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Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

Schulz C, Paus M, Frey K, Schmid R, Kohl Z, Mennerich D, Winkler J, Gillardon F - PLoS ONE (2011)

Bottom Line: LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation.Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.

ABSTRACT

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/principal findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

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Western blot analysis of Nanog protein levels in cell lysates.(A) Protein expression of the pluripotency marker Nanog decreases more rapidly during retinoic acid-induced neuronal differentiation in LRRK2+/− cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls. The immunoblots shown are representative of three independent experiments. (B) Nanog immunoreactive bands were quantified by densitometry and normalized to GAPDH loading control. Bars represent mean ± SD (n = 3). Asterisk (*) indicates statistical significant (p<0.05) difference.
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pone-0020820-g004: Western blot analysis of Nanog protein levels in cell lysates.(A) Protein expression of the pluripotency marker Nanog decreases more rapidly during retinoic acid-induced neuronal differentiation in LRRK2+/− cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls. The immunoblots shown are representative of three independent experiments. (B) Nanog immunoreactive bands were quantified by densitometry and normalized to GAPDH loading control. Bars represent mean ± SD (n = 3). Asterisk (*) indicates statistical significant (p<0.05) difference.

Mentions: In order to confirm alterations in mRNA expression on the protein level, protein extracts from cultured cells were assessed by Western blotting. The pluripotency transcription factor Nanog was still detectable in extracts from wildtype neurons 7 d after retinoic acid-induced differentiation, whereas Nanog protein was not detected in LRRK2+/− neurons (Figure 4). On the other hand, protein levels of the ionotropic glutamate receptor 5 were significantly increased by 117.5±9.1% (Figure 5). Additionally, Western blotting revealed a decline in ezrin/radixin/moesin protein levels by 52.3±6.1% (Figure 6) and in 4E-BP1 by 81.0±7.7% in LRRK2+/− ES cell-derived neurons, whereas protein expression of eukaryotic translation initiation factor 4E (eIF4E), the interaction partner of 4E-BP1, remained unaltered (Figure 5).


Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

Schulz C, Paus M, Frey K, Schmid R, Kohl Z, Mennerich D, Winkler J, Gillardon F - PLoS ONE (2011)

Western blot analysis of Nanog protein levels in cell lysates.(A) Protein expression of the pluripotency marker Nanog decreases more rapidly during retinoic acid-induced neuronal differentiation in LRRK2+/− cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls. The immunoblots shown are representative of three independent experiments. (B) Nanog immunoreactive bands were quantified by densitometry and normalized to GAPDH loading control. Bars represent mean ± SD (n = 3). Asterisk (*) indicates statistical significant (p<0.05) difference.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111438&req=5

pone-0020820-g004: Western blot analysis of Nanog protein levels in cell lysates.(A) Protein expression of the pluripotency marker Nanog decreases more rapidly during retinoic acid-induced neuronal differentiation in LRRK2+/− cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls. The immunoblots shown are representative of three independent experiments. (B) Nanog immunoreactive bands were quantified by densitometry and normalized to GAPDH loading control. Bars represent mean ± SD (n = 3). Asterisk (*) indicates statistical significant (p<0.05) difference.
Mentions: In order to confirm alterations in mRNA expression on the protein level, protein extracts from cultured cells were assessed by Western blotting. The pluripotency transcription factor Nanog was still detectable in extracts from wildtype neurons 7 d after retinoic acid-induced differentiation, whereas Nanog protein was not detected in LRRK2+/− neurons (Figure 4). On the other hand, protein levels of the ionotropic glutamate receptor 5 were significantly increased by 117.5±9.1% (Figure 5). Additionally, Western blotting revealed a decline in ezrin/radixin/moesin protein levels by 52.3±6.1% (Figure 6) and in 4E-BP1 by 81.0±7.7% in LRRK2+/− ES cell-derived neurons, whereas protein expression of eukaryotic translation initiation factor 4E (eIF4E), the interaction partner of 4E-BP1, remained unaltered (Figure 5).

Bottom Line: LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation.Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.

ABSTRACT

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/principal findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus