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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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Differential activation of Erk1/2 in mesDA neurons. (A-D, G-K) Immunohistochemistry of E12 ventral midbrain cell culture stained against TH (green), total Erk1/2 protein (red) (A, B) and phosphorylated Erk1/2 (red) (C-D, G-K). (A-D) While Erk1/2 protein is present in mesDA neurons of both genotypes (A, B), it is only phosphorylated in En2-/- mesDA neurons (C) and not in the En1-/-;En2-/- (EnDM) counterparts (D). (E-I) Erk1/2 becomes activated in EnDM mesDA neurons after treatment with the survival-inducing neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3, or after silencing of P75NTR (RNA interference (RNAi)) (G-J), but not when glial cell line-derived neurotrophic factor (GDNF) is applied (I). (E) Western blot of E12 ventral midbrain tissue confirms the immunohistochemical finding of differential phosphorylation between genotypes and shows that neither AKT, part of the phosphotidyl inositol-3 kinase pathway, nor other mitogen-activated protein kinases, such as JNK and P38, are differentially activated. (F) Quantification of phosphorylated Erk1/2 in western blot normalized against En2-/- tissue. (L) Number of TH-positive cells in EnDM and En2-/- ventral midbrain cultures after 72 hours, treated with the 400 nM Mek inhibitor U0126 in conjunction with BDNF, Penetratin-coupled P75NTR double-stranded RNA oligonucleotides and the P75NTR inhibiting antibody (Rex). Numbers are normalized against untreated cultures at 24 hours. The rescue effect is significantly reduced when the EnDM cultures are treated with the Erk1/2 inhibitor. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.
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Figure 3: Differential activation of Erk1/2 in mesDA neurons. (A-D, G-K) Immunohistochemistry of E12 ventral midbrain cell culture stained against TH (green), total Erk1/2 protein (red) (A, B) and phosphorylated Erk1/2 (red) (C-D, G-K). (A-D) While Erk1/2 protein is present in mesDA neurons of both genotypes (A, B), it is only phosphorylated in En2-/- mesDA neurons (C) and not in the En1-/-;En2-/- (EnDM) counterparts (D). (E-I) Erk1/2 becomes activated in EnDM mesDA neurons after treatment with the survival-inducing neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3, or after silencing of P75NTR (RNA interference (RNAi)) (G-J), but not when glial cell line-derived neurotrophic factor (GDNF) is applied (I). (E) Western blot of E12 ventral midbrain tissue confirms the immunohistochemical finding of differential phosphorylation between genotypes and shows that neither AKT, part of the phosphotidyl inositol-3 kinase pathway, nor other mitogen-activated protein kinases, such as JNK and P38, are differentially activated. (F) Quantification of phosphorylated Erk1/2 in western blot normalized against En2-/- tissue. (L) Number of TH-positive cells in EnDM and En2-/- ventral midbrain cultures after 72 hours, treated with the 400 nM Mek inhibitor U0126 in conjunction with BDNF, Penetratin-coupled P75NTR double-stranded RNA oligonucleotides and the P75NTR inhibiting antibody (Rex). Numbers are normalized against untreated cultures at 24 hours. The rescue effect is significantly reduced when the EnDM cultures are treated with the Erk1/2 inhibitor. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.

Mentions: The up-regulation of P75NTR and the presence of Trk receptors suggested that Engrailed deficiency introduces a neurotrophin requirement to the E12 mesDA neurons that cannot be satisfied at this age, since the neurotrophins specific to TrkB and TrkC – that is, BDNF, NT4 and NT3 – are not expressed in the E12 ventral midbrain as they are in the adult (Figure 2H). To test this hypothesis, we applied saturating concentrations of BDNF, NT4 and NT3 to ventral midbrain cultures. After 72 hours, 50.2 ± 2.9% (p < 0.0001, n = 27), 42.3 ± 10.1% (p < 0.001, n = 9) and 26.0 ± 3.5% (p < 0.001, n = 9), respectively, of the otherwise dying EnDM mesDA neurons were still present in the cultures (Figure 2I). The addition of BDNF to the control littermate cultures demonstrated that this was due to an elevated survival rate and not attributable to a higher rate of precursor cell proliferation (Figure 3L). As expected from the lack of TrkA, application of its ligand, NGF, did not change the survival rate significantly. To test the specificity of BNDF, NT3 and NT4, we applied glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor (GDF)-15 and transforming growth factor (TGF)-β to the mutant cultures, all known survival factors for mesDA neurons [37-39]. Similar to NGF, none of them prevented the death of the Engrailed-deficient mesDA neurons (Figure 2I). Furthermore, the linear dose-response trend-line of the survival effect of BDNF (Figure 2J) indicated a Kd value of approximately 2.5 nM, which corresponds with the reported affinity of BDNF for the TrkB/P75NTRcomplex [40].


