibitor of NFκB (IκB), which include at least 8 members. Upon stimulation, IκB is degraded via a phosphorylation-de‐ pendent proteasome-mediated mechanism and the released NFκB is translocated to the nu‐ cleus where it binds to the κB-sites and regulates the transcription of target genes. The phosphorylation of IκB is regulated by the IκB kinase (IKK) that is activated by its upstream IKK kinases. The classical IKK complex contains 2 catalytic subunits IKK1/2 or IKKα/β and 1 regulatory subunit IKKγ [8, 16]. Three distinct signaling pathways for NFκB activation have been identified: classical (canonical), non-classical (non-canonical, alternative) and atypical pathways, all of them rely on sequentially activated kinases (Figure 1) [17]. The classical pathway involves the activation of classical IKK complex [9]. This pathway generally regu‐ lates the activation of classical NFκB complexes (e.g. p65/p50), in response to a wide range of stimuli such as pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleu‐ kin (IL) 1β, Toll-like receptor agonists (LPS), antigens, etc. The activated IKK complex phos‐ phorylates IκB members (IκBα, IκBβ, IκBε and p105) on a consensus motif DSGFxS (e.g. Ser-32/Ser-36 for IκBα and Ser-19/Ser-23 for IκBβ) and the phosphorylated serines act as binding site for β-TrCP, the substrate recognition subunit of a Skp1-Cullin-F-box (SCF)–type E3 ubiquitin-protein ligase complex, named SCFβ-TrCP. This process, then, leads to the ubiqui‐ tination on specific lysine and the ubiquitinated IκBs are directed to 26S proteasome for full degradation, leaving free NFκB complexes to enter into the nucleus. The kinetics of phos‐ phorylation and degradation of IκBβ or IκBε are much slower than that of IκBα and may reflect different substrate specificities of the IKK complex. The non-classical pathway in‐ volves TNF receptor associated factor 3 (TRAF3)-mediated activation of the NFκB-inducing kinase (NIK) and IKKα [18, 19]. Activated IKKα phosphorylates p100 on specific serine resi‐ dues. After phosphorylation, p100 is ubiquitinated by SCFβ-TrCP E3 ligase and cleaved by 19S proteasome, instead of completely degraded by 26S proteasome, to generate the NFκB subu‐ nit p52. This process is generally slower than the activation of the classical pathway and leads to a delayed activation of nuclear p52-containing complexes, such as RelB/p52. The mechanisms of p52 generation are either constitutive (by cotranslational processing) or in‐ ducible (by post-translational cleavage). The non-classical pathway is triggered by some par‐ ticular members of TNF family, such as Lymphotoxin (LT) β, B-cell activation factor (BAFF), CD40 ligand (CD40L). The function of classical pathway has been well investigated but nonclassical pathway remains in its infancy. In the following discussion, the role of NFκB sig‐ naling in the nervous system relates primarily to classical pathway.

In a mouse inducible IκBα transgenic model, NFκB in NSCs/NPCs is necessary for axogene‐

