The novel protein MANI modulates neurogenesis and neurite-cone growth.
To date, three myelin-associated proteins [Nogo or reticulon 4 (RTN4), myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG)] are known to inhibit axonal regeneration via activation of the neuronal glycosylphosphatidylinositol-anchored Nogo receptor [NgR, together with p75 neurotrophin receptor (p75NTR) and Lingo-1].We show that knockdown of Cdc27, a component of the anaphase-promoting complex (APC), leads to enhanced neurite outgrowth.Our finding describes the novel MANI-Cdc27-APC pathway as an important cascade that prevents neurons from extending axons, thus providing implications for the potential treatment of neurodegenerative diseases.
Affiliation: Department of Molecular and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, Singapore.
- Growth Cones/metabolism*
- Membrane Proteins/genetics/metabolism*
- Nerve Tissue Proteins/genetics/metabolism*
- Aged, 80 and over
- Alzheimer Disease/genetics/metabolism
- Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
- Blotting, Western
- Cell Cycle Proteins/genetics/metabolism
- Cell Line, Tumor
- Gene Expression
- HeLa Cells
- Mice, Inbred C57BL
- Neural Stem Cells/metabolism
- PC12 Cells
- Parkinson Disease/genetics/metabolism
- Protein Binding
- RNA Interference
- Reverse Transcriptase Polymerase Chain Reaction
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fig08: Mani interacts with Cdc27 and acts as a positive effector of Cdc27. CoIP of Mani and Cdc27. Cell lysate was obtained from PC12 cells. Western blot was performed with an anti-Cdc27 (lane 1, 3, 4) or anti-Mani (lane 2, 5) antibody. Lanes 1–2: total PC12 cell lysates only and using an anti-Cdc27 (lane 1) or anti-Mani (lane 2) antibody for Western blot detection respectively; lanes 3 and 5: CoIP products using an anti-Mani antibody (Western blot detection with anti-Cdc27 antibody (lane 3) or an anti-Mani antibody [lane 5)], lane 4: CoIP product using control serum (detection with anti-Cdc27 antibody) (A). Co-localization (yellow) of Mani (green) and Cdc27 (red) expression in PC12 cells. Cells were differentiated with Ngf for 10 days and prepared for ICC. Purple = nuclear DAPI stain. Scale bar = 50 μm (B). Western blot analysis of Cdc27 knockdown (Cdc27-ko) by siRNA in PC12 cells. C = control (mock-(empty siRNA-GFP vector)-transfected), −Cdc27 = cdc27 siRNA, +Mani = Mani-transfected (C). Effect of Cdc27-ko by Cdc27-siRNA on Ngf-induced (10 days) neurite outgrowth in PC12 cells. Control cells were transfected with GFP (mock-(empty siRNA-GFP vector) only. Whereas Cdc27-ko cells demonstrated longer neurites compared with control cells, the response of Mani-transfected (+Mani) cells was poor. Scale bars = 50 μm (D). Quantitative neurite outgrowth estimation demonstrates that Cdc27-ko by siRNA resulted in longer neurites upon Ngf stimulation. Cells were treated as in (D). Data represent mean ± S.D. of three independent experiments (n = 3 data sets), each performed in duplicates (*P < 0.05 compared with controls and GFP-transfected cells) (E). Western blot analysis of control (C = GFP transfected) and Mani-transfected PC12 cells synchronized for 38 hrs with aphidicolin (+38) prior to incubation for an additional 24 and 48 hrs without aphidicolin (−24, −48), respectively (F). Western blot analysis of control and Mani-transfected cells, which were first synchronized with aphidicolin and then stimulated with Ngf for the indicated times (G). Effect of Mani on the cell cycle. Cell cycle analysis was performed with PC12 control cells (transfected with GFP only = C) and PC12 cells transfected with Mani (= Mani). The DNA content is represented by propidium iodide (PI) fluorescence intensity for 1 × 104 cells under each condition. Mani-transfected cells (both PC12 and NSCs) have a tremendous capacity to cross the G1/S and G2/M restriction points. The percentage of cells in the various cell cycle phases for the indicated experimental conditions, as indicated, is shown (H–K). Cell cycle analysis of unsynchronized control (= C, mock/GFP-transfected cells.) and Mani-transfected (= Mani) PC12 cells (H). Cell cycle analysis of synchronized PC12 cells treated for 38 hrs (∼1 cell cycle generation) with aphidicolin before allowing the cells to re-enter the cycle for 24 hrs (I). Cell cycle analysis as in (H), in which the cells were allowed to re-enter the cell cycle for 48 hrs (J). Cell cycle analysis of unsynchronized control (= C) and Mani-transfected (= Mani) NSCs. Evaluation of the cell cycle analysis performed in H-K reveals that Mani drives the cells out of G1/G0, beyond the restriction point, into the G2/M phase. Data represent mean ± S.D. of four independent experiments (n = 4 data sets), each performed in duplicates (*P < 0.05 compared with controls [C]) (K).
Because Mani promoted higher differentiation in NSCs, the first hypothesis is less likely to be the reason. To test this, we fathomed the Ngf signalling pathways involved in the differentiation of PC12 cells in both Mani- and mock-transfected cells. Mani-transfected PC12 cells demonstrated higher expression and activity of proteins involved in neuronal differentiation and survival, such as phosphorylated Mapk1/3 and Akt (Fig. 7B), like NSCs. Treatment of PC12 cells with Ngf has been shown to result in Ngf-receptor (Ntrk1)-dependent Stat3 (signal transducers and activators of transcription protein) activation, which in turn positively regulates the expression of Ccnd1  initiating neurite outgrowth. Additionally, Mani-transfected PC12 cells displayed reduced levels of phosphorylated Stat3 (pStat3) (Fig. 7B) and Ccnd1 (Fig. 8G) upon Ngf stimulation indicating that Mani negatively regulates Stat3 activation. It has been reported that negative regulation of Stat3 phosphorylation is necessary for neurogenesis confirming that Mani-transfected cells are more prone to differentiate into neurons (Figs 5 and 7B) [19, 25]. Thus, concluding that Mani does not inhibit differentiation; rejecting our first hypothesis.