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)

Differential activation of Erk1/2 in mesDA neurons. (A-D, G-K) Immunohistochemistry of E12 ventral midbrain cell culture stained against TH (green), total Erk1/2 protein (red) (A, B) and phosphorylated Erk1/2 (red) (C-D, G-K). (A-D) While Erk1/2 protein is present in mesDA neurons of both genotypes (A, B), it is only phosphorylated in En2-/- mesDA neurons (C) and not in the En1-/-;En2-/- (EnDM) counterparts (D). (E-I) Erk1/2 becomes activated in EnDM mesDA neurons after treatment with the survival-inducing neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3, or after silencing of P75NTR (RNA interference (RNAi)) (G-J), but not when glial cell line-derived neurotrophic factor (GDNF) is applied (I). (E) Western blot of E12 ventral midbrain tissue confirms the immunohistochemical finding of differential phosphorylation between genotypes and shows that neither AKT, part of the phosphotidyl inositol-3 kinase pathway, nor other mitogen-activated protein kinases, such as JNK and P38, are differentially activated. (F) Quantification of phosphorylated Erk1/2 in western blot normalized against En2-/- tissue. (L) Number of TH-positive cells in EnDM and En2-/- ventral midbrain cultures after 72 hours, treated with the 400 nM Mek inhibitor U0126 in conjunction with BDNF, Penetratin-coupled P75NTR double-stranded RNA oligonucleotides and the P75NTR inhibiting antibody (Rex). Numbers are normalized against untreated cultures at 24 hours. The rescue effect is significantly reduced when the EnDM cultures are treated with the Erk1/2 inhibitor. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Differential activation of Erk1/2 in mesDA neurons. (A-D, G-K) Immunohistochemistry of E12 ventral midbrain cell culture stained against TH (green), total Erk1/2 protein (red) (A, B) and phosphorylated Erk1/2 (red) (C-D, G-K). (A-D) While Erk1/2 protein is present in mesDA neurons of both genotypes (A, B), it is only phosphorylated in En2-/- mesDA neurons (C) and not in the En1-/-;En2-/- (EnDM) counterparts (D). (E-I) Erk1/2 becomes activated in EnDM mesDA neurons after treatment with the survival-inducing neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)4 and NT3, or after silencing of P75NTR (RNA interference (RNAi)) (G-J), but not when glial cell line-derived neurotrophic factor (GDNF) is applied (I). (E) Western blot of E12 ventral midbrain tissue confirms the immunohistochemical finding of differential phosphorylation between genotypes and shows that neither AKT, part of the phosphotidyl inositol-3 kinase pathway, nor other mitogen-activated protein kinases, such as JNK and P38, are differentially activated. (F) Quantification of phosphorylated Erk1/2 in western blot normalized against En2-/- tissue. (L) Number of TH-positive cells in EnDM and En2-/- ventral midbrain cultures after 72 hours, treated with the 400 nM Mek inhibitor U0126 in conjunction with BDNF, Penetratin-coupled P75NTR double-stranded RNA oligonucleotides and the P75NTR inhibiting antibody (Rex). Numbers are normalized against untreated cultures at 24 hours. The rescue effect is significantly reduced when the EnDM cultures are treated with the Erk1/2 inhibitor. Scale bars: 25 μm. Error bars indicate standard error. Ctl, control.
Mentions: The up-regulation of P75NTR and the presence of Trk receptors suggested that Engrailed deficiency introduces a neurotrophin requirement to the E12 mesDA neurons that cannot be satisfied at this age, since the neurotrophins specific to TrkB and TrkC – that is, BDNF, NT4 and NT3 – are not expressed in the E12 ventral midbrain as they are in the adult (Figure 2H). To test this hypothesis, we applied saturating concentrations of BDNF, NT4 and NT3 to ventral midbrain cultures. After 72 hours, 50.2 ± 2.9% (p < 0.0001, n = 27), 42.3 ± 10.1% (p < 0.001, n = 9) and 26.0 ± 3.5% (p < 0.001, n = 9), respectively, of the otherwise dying EnDM mesDA neurons were still present in the cultures (Figure 2I). The addition of BDNF to the control littermate cultures demonstrated that this was due to an elevated survival rate and not attributable to a higher rate of precursor cell proliferation (Figure 3L). As expected from the lack of TrkA, application of its ligand, NGF, did not change the survival rate significantly. To test the specificity of BNDF, NT3 and NT4, we applied glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor (GDF)-15 and transforming growth factor (TGF)-β to the mutant cultures, all known survival factors for mesDA neurons [37-39]. Similar to NGF, none of them prevented the death of the Engrailed-deficient mesDA neurons (Figure 2I). Furthermore, the linear dose-response trend-line of the survival effect of BDNF (Figure 2J) indicated a Kd value of approximately 2.5 nM, which corresponds with the reported affinity of BDNF for the TrkB/P75NTRcomplex [40].

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