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Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression.

Alavian KN, Sgadò P, Alberi L, Subramaniam S, Simon HH - Neural Dev (2009)

Bottom Line: The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability.In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins.Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Neuroscience, Department of Neuroanatomy, Ruprecht-Karls-Universität, Heidelberg, Germany. kambiz.alavian@gmail.com

ABSTRACT

Background: The homeodomain transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic (mesDA) neurons in a cell-autonomous and gene-dose-dependent manner. Homozygote mutant mice, deficient of both genes (En1-/-;En2-/-), die at birth and exhibit a loss of all mesDA neurons by mid-gestation. In heterozygote animals (En1+/-;En2-/-), which are viable and fertile, postnatal maintenance of the nigrostriatal dopaminergic system is afflicted, leading to a progressive degeneration specific to this subpopulation and Parkinson's disease-like molecular and behavioral deficits.

Results: In this work, we show that the dose of Engrailed is inversely correlated to the expression level of the pan-neurotrophin receptor gene P75NTR (Ngfr). Loss of mesDA neurons in the Engrailed- mutant embryos is caused by elevated expression of this neurotrophin receptor: Unusually, in this case, the cell death signal of P75NTR is mediated by suppression of Erk1/2 (extracellular-signal-regulated kinase 1/2) activity. The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability. In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins.

Conclusion: Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult. The similarities to the disease etiology in combination with the nigral phenotype of En1+/-;En2-/- mice suggests that haplotype variations in the Engrailed genes and/or P75NTR that alter their expression levels could, in part, determine susceptibility to Parkinson's disease.

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Elevated P75NTR expression is causal for cell death. (A) Quantitative RT-PCR of ventral midbrain tissue (VM) derived from EnDM (En1-/-;En2-/-) and EnHT E12 embryos, and of En1-expressing N2A cells inducible by doxycycline (Dox). P75NTR expression is inversely correlated with En1 expression levels in tissues and cell lines. (B, C) Western blot analysis of the ventral midbrain tissue shows the same relationship between P75NTR protein levels and En1 expression. Each active En1 allele decreases the P75NTR expression level (n = 3, p = 0.002). (D) Ventral midbrain cultures derived from EnDM and En2-/- embryos. Silencing of P75NTR by double-stranded RNA oligos and application of P75NTR-inhibiting antibody (Rex) increases the survival rate of EnDM mesDA neurons compared to untreated control (Ctl) or after treatment with scrambled RNA oligos (n ≥ 6, p < 0.01). Error bars indicate standard error.
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Figure 1: Elevated P75NTR expression is causal for cell death. (A) Quantitative RT-PCR of ventral midbrain tissue (VM) derived from EnDM (En1-/-;En2-/-) and EnHT E12 embryos, and of En1-expressing N2A cells inducible by doxycycline (Dox). P75NTR expression is inversely correlated with En1 expression levels in tissues and cell lines. (B, C) Western blot analysis of the ventral midbrain tissue shows the same relationship between P75NTR protein levels and En1 expression. Each active En1 allele decreases the P75NTR expression level (n = 3, p = 0.002). (D) Ventral midbrain cultures derived from EnDM and En2-/- embryos. Silencing of P75NTR by double-stranded RNA oligos and application of P75NTR-inhibiting antibody (Rex) increases the survival rate of EnDM mesDA neurons compared to untreated control (Ctl) or after treatment with scrambled RNA oligos (n ≥ 6, p < 0.01). Error bars indicate standard error.

Mentions: A genome-wide expression analysis, using microarrays on En1 inducible N2A cell line, identified the NGF receptor P75NTR as downstream of the Engrailed transcription factors (data not shown). To confirm the microarray, we examined by quantitative RT-PCR the levels of P75NTR in the cell line as well as in ventral midbrain tissue in relationship to En1 expression. As a result of En1 induction in N2A cells, the endogenous expression of P75NTR decreased by 10-fold (9.9 ± 1.9%, p < 0.001, n = 8). Likewise, the ventral midbrain tissue, derived from control littermates (En2-/-) expressed almost 2.5-fold less P75NTR (42% ± 14.0%, p < 0.001, n = 6) than EnDM mutants (Figure 1A). The western blot analysis confirmed the latter results; ventral midbrain from En2-/- and EnHT embryos contained 74.4 ± 4.4% (p < 0.001, n = 3) and 60.2 ± 8.8% (p = 0.002, n = 3) less P75NTR protein, respectively, than the same tissue from EnDM littermates (Figure 1B,C).


Elevated P75NTR expression causes death of engrailed-deficient midbrain dopaminergic neurons by Erk1/2 suppression.