**IRAK**

**MyD88**

**TOLLIP**

**TLRs**

P

**Transcription**

P

**IKK-**<sup>a</sup> <sup>P</sup> <sup>P</sup>

P

**p100**

**p100**

**NIK**

**TRAF3**

**BAFFR**

**Ub UbUb**

**C-Terminal Degradation**

NFκB Signaling Directs Neuronal Fate Decision

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199

P

**LT-**b

**IKK**b

**<sup>I</sup>**k**Bs <sup>I</sup>**k**Bs**

P

P

P P

**Figure 1.** Classical and non-classical signaling pathways of NFκB activation.

**IL-1**

**IL-1R**

**IRAK**

<sup>P</sup> <sup>P</sup> <sup>P</sup> <sup>P</sup>

**Transcription**

In the zones of active neurogenesis in both postnatal and adult mouse brain, various mem‐ bers of the NFκB family are highly expressed [29], indicating for the first time that NFκB is actively involved in the proliferation, migration and differentiation of adult NSCs/NPCs [30, 31]. The presence of NFκB in adult neurogenic zone is further validated by the studies using immunofluorescent microscopy [32, 33]. Direct evidence for the *in vivo* effect of NFκB signal‐ ing on the proliferation of NSCs/NPCs derives from p65 and p50 double knockout mice [34] as well as overexpression of super inhibitor IκBα mutant in NSCs/NPCs [35, 36, 37, 38]. Lit‐ tle is known about the role of NFκB signaling in regulating neural differentiation of NSCs/ NPCs. A recent study demonstrates that toll-like receptor 2 (TLR2) induces neuronal differ‐ entiation via protein kinase C (PKC)-dependent activation of NFκB whereas TLR4 inhibites proliferation and neuronal differentiation of NPCs [30]. In p50-deficient mice, the neuronal differentiation of adult hippocampal NSCs is reduced by 50% while the proliferation does not change [39]. In our recent study, we demonstrate that NFκB signaling regulates the early differentiation of NSCs [32]. During early differentiation of NSCs, NFκB signaling becomes activated [32]. Addition of TNFα to activate NFκB signaling under proliferation conditions induces neural differentiation of NSCs/NPCs [32, 40, 41]. TNF-like weak inducer of apopto‐ sis (TWEAK) induces neuronal differentiation of NSCs/NPCs, under proliferation condition, through NFκB-dependent down-regulation of Hes1 that prevents neuronal differentiation

**MyD88**

**TRAF6**

**TNFR TNF**

**TRAF2/5**

**TAK1**

P **Ub Ub**

**Ub**

**I**k**Bs Degradation**

sis and maturation [21].

In adult nervous system, NFκB signaling plays a sword-edge role after injuries or diseases [15, 20, 21, 22, 23]. The final outcome is attributable to the cell types, disease stages, and tar‐ get genes. In most cases, NFκB signaling in immune cells, microglia/macrophage and astro‐ cytes is neurodestructive due to overwhelming production of inflammatory mediators and neurotoxic molecules [22, 23]. However, neuronal NFκB signaling is neuroprotective via its crucial role in maintaining neuronal survival, synaptogenesis, neural plasticity, learning and memory [22, 23, 24, 25]. Recent studies demonstrate a striking enrichment of phosphorylat‐ ed IκBα and IKK in the axon initial segment [26, 27] and the nodes of Ranvier [28], suggest‐ ing a novel role of NFκB signaling in regulating axonal polarity and initial axonal formation. In a mouse inducible IκBα transgenic model, NFκB in NSCs/NPCs is necessary for axogene‐ sis and maturation [21].

