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Absence of Ret signaling in mice causes progressive and late degeneration of the nigrostriatal system.

Kramer ER, Aron L, Ramakers GM, Seitz S, Zhuang X, Beyer K, Smidt MP, Klein R - PLoS Biol. (2007)

Bottom Line: Support of ageing neurons by endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration.We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation.These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max-Planck Institute of Neurobiology, Martinsried, Germany. ekramer@neuro.mpg.de

ABSTRACT
Support of ageing neurons by endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.

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Conditional Ablation of Ret Expression in the Nigrostriatal System(A–D) Recombination efficiency of DAT-Cre mice crossed with Rosa26R lacZ reporter mice (DAT-Rosa26R). β-galactosidase (X-Gal) activity (blue) in coronal brain sections of DAT-Rosa26R transgenic mice at embryonic day E15.5 (A) and at 3-mo postnatal (B). Anti- TH (C) and anti–β-galactosidase (β-Gal) (D) immunostaining in adjacent brain sections of 2-y-old DAT-Rosa26R mice. Cre activity is restricted to SNpc and VTA.(E–J) Immunohistochemical detection of TH (E, G, and I) and Ret (F, H, and J) in adjacent coronal brain sections of 3-mo-old wild-type (wt), DAT-Retlx/lx, and Nes-Retlx/lx mice. Note the nearly complete removal of Ret immunoreactivity in SNpc and VTA of DAT-Retlx/lx and Nes-Retlx/lx mice.(K and L) Western blot analysis of Ret protein levels in protein lysates from SNpc (K) and striatum (L) of 3-mo-old control (Retlx/+and DAT-Retlx/+) and DAT-Retlx/lx mutant mice. Immunoblots were reprobed with anti–α-tubulin antibodies as loading controls.
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pbio-0050039-g001: Conditional Ablation of Ret Expression in the Nigrostriatal System(A–D) Recombination efficiency of DAT-Cre mice crossed with Rosa26R lacZ reporter mice (DAT-Rosa26R). β-galactosidase (X-Gal) activity (blue) in coronal brain sections of DAT-Rosa26R transgenic mice at embryonic day E15.5 (A) and at 3-mo postnatal (B). Anti- TH (C) and anti–β-galactosidase (β-Gal) (D) immunostaining in adjacent brain sections of 2-y-old DAT-Rosa26R mice. Cre activity is restricted to SNpc and VTA.(E–J) Immunohistochemical detection of TH (E, G, and I) and Ret (F, H, and J) in adjacent coronal brain sections of 3-mo-old wild-type (wt), DAT-Retlx/lx, and Nes-Retlx/lx mice. Note the nearly complete removal of Ret immunoreactivity in SNpc and VTA of DAT-Retlx/lx and Nes-Retlx/lx mice.(K and L) Western blot analysis of Ret protein levels in protein lysates from SNpc (K) and striatum (L) of 3-mo-old control (Retlx/+and DAT-Retlx/+) and DAT-Retlx/lx mutant mice. Immunoblots were reprobed with anti–α-tubulin antibodies as loading controls.

Mentions: To disrupt the genes encoding Ret and TrkB in a regionally selective manner, we used mice with floxed alleles of Ret [24] and TrkB [25] in combination with Dopamine transporter (DAT)-Cre mice [26] (DAT-Retlx/lx and DAT-TrkBlx/lx mice, respectively) and Nestin-Cre mice [27] (Nes-Retlx/lx mutants). DAT-Cre mice had been generated by knocking Cre into the 5′ UTR region of the endogenous mouse DAT locus [26], whereas Nestin-Cre mice express Cre from a transgene [27]. For DAT-Cre mice, it was previously shown that virtually all (95%) of tyrosine hydroxylase (TH)-positive cells in SNpc and the nearby VTA regions express Cre and show Cre-mediated recombination in adult mice, whereas weak lacZ reporter activity was seen in DA neurons of the olfactory bulb and hypothalamus. No reporter activity was seen in the striatum [26]. We confirmed these observations and extended the analysis to other time points, including embryonic day 15 and 2-y postnatal (Figure 1A–1D). Our results indicate that DAT-Cre–mediated recombination is region selective from late embryonic stages to aged mice. Ret expression is high in the SNpc and VTA of adult control mice (Figure 1E and 1F) and is efficiently removed in DAT-Retlx/lx and Nestin-Retlx/lx mice (Figure 1G–1J). Western blot analysis of Ret protein revealed a nearly complete loss of the protein in SN of Dat-Retlx/lx mice, and complete loss of Ret in the striatum of DAT-Retlx/lx mice (Figure 1K and 1L). TrkB expression is found in nigral DA neurons [17], but also in other neuronal populations in the entire ventral midbrain (unpublished data). The conditional trkBlx allele has previously been used for region-specific removal of TrkB in several studies [25,28], indicating that this locus can efficiently be modified by Cre recombination. We were unable to visualize loss of TrkB by immunostaining and Western blotting in DAT-TrkBlx/lx mice, because the TH-positive subpopulation expresses low amounts of TrkB and is a minor population within the TrkB expression domain. However, we detected the recombined TrkBlx allele by PCR specifically in SNpc, but not in striatum, of DAT-TrkBlx/lx mice (Figure S1A). In addition, using laser capture microdissection combined with single-cell RT-PCR, we found a 65% decrease of TrkB mRNA–positive DA neurons in the SNpc in DAT-TrkBlx/lx mice compared to controls (Figure S1B–S1D).


