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The novel protein MANI modulates neurogenesis and neurite-cone growth.

Mishra M, Akatsu H, Heese K - J. Cell. Mol. Med. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, Singapore.

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Mani induces NSCs differentiation and neurogenesis. FACS purified neurospheres overexpressing Mani grown for three passages (3P, 1P ∼4 days) in Egf. Controls (C) were mock/GFP-transfected cells. Scale bar = 100 μm (A). Western blot analysis of Mani-transfected cells (NSC-Mani, E14, 3P) showed no changes in the glial marker Gfap, but a significant increase in Tubb3 and Th expression, whereas Nestin (Nes) was down-regulated and other neuronal markers (Chat, Gad [not shown]) remained unchanged, indicating that Mani induced a specific differentiation into catecholaminergic neurons (B). Comparison of Mani expression under different conditions, as indicated (NSCs were grown in the presence of Egf or Ngf or brain-derived neurotrophic factor for 3P, N = primary neuro-progenitor/neuron culture; compare with Fig. 2B) (C). Expression of neural Tubb3 in Mani-transfected NSCs (Mani, left) and control (mock-(GFP)-transfected) NSCs (C, right) upon differentiation in NB/B27 without Egf. Scale bar = 100 μm (D). Expression of Mbp in Mani-transfected and differentiated (in NB/B27) NSCs. Left panel: phase contrast microscopy. Right panel: Fluorescence microscopy of an Mbp-stained cell. The small panel shows the Western blot analysis of Mbp expression in Mani-transfected (= Mani) cells compared with mock/GFP-transfected (= C) NSCs. Scale bar = 50 μm (E). Mani-transfected NSCs were grown for 3P prior to exposure to differentiation medium (in NB/B27). Cells were then stained for Mani and Mtap2 to demonstrate the neuronal character of differentiated cells. Green = Mani, red = Mtap2, blue = nuclear DAPI stain. Scale bar = 100 μm (F).
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fig05: Mani induces NSCs differentiation and neurogenesis. FACS purified neurospheres overexpressing Mani grown for three passages (3P, 1P ∼4 days) in Egf. Controls (C) were mock/GFP-transfected cells. Scale bar = 100 μm (A). Western blot analysis of Mani-transfected cells (NSC-Mani, E14, 3P) showed no changes in the glial marker Gfap, but a significant increase in Tubb3 and Th expression, whereas Nestin (Nes) was down-regulated and other neuronal markers (Chat, Gad [not shown]) remained unchanged, indicating that Mani induced a specific differentiation into catecholaminergic neurons (B). Comparison of Mani expression under different conditions, as indicated (NSCs were grown in the presence of Egf or Ngf or brain-derived neurotrophic factor for 3P, N = primary neuro-progenitor/neuron culture; compare with Fig. 2B) (C). Expression of neural Tubb3 in Mani-transfected NSCs (Mani, left) and control (mock-(GFP)-transfected) NSCs (C, right) upon differentiation in NB/B27 without Egf. Scale bar = 100 μm (D). Expression of Mbp in Mani-transfected and differentiated (in NB/B27) NSCs. Left panel: phase contrast microscopy. Right panel: Fluorescence microscopy of an Mbp-stained cell. The small panel shows the Western blot analysis of Mbp expression in Mani-transfected (= Mani) cells compared with mock/GFP-transfected (= C) NSCs. Scale bar = 50 μm (E). Mani-transfected NSCs were grown for 3P prior to exposure to differentiation medium (in NB/B27). Cells were then stained for Mani and Mtap2 to demonstrate the neuronal character of differentiated cells. Green = Mani, red = Mtap2, blue = nuclear DAPI stain. Scale bar = 100 μm (F).

Mentions: For additional functional investigations of Mani, we continued our studies with NSCs (E14) (Fig. 5). As shown in (Fig. 5B), Mani-transfected neurospheres continuously cultured in the presence of Egf (epidermal growth factor) demonstrated a moderate change in Gfap (glial fibrillary acidic protein, a glial cell marker) expression, but evidently higher expression levels of the neuronal marker Tubb3 (neuron-specific β-class-III tubulin, Tuj1). Additionally, the stem cell marker Nestin was significantly reduced, indicating that Mani drives NSCs towards neurogenesis, even in the presence of the mitogen Egf (Fig. 5B). The increase in Tubb3 in Mani-transfected NSCs led us to the identification of the differentiating neuronal subtype. Therefore, we proceeded to check the expression levels of various neuronal markers, such as Chat (choline acetyltransferase, marker for cholinergic neurons), Gad (glutamate decarboxylase, marker for GABAergic neurons) and Th (marker for catecholaminergic neurons). Of utmost interest is our finding that Mani induced a striking up-regulation of Th (Fig. 5B) correlating with our findings of high expression of Mani in the catecholaminergic PC12 cells and Th+ areas in the CNS (Figs 2, 4, S6 and S7). In contrast, other neuro-specific markers, such as Chat or Gad (data not shown), remained unchanged.