**Figure 1.** Classical and non-classical signaling pathways of NFκB activation.

which form various combination of homodimers or heterodimers [8, 16]. In non-stimulated cells, the NFκB dimer is sequestered in the cytoplasm by the Inhibitor of NFκB (IκB), which include at least 8 members. Upon stimulation, IκB is degraded via a phosphorylation-de‐ pendent proteasome-mediated mechanism and the released NFκB is translocated to the nu‐ cleus where it binds to the κB-sites and regulates the transcription of target genes. The phosphorylation of IκB is regulated by the IκB kinase (IKK) that is activated by its upstream IKK kinases. The classical IKK complex contains 2 catalytic subunits IKK1/2 or IKKα/β and 1 regulatory subunit IKKγ [8, 16]. Three distinct signaling pathways for NFκB activation have been identified: classical (canonical), non-classical (non-canonical, alternative) and atypical pathways, all of them rely on sequentially activated kinases (Figure 1) [17]. The classical pathway involves the activation of classical IKK complex [9]. This pathway generally regu‐ lates the activation of classical NFκB complexes (e.g. p65/p50), in response to a wide range of stimuli such as pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleu‐ kin (IL) 1β, Toll-like receptor agonists (LPS), antigens, etc. The activated IKK complex phos‐ phorylates IκB members (IκBα, IκBβ, IκBε and p105) on a consensus motif DSGFxS (e.g. Ser-32/Ser-36 for IκBα and Ser-19/Ser-23 for IκBβ) and the phosphorylated serines act as binding site for β-TrCP, the substrate recognition subunit of a Skp1-Cullin-F-box (SCF)–type E3 ubiquitin-protein ligase complex, named SCFβ-TrCP. This process, then, leads to the ubiqui‐ tination on specific lysine and the ubiquitinated IκBs are directed to 26S proteasome for full degradation, leaving free NFκB complexes to enter into the nucleus. The kinetics of phos‐ phorylation and degradation of IκBβ or IκBε are much slower than that of IκBα and may reflect different substrate specificities of the IKK complex. The non-classical pathway in‐ volves TNF receptor associated factor 3 (TRAF3)-mediated activation of the NFκB-inducing kinase (NIK) and IKKα [18, 19]. Activated IKKα phosphorylates p100 on specific serine resi‐ dues. After phosphorylation, p100 is ubiquitinated by SCFβ-TrCP E3 ligase and cleaved by 19S proteasome, instead of completely degraded by 26S proteasome, to generate the NFκB subu‐ nit p52. This process is generally slower than the activation of the classical pathway and leads to a delayed activation of nuclear p52-containing complexes, such as RelB/p52. The mechanisms of p52 generation are either constitutive (by cotranslational processing) or in‐ ducible (by post-translational cleavage). The non-classical pathway is triggered by some par‐ ticular members of TNF family, such as Lymphotoxin (LT) β, B-cell activation factor (BAFF), CD40 ligand (CD40L). The function of classical pathway has been well investigated but nonclassical pathway remains in its infancy. In the following discussion, the role of NFκB sig‐

198 Trends in Cell Signaling Pathways in Neuronal Fate Decision

naling in the nervous system relates primarily to classical pathway.

In adult nervous system, NFκB signaling plays a sword-edge role after injuries or diseases [15, 20, 21, 22, 23]. The final outcome is attributable to the cell types, disease stages, and tar‐ get genes. In most cases, NFκB signaling in immune cells, microglia/macrophage and astro‐ cytes is neurodestructive due to overwhelming production of inflammatory mediators and neurotoxic molecules [22, 23]. However, neuronal NFκB signaling is neuroprotective via its crucial role in maintaining neuronal survival, synaptogenesis, neural plasticity, learning and memory [22, 23, 24, 25]. Recent studies demonstrate a striking enrichment of phosphorylat‐ ed IκBα and IKK in the axon initial segment [26, 27] and the nodes of Ranvier [28], suggest‐ ing a novel role of NFκB signaling in regulating axonal polarity and initial axonal formation.

In the zones of active neurogenesis in both postnatal and adult mouse brain, various mem‐ bers of the NFκB family are highly expressed [29], indicating for the first time that NFκB is actively involved in the proliferation, migration and differentiation of adult NSCs/NPCs [30, 31]. The presence of NFκB in adult neurogenic zone is further validated by the studies using immunofluorescent microscopy [32, 33]. Direct evidence for the *in vivo* effect of NFκB signal‐ ing on the proliferation of NSCs/NPCs derives from p65 and p50 double knockout mice [34] as well as overexpression of super inhibitor IκBα mutant in NSCs/NPCs [35, 36, 37, 38]. Lit‐ tle is known about the role of NFκB signaling in regulating neural differentiation of NSCs/ NPCs. A recent study demonstrates that toll-like receptor 2 (TLR2) induces neuronal differ‐ entiation via protein kinase C (PKC)-dependent activation of NFκB whereas TLR4 inhibites proliferation and neuronal differentiation of NPCs [30]. In p50-deficient mice, the neuronal differentiation of adult hippocampal NSCs is reduced by 50% while the proliferation does not change [39]. In our recent study, we demonstrate that NFκB signaling regulates the early differentiation of NSCs [32]. During early differentiation of NSCs, NFκB signaling becomes activated [32]. Addition of TNFα to activate NFκB signaling under proliferation conditions induces neural differentiation of NSCs/NPCs [32, 40, 41]. TNF-like weak inducer of apopto‐ sis (TWEAK) induces neuronal differentiation of NSCs/NPCs, under proliferation condition, through NFκB-dependent down-regulation of Hes1 that prevents neuronal differentiation [42]. Selective inhibition of classical NFκB signaling by various pharmacologic inhibitors, small interfering RNA and NSC-specific transgene dominant-negative IκBα retain the tripo‐ tential ability of differentiation and restore or enhance self-renewal capability of NSCs, sug‐ gesting that NFκB signaling is essential for early neural differentiation [32]. The critical role of NFκB in the initial differentiation step of NSCs highlights a novel molecular mechanism for neurogenesis. We hypothesize that moderate activation of NFκB signaling promotes NSC differentiation into NPCs and maintains a continuous source for adult neurogenesis under physiological conditions. However, persistent and repeated overactivation of NFκB signaling in NSCs may exhaust NSC pool and thus lead to reduced neurogenesis as seen in aging patients [43, 44] and chronic stress [45].

