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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 LRRK2 protein levels in cell lysates.(A) LRRK2 (and one fragment) is expressed in cultured mouse embronic stem cells (ESC), and its expression increases during retinoic acid-induced neuronal differentiation. LRRK2 levels in LRRK2+/− cells are about 50% lower than in wildtype (wt) cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls and indicate that lower protein amounts were loaded in the LRRK2+/− ESC-derived neuron sample. The immunoblots shown are representative of three independent experiments. (B) Full-length LRRK2 protein bands were quantified by densitometry and normalized to GAPDH loading control using Quantity One software. Bars represent mean ± SD (n = 3).
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pone-0020820-g001: Western blot analysis of LRRK2 protein levels in cell lysates.(A) LRRK2 (and one fragment) is expressed in cultured mouse embronic stem cells (ESC), and its expression increases during retinoic acid-induced neuronal differentiation. LRRK2 levels in LRRK2+/− cells are about 50% lower than in wildtype (wt) cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls and indicate that lower protein amounts were loaded in the LRRK2+/− ESC-derived neuron sample. The immunoblots shown are representative of three independent experiments. (B) Full-length LRRK2 protein bands were quantified by densitometry and normalized to GAPDH loading control using Quantity One software. Bars represent mean ± SD (n = 3).

Mentions: By homologous recombination of the targeting vector at the exon 2 region of the mouse genomic LRRK2 locus, heterozygous LRRK2+/− C57BL/6N mouse embryonic stem (ES) cells were generated. Two ES cell clones, where homologous recombination was verified by Southern blot analysis (Figure S1), were used for further characterisation. Quantitative RT-PCR revealed a significant reduction of LRRK2 mRNA levels to 55.9±4.3% compared to wildtype C57BL/6N ES cells. We also analysed expression levels of LRRK2 protein at different stages of neuronal differentiation by Western blotting using a validated anti-LRRK2 antibody. Densitometric quantification of the 275 kDa full-length LRRK2 band indicated that LRRK2 protein is expressed in cultured murine ES cells (Figure 1). Protein levels significantly increased in embryoid bodies and remained elevated in ES cell-derived neurons. In line with the LRRK2 mRNA data mentioned above, LRRK2 protein levels in LRRK2+/− cells were about 50% lower than in wildtype cells at all stages (Figure 1). Transduction of LRRK2-deficient embryonic ES cells with an adenoviral type 5 expression vector in order to reconstitute LRRK2 expression caused cell death as has been shown in other cell culture models overexpressing LRRK2 (data not shown).


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 LRRK2 protein levels in cell lysates.(A) LRRK2 (and one fragment) is expressed in cultured mouse embronic stem cells (ESC), and its expression increases during retinoic acid-induced neuronal differentiation. LRRK2 levels in LRRK2+/− cells are about 50% lower than in wildtype (wt) cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls and indicate that lower protein amounts were loaded in the LRRK2+/− ESC-derived neuron sample. The immunoblots shown are representative of three independent experiments. (B) Full-length LRRK2 protein bands were quantified by densitometry and normalized to GAPDH loading control using Quantity One software. Bars represent mean ± SD (n = 3).
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Related In: Results  -  Collection

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

pone-0020820-g001: Western blot analysis of LRRK2 protein levels in cell lysates.(A) LRRK2 (and one fragment) is expressed in cultured mouse embronic stem cells (ESC), and its expression increases during retinoic acid-induced neuronal differentiation. LRRK2 levels in LRRK2+/− cells are about 50% lower than in wildtype (wt) cells. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were co-detected as gel loading controls and indicate that lower protein amounts were loaded in the LRRK2+/− ESC-derived neuron sample. The immunoblots shown are representative of three independent experiments. (B) Full-length LRRK2 protein bands were quantified by densitometry and normalized to GAPDH loading control using Quantity One software. Bars represent mean ± SD (n = 3).
Mentions: By homologous recombination of the targeting vector at the exon 2 region of the mouse genomic LRRK2 locus, heterozygous LRRK2+/− C57BL/6N mouse embryonic stem (ES) cells were generated. Two ES cell clones, where homologous recombination was verified by Southern blot analysis (Figure S1), were used for further characterisation. Quantitative RT-PCR revealed a significant reduction of LRRK2 mRNA levels to 55.9±4.3% compared to wildtype C57BL/6N ES cells. We also analysed expression levels of LRRK2 protein at different stages of neuronal differentiation by Western blotting using a validated anti-LRRK2 antibody. Densitometric quantification of the 275 kDa full-length LRRK2 band indicated that LRRK2 protein is expressed in cultured murine ES cells (Figure 1). Protein levels significantly increased in embryoid bodies and remained elevated in ES cell-derived neurons. In line with the LRRK2 mRNA data mentioned above, LRRK2 protein levels in LRRK2+/− cells were about 50% lower than in wildtype cells at all stages (Figure 1). Transduction of LRRK2-deficient embryonic ES cells with an adenoviral type 5 expression vector in order to reconstitute LRRK2 expression caused cell death as has been shown in other cell culture models overexpressing LRRK2 (data not shown).

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