The novel protein MANI modulates neurogenesis and neurite-cone growth.

Mishra M, Akatsu H, Heese K - J. Cell. Mol. Med. (2011)

Mani induces NSCs differentiation and neurogenesis. FACS purified neurospheres overexpressing Mani grown for three passages (3P, 1P ∼4 days) in Egf. Controls (C) were mock/GFP-transfected cells. Scale bar = 100 μm (A). Western blot analysis of Mani-transfected cells (NSC-Mani, E14, 3P) showed no changes in the glial marker Gfap, but a significant increase in Tubb3 and Th expression, whereas Nestin (Nes) was down-regulated and other neuronal markers (Chat, Gad [not shown]) remained unchanged, indicating that Mani induced a specific differentiation into catecholaminergic neurons (B). Comparison of Mani expression under different conditions, as indicated (NSCs were grown in the presence of Egf or Ngf or brain-derived neurotrophic factor for 3P, N = primary neuro-progenitor/neuron culture; compare with Fig. 2B) (C). Expression of neural Tubb3 in Mani-transfected NSCs (Mani, left) and control (mock-(GFP)-transfected) NSCs (C, right) upon differentiation in NB/B27 without Egf. Scale bar = 100 μm (D). Expression of Mbp in Mani-transfected and differentiated (in NB/B27) NSCs. Left panel: phase contrast microscopy. Right panel: Fluorescence microscopy of an Mbp-stained cell. The small panel shows the Western blot analysis of Mbp expression in Mani-transfected (= Mani) cells compared with mock/GFP-transfected (= C) NSCs. Scale bar = 50 μm (E). Mani-transfected NSCs were grown for 3P prior to exposure to differentiation medium (in NB/B27). Cells were then stained for Mani and Mtap2 to demonstrate the neuronal character of differentiated cells. Green = Mani, red = Mtap2, blue = nuclear DAPI stain. Scale bar = 100 μm (F).
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fig05: Mani induces NSCs differentiation and neurogenesis. FACS purified neurospheres overexpressing Mani grown for three passages (3P, 1P ∼4 days) in Egf. Controls (C) were mock/GFP-transfected cells. Scale bar = 100 μm (A). Western blot analysis of Mani-transfected cells (NSC-Mani, E14, 3P) showed no changes in the glial marker Gfap, but a significant increase in Tubb3 and Th expression, whereas Nestin (Nes) was down-regulated and other neuronal markers (Chat, Gad [not shown]) remained unchanged, indicating that Mani induced a specific differentiation into catecholaminergic neurons (B). Comparison of Mani expression under different conditions, as indicated (NSCs were grown in the presence of Egf or Ngf or brain-derived neurotrophic factor for 3P, N = primary neuro-progenitor/neuron culture; compare with Fig. 2B) (C). Expression of neural Tubb3 in Mani-transfected NSCs (Mani, left) and control (mock-(GFP)-transfected) NSCs (C, right) upon differentiation in NB/B27 without Egf. Scale bar = 100 μm (D). Expression of Mbp in Mani-transfected and differentiated (in NB/B27) NSCs. Left panel: phase contrast microscopy. Right panel: Fluorescence microscopy of an Mbp-stained cell. The small panel shows the Western blot analysis of Mbp expression in Mani-transfected (= Mani) cells compared with mock/GFP-transfected (= C) NSCs. Scale bar = 50 μm (E). Mani-transfected NSCs were grown for 3P prior to exposure to differentiation medium (in NB/B27). Cells were then stained for Mani and Mtap2 to demonstrate the neuronal character of differentiated cells. Green = Mani, red = Mtap2, blue = nuclear DAPI stain. Scale bar = 100 μm (F).
Mentions: For additional functional investigations of Mani, we continued our studies with NSCs (E14) (Fig. 5). As shown in (Fig. 5B), Mani-transfected neurospheres continuously cultured in the presence of Egf (epidermal growth factor) demonstrated a moderate change in Gfap (glial fibrillary acidic protein, a glial cell marker) expression, but evidently higher expression levels of the neuronal marker Tubb3 (neuron-specific β-class-III tubulin, Tuj1). Additionally, the stem cell marker Nestin was significantly reduced, indicating that Mani drives NSCs towards neurogenesis, even in the presence of the mitogen Egf (Fig. 5B). The increase in Tubb3 in Mani-transfected NSCs led us to the identification of the differentiating neuronal subtype. Therefore, we proceeded to check the expression levels of various neuronal markers, such as Chat (choline acetyltransferase, marker for cholinergic neurons), Gad (glutamate decarboxylase, marker for GABAergic neurons) and Th (marker for catecholaminergic neurons). Of utmost interest is our finding that Mani induced a striking up-regulation of Th (Fig. 5B) correlating with our findings of high expression of Mani in the catecholaminergic PC12 cells and Th+ areas in the CNS (Figs 2, 4, S6 and S7). In contrast, other neuro-specific markers, such as Chat or Gad (data not shown), remained unchanged.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, Singapore.

Show MeSH
Related in: MedlinePlus