To further test this hypothesis, we generated double transgenic mice expressing constitu‐ tively active form of IKKβ (IKKβCA) [46, 47] driven by the promoter of glial fibrillary acid protein (GFAP) by crossbreeding GFAP-Cre mice (Jackson Lab, 004600) with Rosa26- StopFloxed-IKKβCA mice (Jackson Lab, 008242). *In vitro* studies using the NSCs/NPCs cultured from the brain of GFAP-IKKβCA mice validated the over-activation of NFκB signaling (Fig‐ ure 2), the loss of NSCs during passage as determined by the reduced number of GFAP<sup>+</sup> / Nestin+ NSCs (Figure 3) as well as the inhibition of NSC selfrenewing and tripotential ca‐ pacity (Figure 4) [32]. The *in vivo* effect of persistent over-activation of NFκB on GFAP<sup>+</sup> NSCs and their progeny in brain neurogenic zones of adult animals and their correlations with aging are currently under investigation.

**GFAP Nestin**

**400x**

**WT**

**Merge**

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**GFAP Nestin Merge TG**

**Figure 3.** Over-activation of NFκB signaling in cultured brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice reduced the number of GFAP+/Nestin+ neural stem cells (Arrow). Passage 2 neurospheres cultured from brain subven‐ tricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) 5-week-old mouse were dissociated into single cells. Cells were plated in matrigel-coated 8-well chamber slide and cultured under proliferation media containing 20 ng/mL of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 3 d. After fixation for 10 min at room temperature with 4% paraformaldehyde, the cells were immunostained simultaneously with goat anti-GFAP polyclonal antibody and mouse anti-Nestin monoclonal antibody followed by donkey anti-goat *Alexa Fluor*® 488 and donkey anti-mouse *Alexa Fluor*® 594 secondary antibodies. The nuclei were counterstained with Hoechst 33258.

**Figure 2.** Over-activation of NFκB signaling in brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice deter‐ mined by Western blot analysis (A) and adenovirus-mediated NFκB-luciferase reporter assay (B). *A.* Whole cell lysates of primary neurospheres cultured from brain subventricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) adult mouse were immunoblotted with antibodies against phosphorylated p65 (Ser-536) or β-actin (as loading control). *B.* Dissociated neural stem/progenitor cells were plated on 96-well plate and infected with adenovirus carry‐ ing NFκB *firefly*-luciferase at 50 multiplicity of infection (MOI) for 24 h. Luciferase activity was measured with *OneGlo*™ luciferase assay and cell viability was determined with *CellTiter*-*Glo*™ luminescent assay. Data are expressed as relative fold change after cell number normalization. \*\* p<0.01 indicates statistical significance from WT control.

