Phosphatidylinositol 4-phosphate 5-kinase a negatively regulates nerve growth factor-induced neurite outgrowth in PC12 cells

Tian Liu,이상윤 생화학분자생물학회 2013 Experimental and molecular medicine Vol.45 No.3

Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Ka, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Ka in PC12 rat pheochromocytoma cells by stable expression of PIP5Ka microRNA that significantly reduced PIP5Ka expression and PIP2level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Ka-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Ka into PIP5Ka KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Ka KD, but was attenuated by the reintroduction of PIP5Ka. Moreover, exogenously applied PIP2 to PIP5Ka KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Ka acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.

BMB Reports : Nuclear Rac1 regulates the bFGF-induced neurite outgrowth in PC12 cells

( Eung Gook Kim ),( Eun Young Shin ) 생화학분자생물학회 2013 BMB Reports Vol.46 No.12

Rac1 plays a key role in neurite outgrowth via reorganization of the actin cytoskeleton. The molecular mechanisms underlying Rac1-mediated actin dynamics in the cytosol and plasma membrane have been intensively studied, but the nuclear function of Rac1 in neurite outgrowth has not yet been addressed. Using subcellular fractionation and immunocytochemistry, we sought to explore the role of nuclear Rac1 in neurite outgrowth. bFGF, a strong agonist for neurite outgrowth in PC12 cells, stimulated the nuclear accumulation of an active form of Rac1. Rac1-PBR (Q) mutant, in which six basic residues in the polybasic region at the C-terminus were replaced by glutamine, didn`t accumulate in the nucleus. In comparison with control cells, cells expressing this mutant form of Rac1 displayed a marked defect in extending neurites that was concomitant with reduced expression of MAP2 and MEK-1. These results suggest that Rac1 translocation to the nucleus functionally correlates with bFGF-induced neurite outgrowth. [BMB Reports 2013; 46(12): 617-622]

Coordinate Regulation of Neurite Outgrowth by LRRK2 and Its Interactor, Rab5

허혜영,김광수,설원기 한국뇌신경과학회 2010 Experimental Neurobiology Vol.19 No.2

Neurite outgrowth and its maintenance are essential aspects of neuronal cells for their connectivity and communication with other neurons. Recent studies showed that over-expression of either leucine-rich repeat kinase 2 (LRRK2), whose mutations are associated with familial Parkinson's disease (PD), or Rab5b, an early endosomal marker protein, induces reduction in neurite length. Based on our recent findings that LRRK2 co-localizes and interacts with Rab5, we tested the hypothesis that LRRK2 and Rab5 may functionally interplay while controlling neurite outgrowth. Firstly, we confirmed previous reports that over-expression of either the LRRK2 PD-specific G2019S mutant or the Rab5 constitutively active Q79L mutant, but not of dominant negative N133I mutant, significantly reduces neurite outgrowth. Secondly, when over-expression of either LRRK2 wild type (WT) or G2019S was accompanied with over-expression of one of the Rab5 variants (WT, Q79L and N133I), or with down-regulation of Rab5, the reduction extent of its neurite length was similar to that of cells over-expressing LRRK2 alone, regardless of Rab5's status. Finally, we observed similar patterns of neurite length regulation in embryonic rat hippocampal neuron cultures. Taken together, our results suggest that LRRK2 and Rab5 functionally coordinate their regulation of neurite outgrowth and that LRRK2 is a more critical factor than Rab5.

Neurites from PC12 cells are connected to each other by synapse-like structures

Jeon, Chan-Young,Jin, Jae-Kwang,Koh, Young-Ho,Chun, Wook,Choi, Ihn-Geun,Kown, Hyung-Joo,Kim, Yong-Sun,Park, Jae-Bong Wiley Subscription Services, Inc., A Wiley Company 2010 Synapse Vol.64 No.10

<P>PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin-coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno-staining method. Tau-1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP-2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse-like structures. Tat-C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat-C3 toxin together with neurotrophins also exhibited synapse-like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse-like structures in PC12 cells. Synapse 64:765–772, 2010. © 2010 Wiley-Liss, Inc.</P>

SGTb regulates a surface localization of a guidance receptor BOC to promote neurite outgrowth

Vuong, Tuan Anh,Lee, Sang-Jin,Leem, Young-Eun,Lee, Jae-Rin,Bae, Gyu-Un,Kang, Jong-Sun Elsevier 2019 Cellular signalling Vol.55 No.-

<P><B>Abstract</B></P> <P>Neuritogenesis is a critical event for neuronal differentiation and neuronal circuitry formation during neuronal development and regeneration. Our previous study revealed a critical role of a guidance receptor BOC in a neuronal differentiation and neurite outgrowth. However, regulatory mechanisms for BOC signaling pathway remain largely unexplored. In the current study, we have identified Small glutamine-rich tetratricopeptide repeat (TPR)-containing b (SGTb) as a BOC interacting protein through yeast two-hybrid screening. Like BOC, SGTb is highly expressed in brain and P19 embryonal carcinoma (EC) cells differentiated into neuronal cells. BOC and SGTb proteins co-precipitate in mouse brain and differentiated P19 EC cells. Furthermore, BOC and SGTb co-localize in neurites and especially are concentrated at the tip of neurites in various neuronal cells. SGTb depletion attenuates neuronal differentiation of P19 cells through reduction of the surface level of BOC. Additionally, SGTb depletion causes BOC localization at neurite tip, coinciding with decreased p-JNK levels critical for actin cytoskeleton remodeling. The overexpression of SGTb or BOC restores JNK activation in BOC or SGTb-depleted cells, respectively. Finally, SGTb elevates the level of surface-resident BOC in BOC-depleted cells, restoring JNK activation. Taken together, our data suggest that SGTb interacts with BOC and regulates its surface level and consequent JNK activation, thereby promoting neuronal differentiation and neurite outgrowth.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The TPR-region of SGTb is critical for the interaction with the intracellular region of BOC. </LI> <LI> SGTb-BOC was colocalized at the tip of neurites during neuronal differentiation. </LI> <LI> SGTb restores neuronal differentiation and JNK activity in BOC-depleted cells. </LI> <LI> SGTb regulates BOC's localization at the tip of neurite and JNK activation. </LI> <LI> SGTb promotes neurogenesis and neurite outgrowth through BOC-mediated JNK activation. </LI> </UL> </P>

Induction of Neurite Outgrowth by (-)-(7R, 8S)-Dihydrodehyd­rodiconiferyl Alcohol from PC12 Cells

Shin Jung Soo,Kim Yu Mi,Hong Seong Su,Kang Ho Sang,Yang Yoo Jung,Lee Don Koo,Hwang Bang Yeon,Ro Jai Seup,Lee Myung Koo The Pharmaceutical Society of Korea 2005 Archives of Pharmacal Research Vol.28 No.12

A lignan derivative, (-)-(7R, 8S)-dihydrodehydrodiconiferyl alcohol (DHDA), was isolated from Kalopanax septemlobus L. and was observed to have neuritogenic activity. DHDA at 50 $\mu$M caused a marked induction of neurite outgrowth and an enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. However, it did not exhibit any neurotrophic action. At 50 $\mu$M, DHDA enhanced NGF-induced neurite-bearing activity. This activity was partially blocked by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and by GF109203X, a protein kinase C (PKC) inhibitor. These results suggest that DHDA can induce neurite outgrowth and enhance NGF-induced neurite outgrowth from PC12 cells by amplifying up-stream steps such as MAPK and PKC.

