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Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

Santos DM, Xavier JM, Morgado AL, Solá S, Rodrigues CM - PLoS ONE (2012)

Bottom Line: This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium.Calpain 2 silencing elicited decreased levels of GFAP.These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.

ABSTRACT
Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

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Related in: MedlinePlus

Neuronal and astrocytic phenotypes after induction of neural differentiation.β-III Tubulin and GFAP expression was evaluated in NS-TGFP cells at different times of differentiation as described in Materials and Methods. (A) Representative immunoblots (top) and corresponding densitometry analysis (bottom) showing an increase in β-III Tubulin and GFAP protein levels throughout neural differentiation. Results are expressed as mean ± SEM arbitrary units for at least three independent experiments. §p<0.05 and †p<0.001 from undifferentiated cells. (B) Immunofluorescence detection of neuronal and astrocytic morphology in cells co-stained with anti-β-III Tubulin and anti-GFAP antibodies after 4 days of neural differentiation. Scale bar, 20 µm.
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pone-0033468-g005: Neuronal and astrocytic phenotypes after induction of neural differentiation.β-III Tubulin and GFAP expression was evaluated in NS-TGFP cells at different times of differentiation as described in Materials and Methods. (A) Representative immunoblots (top) and corresponding densitometry analysis (bottom) showing an increase in β-III Tubulin and GFAP protein levels throughout neural differentiation. Results are expressed as mean ± SEM arbitrary units for at least three independent experiments. §p<0.05 and †p<0.001 from undifferentiated cells. (B) Immunofluorescence detection of neuronal and astrocytic morphology in cells co-stained with anti-β-III Tubulin and anti-GFAP antibodies after 4 days of neural differentiation. Scale bar, 20 µm.

Mentions: To further explore the mechanism by which calpains regulate neural differentiation, we transfected NS-TGFP cells with calpastatin, an endogenous specific calpain inhibitor [17], and then induced neural differentiation. Cells were grown under differentiation conditions for 48 h and the expression of neuronal and glial differentiation markers β-III Tubulin and glial fibrillary acidic protein (GFAP) was accessed at several time-points. As expected, both neural markers increased throughout differentiation, starting at 24 h and peaking at 48 h (Figure 5A). Cells developed typical neuronal and astrocytic morphologies, accompanied by the expression of β-III Tubulin and GFAP, respectively, at 2 days (data not shown) and 4 days in culture (Figure 5B).


Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

Santos DM, Xavier JM, Morgado AL, Solá S, Rodrigues CM - PLoS ONE (2012)

Neuronal and astrocytic phenotypes after induction of neural differentiation.β-III Tubulin and GFAP expression was evaluated in NS-TGFP cells at different times of differentiation as described in Materials and Methods. (A) Representative immunoblots (top) and corresponding densitometry analysis (bottom) showing an increase in β-III Tubulin and GFAP protein levels throughout neural differentiation. Results are expressed as mean ± SEM arbitrary units for at least three independent experiments. §p<0.05 and †p<0.001 from undifferentiated cells. (B) Immunofluorescence detection of neuronal and astrocytic morphology in cells co-stained with anti-β-III Tubulin and anti-GFAP antibodies after 4 days of neural differentiation. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303840&req=5

pone-0033468-g005: Neuronal and astrocytic phenotypes after induction of neural differentiation.β-III Tubulin and GFAP expression was evaluated in NS-TGFP cells at different times of differentiation as described in Materials and Methods. (A) Representative immunoblots (top) and corresponding densitometry analysis (bottom) showing an increase in β-III Tubulin and GFAP protein levels throughout neural differentiation. Results are expressed as mean ± SEM arbitrary units for at least three independent experiments. §p<0.05 and †p<0.001 from undifferentiated cells. (B) Immunofluorescence detection of neuronal and astrocytic morphology in cells co-stained with anti-β-III Tubulin and anti-GFAP antibodies after 4 days of neural differentiation. Scale bar, 20 µm.
Mentions: To further explore the mechanism by which calpains regulate neural differentiation, we transfected NS-TGFP cells with calpastatin, an endogenous specific calpain inhibitor [17], and then induced neural differentiation. Cells were grown under differentiation conditions for 48 h and the expression of neuronal and glial differentiation markers β-III Tubulin and glial fibrillary acidic protein (GFAP) was accessed at several time-points. As expected, both neural markers increased throughout differentiation, starting at 24 h and peaking at 48 h (Figure 5A). Cells developed typical neuronal and astrocytic morphologies, accompanied by the expression of β-III Tubulin and GFAP, respectively, at 2 days (data not shown) and 4 days in culture (Figure 5B).

Bottom Line: This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium.Calpain 2 silencing elicited decreased levels of GFAP.These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal.

ABSTRACT
Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

Show MeSH
Related in: MedlinePlus