[42]. Selective inhibition of classical NFκB signaling by various pharmacologic inhibitors, small interfering RNA and NSC-specific transgene dominant-negative IκBα retain the tripo‐ tential ability of differentiation and restore or enhance self-renewal capability of NSCs, sug‐ gesting that NFκB signaling is essential for early neural differentiation [32]. The critical role of NFκB in the initial differentiation step of NSCs highlights a novel molecular mechanism for neurogenesis. We hypothesize that moderate activation of NFκB signaling promotes NSC differentiation into NPCs and maintains a continuous source for adult neurogenesis under physiological conditions. However, persistent and repeated overactivation of NFκB signaling in NSCs may exhaust NSC pool and thus lead to reduced neurogenesis as seen in

To further test this hypothesis, we generated double transgenic mice expressing constitu‐ tively active form of IKKβ (IKKβCA) [46, 47] driven by the promoter of glial fibrillary acid protein (GFAP) by crossbreeding GFAP-Cre mice (Jackson Lab, 004600) with Rosa26- StopFloxed-IKKβCA mice (Jackson Lab, 008242). *In vitro* studies using the NSCs/NPCs cultured from the brain of GFAP-IKKβCA mice validated the over-activation of NFκB signaling (Fig‐ ure 2), the loss of NSCs during passage as determined by the reduced number of GFAP<sup>+</sup>

NSCs (Figure 3) as well as the inhibition of NSC selfrenewing and tripotential ca‐

pacity (Figure 4) [32]. The *in vivo* effect of persistent over-activation of NFκB on GFAP<sup>+</sup> NSCs and their progeny in brain neurogenic zones of adult animals and their correlations

**Figure 2.** Over-activation of NFκB signaling in brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice deter‐ mined by Western blot analysis (A) and adenovirus-mediated NFκB-luciferase reporter assay (B). *A.* Whole cell lysates of primary neurospheres cultured from brain subventricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) adult mouse were immunoblotted with antibodies against phosphorylated p65 (Ser-536) or β-actin (as loading control). *B.* Dissociated neural stem/progenitor cells were plated on 96-well plate and infected with adenovirus carry‐ ing NFκB *firefly*-luciferase at 50 multiplicity of infection (MOI) for 24 h. Luciferase activity was measured with *OneGlo*™ luciferase assay and cell viability was determined with *CellTiter*-*Glo*™ luminescent assay. Data are expressed as relative

fold change after cell number normalization. \*\* p<0.01 indicates statistical significance from WT control.

/

aging patients [43, 44] and chronic stress [45].

200 Trends in Cell Signaling Pathways in Neuronal Fate Decision

with aging are currently under investigation.

Nestin+

**Figure 3.** Over-activation of NFκB signaling in cultured brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice reduced the number of GFAP+/Nestin+ neural stem cells (Arrow). Passage 2 neurospheres cultured from brain subven‐ tricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) 5-week-old mouse were dissociated into single cells. Cells were plated in matrigel-coated 8-well chamber slide and cultured under proliferation media containing 20 ng/mL of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 3 d. After fixation for 10 min at room temperature with 4% paraformaldehyde, the cells were immunostained simultaneously with goat anti-GFAP polyclonal antibody and mouse anti-Nestin monoclonal antibody followed by donkey anti-goat *Alexa Fluor*® 488 and donkey anti-mouse *Alexa Fluor*® 594 secondary antibodies. The nuclei were counterstained with Hoechst 33258.

and tensin homolog deleted on chromosome 10), and the Drosophila membrane-associated protein Numb homologs, Numb and Numblike [48]. Neuronal fate decision also relies on the intrinsic proneuronal genes in NECs/NSCs/NPCs [49]. The proneuronal factors specify distinct neuronal identities in different regions of the nervous system [49, 50]. Transcription‐ al activation and epigenic modification of the proneuronal genes are essential for neuronal lineage progression [51]. Little is known about the effect of NFκB signaling on the expres‐ sion or function of proneuronal factors during neurogenesis. The Hes family plays key but opposing role in regulating neurodevelopment. Hes1 and Hes5 are activated by Notch sig‐ naling and repress the expression of proneuronal factors such as Mash1, Neurogenin, Math and NeuroD [52, 53, 54]. In contrast, Hes6 promotes neuronal differentiation but inhibits as‐ trocyte differentiation [55, 56]. Notch signaling is regulated by NFκB signaling, and thus it is speculated that NFκB signaling may regulate the expression of proneuronal genes during neural induction and neurogenesis [57, 58, 59]. The tripartite motif-containing protein 32 (Trim32) promotes asymmetric dividing and neuronal differentiation of NSCs/NPCs by reg‐ ulating protein degradation and microRNA activity [60, 61], and enhancing retinoic acid re‐ ceptor-mediated transcription [62]. Our studies demonstrated that NFκB inhibition blocks the asymmetric distribution of Trim32 and maintain NSC selfrenewal [32], implying that NFκB signaling may initiate neuronal differentiation through suppressing Trim32 function.