Disrupted-in-schizophrenia 1 (DISC1) Regulates Dysbindin Function by Enhancing Its Stability

Lee, Seol-Ae,Kim, Seong-Mo,Suh, Bo Kyoung,Sun, Hwa-Young,Park, Young-Un,Hong, Ji-Ho,Park, Cana,Nguyen, Minh Dang,Nagata, Koh-ichi,Yoo, Joo-Yeon,Park, Sang Ki American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.11

<P>Dysbindin and DISC1 are schizophrenia susceptibility factors playing roles in neuronal development. Here we show that the physical interaction between dysbindin and DISC1 is critical for the stability of dysbindin and for the process of neurite outgrowth. We found that DISC1 forms a complex with dysbindin and increases its stability in association with a reduction in ubiquitylation. Furthermore, knockdown of DISC1 or expression of a deletion mutant, DISC1 lacking amino acid residues 403–504 of DISC1 (DISC1<SUP>Δ403–504</SUP>), effectively decreased levels of endogenous dysbindin. Finally, the neurite outgrowth defect induced by knockdown of DISC1 was partially reversed by coexpression of dysbindin. Taken together, these results indicate that dysbindin and DISC1 form a physiologically functional complex that is essential for normal neurite outgrowth.</P>

Nuclear Akt promotes neurite outgrowth in the early stage of neuritogenesis

( Ji Hye Park ),( Sang Bae Lee ),( Kyung Hoon Lee ),( Jee Yin Ahn ) 생화학분자생물학회 2012 BMB Reports Vol.45 No.9

In addition to its pivotal role in neuronal survival, PI3K/Akt signaling is integral to neuronal differentiation and neurite outgrowth. However, the exact role of Akt in neuronal differentiation is still controversial. Here, we found that nuclear expression of CA-Akt resulted in unusual rapid neurite outgrowth and overexpression of KD-Akt caused multiple dendrite growth without specific axon elongation. Moreover, microarray data revealed that the expression of FOXQ1 expression was about 10-fold higher in cells with nuclear, active Akt than in control cells. Quantitative real-time PCR analysis showed that mRNA levels were upregulated in NLS-CA-Akt cells as compared to KD or EV cells. Furthermore, our FACS analysis demonstrated that overexpression of NLS-CA-Akt accumulate cells in the G1 phase within 24 h, fitting with the rapid sprouting of neuritis. Thus, our data implied that at least in this early time frame, the overexpression of nuclear, active Akt forced cells into neurite development through probably FOXQ1regulation. [BMB Reports 2012; 45(9): 521-525]

Iron Enhances NGF-induced Neurite Outgrowth in PC12 Cells

Young-eun Yoo,정준모,홍진희,허규정,오억수 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

Rat pheochromocytoma 12 (PC12) cells undergo neu-ronal differentiation in response to nerve growth fac-tor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular sig-nal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting ef-fect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF- induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.

Effects of (+)-Eudesmin from the Stem Bark of Magnolia kobus DC. var. borealis Sarg. on Neurite Outgrowth in PC12 Cells

Yang, Yoo-Jung,Park, Jae-In,Lee, Hak-Ju,Seo, Seon-Mi,Lee, Oh-Kyu,Choi, Don-Ha,Paik, Ki-Hyon,Lee, Myung-Koo The Pharmaceutical Society of Korea 2006 Archives of Pharmacal Research Vol.29 No.12

(+)-Eudesmin [4,8-bis(3,4-dimethoxyphenyl)-3,7 -dioxabicyclo[3.3.0]octane] was isolated from the stem bark of Magnolia kobus DC. var. borealis Sarg. and found to have neuritogenic activity. $50\;{\mu}M$ (+)-eudesmin induced neurite outgrowth and enhanced nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. At this concentration, (+)-eudesmin also enhanced NGF-induced neurite-bearing activity and this activity was partially blocked by various protein kinase inhibitors. These included PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor. GF109203X, a protein kinase C (PKC) inhibitor and H89, a protein kinase A (PKA) inhibitor. These results suggest that (+)-eudesmin can induce neurite outgrowth from PC12 cells by stimulating up-stream MAPK, PKC and PKA pathways.