Alavian KN, Sgadò P, Alberi L, Subramaniam S, Simon HH - Neural Dev (2009)

Elevated P75NTR expression is causal for cell death. (A) Quantitative RT-PCR of ventral midbrain tissue (VM) derived from EnDM (En1-/-;En2-/-) and EnHT E12 embryos, and of En1-expressing N2A cells inducible by doxycycline (Dox). P75NTR expression is inversely correlated with En1 expression levels in tissues and cell lines. (B, C) Western blot analysis of the ventral midbrain tissue shows the same relationship between P75NTR protein levels and En1 expression. Each active En1 allele decreases the P75NTR expression level (n = 3, p = 0.002). (D) Ventral midbrain cultures derived from EnDM and En2-/- embryos. Silencing of P75NTR by double-stranded RNA oligos and application of P75NTR-inhibiting antibody (Rex) increases the survival rate of EnDM mesDA neurons compared to untreated control (Ctl) or after treatment with scrambled RNA oligos (n ≥ 6, p < 0.01). Error bars indicate standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667502&req=5

Figure 1: Elevated P75NTR expression is causal for cell death. (A) Quantitative RT-PCR of ventral midbrain tissue (VM) derived from EnDM (En1-/-;En2-/-) and EnHT E12 embryos, and of En1-expressing N2A cells inducible by doxycycline (Dox). P75NTR expression is inversely correlated with En1 expression levels in tissues and cell lines. (B, C) Western blot analysis of the ventral midbrain tissue shows the same relationship between P75NTR protein levels and En1 expression. Each active En1 allele decreases the P75NTR expression level (n = 3, p = 0.002). (D) Ventral midbrain cultures derived from EnDM and En2-/- embryos. Silencing of P75NTR by double-stranded RNA oligos and application of P75NTR-inhibiting antibody (Rex) increases the survival rate of EnDM mesDA neurons compared to untreated control (Ctl) or after treatment with scrambled RNA oligos (n ≥ 6, p < 0.01). Error bars indicate standard error.
Mentions: A genome-wide expression analysis, using microarrays on En1 inducible N2A cell line, identified the NGF receptor P75NTR as downstream of the Engrailed transcription factors (data not shown). To confirm the microarray, we examined by quantitative RT-PCR the levels of P75NTR in the cell line as well as in ventral midbrain tissue in relationship to En1 expression. As a result of En1 induction in N2A cells, the endogenous expression of P75NTR decreased by 10-fold (9.9 ± 1.9%, p < 0.001, n = 8). Likewise, the ventral midbrain tissue, derived from control littermates (En2-/-) expressed almost 2.5-fold less P75NTR (42% ± 14.0%, p < 0.001, n = 6) than EnDM mutants (Figure 1A). The western blot analysis confirmed the latter results; ventral midbrain from En2-/- and EnHT embryos contained 74.4 ± 4.4% (p < 0.001, n = 3) and 60.2 ± 8.8% (p = 0.002, n = 3) less P75NTR protein, respectively, than the same tissue from EnDM littermates (Figure 1B,C).

Bottom Line: The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability.In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins.Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Neuroscience, Department of Neuroanatomy, Ruprecht-Karls-Universität, Heidelberg, Germany. kambiz.alavian@gmail.com

ABSTRACT

Background: The homeodomain transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic (mesDA) neurons in a cell-autonomous and gene-dose-dependent manner. Homozygote mutant mice, deficient of both genes (En1-/-;En2-/-), die at birth and exhibit a loss of all mesDA neurons by mid-gestation. In heterozygote animals (En1+/-;En2-/-), which are viable and fertile, postnatal maintenance of the nigrostriatal dopaminergic system is afflicted, leading to a progressive degeneration specific to this subpopulation and Parkinson's disease-like molecular and behavioral deficits.

Results: In this work, we show that the dose of Engrailed is inversely correlated to the expression level of the pan-neurotrophin receptor gene P75NTR (Ngfr). Loss of mesDA neurons in the Engrailed- mutant embryos is caused by elevated expression of this neurotrophin receptor: Unusually, in this case, the cell death signal of P75NTR is mediated by suppression of Erk1/2 (extracellular-signal-regulated kinase 1/2) activity. The reduction in expression of Engrailed, possibly related to the higher levels of P75NTR, also decreases mitochondrial stability. In particular, the dose of Engrailed determines the sensitivity to cell death induced by the classic Parkinson-model toxin MPTP and to inhibition of the anti-apoptotic members of the Bcl-2 family of proteins.

Conclusion: Our study links the survival function of the Engrailed genes in developing mesDA neurons to the regulation of P75NTR and the sensitivity of these neurons to mitochondrial insult. The similarities to the disease etiology in combination with the nigral phenotype of En1+/-;En2-/- mice suggests that haplotype variations in the Engrailed genes and/or P75NTR that alter their expression levels could, in part, determine susceptibility to Parkinson's disease.

Show MeSH
Related in: MedlinePlus