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**4. Regulation of neural induction and neural plate patterning by NFκB**

NFκB signaling is essential for embryonic development (http://www.bu.edu/nf-kb/gene-re‐ sources/gene-knockouts/) because p65 knockout mice died on E15 and p65/p50 or p65/c-rel double knockout mice died on E13 due to liver degeneration [63, 64]. Such embryonic lethal‐ ity precluded further investigation on the role of NFκB in late embryonic brain develop‐ ment. Additional knockout of TNF receptor 1 (TNFR1) in these p65-null mice rescued embryonic lethality [65], providing an opportunity to investigate the role of NFκB signaling in regulating embryonic neurogenesis [34]. However, the distribution pattern of NSCs/NPCs and cell lineage analysis in neurogenic zones of these mutants have not yet been examined. IKKα/IKKβ double knockout mice died on E12 due to apoptosis of NECs leading to impair‐

Several lines of clinical studies identified the correlation of NFκB signaling defects to vari‐ ous neurodevelopmental disorders. Among 6 genes associated with nonsyndromic autoso‐ mal-recessive mental retardation [67, 68, 69], two, NIK- and IKKβ-binding protein (NIBP) [67, 68, 69] and coiled-coil and C2 domain-containing protein 2A (CC2D1A) [70, 71], have been shown to regulate NFκB signaling through the classical IKKβ pathway, implying the important role of NFκB signaling in mental retardation and possibly other neurodevelop‐ mental diseases. In autism spectrum disorders, activation of NFκB signaling is significantly increased [72, 73, 74], although the role and mechanism of the activated NFκB signaling re‐

**signaling**

ments in neurogenesis [66].

main to be determined.

**Figure 4.** Over-activation of NFκB signaling in cultured brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice led to loss of stemness (selfrenewal and tripotency). *A.* Diagram of modified stemness assay. Passage 2 neurospheres cultured from brain subventricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) 5-week-old mouse were dissociated into single cells for monolayer culture under differentiation media for 24 h. Then dissociated single cells (500 per well) were cultured in semisolid medium containing 20 ng/mL of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 12 d. The clones with more than 300 μm in diameter were picked, dissociated and cultured as neurosphere. The biggest secondary clones were dissociated into single cells plated on matrigel-coated 96-well plate. After 5 days' differentiation, the cells were fixed in 4% paraformaldehyde and cell lineage differentia‐ tions were examined with multi-labeled fluorescent immunocytochemistry using cell type-specific antibodies against neuron (N, Tuji1), astrocytes (A, GFAP) and oligodendrocytes (O, myelin basic protein). *B.* Fraction of primary clones that show different multipotency. Tripotential clone: differentiating into three types of neural cells; bipotential clone: differentiating into either two cell types; monopotential clone: differentiating into one cell type. *C.* The percentage of primary clones with selfrenewal and tripotency over the plated single cells.

#### **3. Regulation of proneuronal genes by NFκB signaling**

At each step of neurogenesis, cells undergo symmetric and asymmetric dividing to maintain stemness and generate daughter progeny. The self-renewal and neuronal fate decision of NECs/NSCs during embryonic neurogenesis are regulated by various transcription factors and their signaling pathways including the nuclear hormone receptor TLX (tailless), the high-mobility-group transcription factor Sox2, the basic helix-loop-helix transcriptional fac‐ tor Hes (hairy and enhancer of split), the tumor suppressor phosphatase Pten (phosphatase and tensin homolog deleted on chromosome 10), and the Drosophila membrane-associated protein Numb homologs, Numb and Numblike [48]. Neuronal fate decision also relies on the intrinsic proneuronal genes in NECs/NSCs/NPCs [49]. The proneuronal factors specify distinct neuronal identities in different regions of the nervous system [49, 50]. Transcription‐ al activation and epigenic modification of the proneuronal genes are essential for neuronal lineage progression [51]. Little is known about the effect of NFκB signaling on the expres‐ sion or function of proneuronal factors during neurogenesis. The Hes family plays key but opposing role in regulating neurodevelopment. Hes1 and Hes5 are activated by Notch sig‐ naling and repress the expression of proneuronal factors such as Mash1, Neurogenin, Math and NeuroD [52, 53, 54]. In contrast, Hes6 promotes neuronal differentiation but inhibits as‐ trocyte differentiation [55, 56]. Notch signaling is regulated by NFκB signaling, and thus it is speculated that NFκB signaling may regulate the expression of proneuronal genes during neural induction and neurogenesis [57, 58, 59]. The tripartite motif-containing protein 32 (Trim32) promotes asymmetric dividing and neuronal differentiation of NSCs/NPCs by reg‐ ulating protein degradation and microRNA activity [60, 61], and enhancing retinoic acid re‐ ceptor-mediated transcription [62]. Our studies demonstrated that NFκB inhibition blocks the asymmetric distribution of Trim32 and maintain NSC selfrenewal [32], implying that NFκB signaling may initiate neuronal differentiation through suppressing Trim32 function.

**Treatment (24 h)**

**Tripotential Bipotential**

primary clones with selfrenewal and tripotency over the plated single cells.

**3. Regulation of proneuronal genes by NFκB signaling**

**Dissociate**

**Monolayer Culture**

202 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**A**

**B**

**Fraction of clones(%)**

**Semi-solid** 

**Monopotential No clones**

**media Culture N/A/O**

**0.0**

**0.2**

**0.4**

**Stemness (%)**

**C**

**WT TG WT TG**

**Figure 4.** Over-activation of NFκB signaling in cultured brain neural stem/progenitor cells from GFAP-Cre-IKKβCA mice led to loss of stemness (selfrenewal and tripotency). *A.* Diagram of modified stemness assay. Passage 2 neurospheres cultured from brain subventricular zones (SVZ) of littermate wild-type (WT) or transgenic (TG) 5-week-old mouse were dissociated into single cells for monolayer culture under differentiation media for 24 h. Then dissociated single cells (500 per well) were cultured in semisolid medium containing 20 ng/mL of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 12 d. The clones with more than 300 μm in diameter were picked, dissociated and cultured as neurosphere. The biggest secondary clones were dissociated into single cells plated on matrigel-coated 96-well plate. After 5 days' differentiation, the cells were fixed in 4% paraformaldehyde and cell lineage differentia‐ tions were examined with multi-labeled fluorescent immunocytochemistry using cell type-specific antibodies against neuron (N, Tuji1), astrocytes (A, GFAP) and oligodendrocytes (O, myelin basic protein). *B.* Fraction of primary clones that show different multipotency. Tripotential clone: differentiating into three types of neural cells; bipotential clone: differentiating into either two cell types; monopotential clone: differentiating into one cell type. *C.* The percentage of

At each step of neurogenesis, cells undergo symmetric and asymmetric dividing to maintain stemness and generate daughter progeny. The self-renewal and neuronal fate decision of NECs/NSCs during embryonic neurogenesis are regulated by various transcription factors and their signaling pathways including the nuclear hormone receptor TLX (tailless), the high-mobility-group transcription factor Sox2, the basic helix-loop-helix transcriptional fac‐ tor Hes (hairy and enhancer of split), the tumor suppressor phosphatase Pten (phosphatase

**0.6**

**0.8**

**Dissociate**

**Primary clones**

**Selfrenewal**

**N/A, N/O, A/O**

**N, A, O**

**Multipotency**

**Secondary clones**