Absence of Ret signaling in mice causes progressive and late degeneration of the nigrostriatal system.

Kramer ER, Aron L, Ramakers GM, Seitz S, Zhuang X, Beyer K, Smidt MP, Klein R - PLoS Biol. (2007)

Conditional Ablation of Ret Expression in the Nigrostriatal System(A–D) Recombination efficiency of DAT-Cre mice crossed with Rosa26R lacZ reporter mice (DAT-Rosa26R). β-galactosidase (X-Gal) activity (blue) in coronal brain sections of DAT-Rosa26R transgenic mice at embryonic day E15.5 (A) and at 3-mo postnatal (B). Anti- TH (C) and anti–β-galactosidase (β-Gal) (D) immunostaining in adjacent brain sections of 2-y-old DAT-Rosa26R mice. Cre activity is restricted to SNpc and VTA.(E–J) Immunohistochemical detection of TH (E, G, and I) and Ret (F, H, and J) in adjacent coronal brain sections of 3-mo-old wild-type (wt), DAT-Retlx/lx, and Nes-Retlx/lx mice. Note the nearly complete removal of Ret immunoreactivity in SNpc and VTA of DAT-Retlx/lx and Nes-Retlx/lx mice.(K and L) Western blot analysis of Ret protein levels in protein lysates from SNpc (K) and striatum (L) of 3-mo-old control (Retlx/+and DAT-Retlx/+) and DAT-Retlx/lx mutant mice. Immunoblots were reprobed with anti–α-tubulin antibodies as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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pbio-0050039-g001: Conditional Ablation of Ret Expression in the Nigrostriatal System(A–D) Recombination efficiency of DAT-Cre mice crossed with Rosa26R lacZ reporter mice (DAT-Rosa26R). β-galactosidase (X-Gal) activity (blue) in coronal brain sections of DAT-Rosa26R transgenic mice at embryonic day E15.5 (A) and at 3-mo postnatal (B). Anti- TH (C) and anti–β-galactosidase (β-Gal) (D) immunostaining in adjacent brain sections of 2-y-old DAT-Rosa26R mice. Cre activity is restricted to SNpc and VTA.(E–J) Immunohistochemical detection of TH (E, G, and I) and Ret (F, H, and J) in adjacent coronal brain sections of 3-mo-old wild-type (wt), DAT-Retlx/lx, and Nes-Retlx/lx mice. Note the nearly complete removal of Ret immunoreactivity in SNpc and VTA of DAT-Retlx/lx and Nes-Retlx/lx mice.(K and L) Western blot analysis of Ret protein levels in protein lysates from SNpc (K) and striatum (L) of 3-mo-old control (Retlx/+and DAT-Retlx/+) and DAT-Retlx/lx mutant mice. Immunoblots were reprobed with anti–α-tubulin antibodies as loading controls.
Mentions: To disrupt the genes encoding Ret and TrkB in a regionally selective manner, we used mice with floxed alleles of Ret [24] and TrkB [25] in combination with Dopamine transporter (DAT)-Cre mice [26] (DAT-Retlx/lx and DAT-TrkBlx/lx mice, respectively) and Nestin-Cre mice [27] (Nes-Retlx/lx mutants). DAT-Cre mice had been generated by knocking Cre into the 5′ UTR region of the endogenous mouse DAT locus [26], whereas Nestin-Cre mice express Cre from a transgene [27]. For DAT-Cre mice, it was previously shown that virtually all (95%) of tyrosine hydroxylase (TH)-positive cells in SNpc and the nearby VTA regions express Cre and show Cre-mediated recombination in adult mice, whereas weak lacZ reporter activity was seen in DA neurons of the olfactory bulb and hypothalamus. No reporter activity was seen in the striatum [26]. We confirmed these observations and extended the analysis to other time points, including embryonic day 15 and 2-y postnatal (Figure 1A–1D). Our results indicate that DAT-Cre–mediated recombination is region selective from late embryonic stages to aged mice. Ret expression is high in the SNpc and VTA of adult control mice (Figure 1E and 1F) and is efficiently removed in DAT-Retlx/lx and Nestin-Retlx/lx mice (Figure 1G–1J). Western blot analysis of Ret protein revealed a nearly complete loss of the protein in SN of Dat-Retlx/lx mice, and complete loss of Ret in the striatum of DAT-Retlx/lx mice (Figure 1K and 1L). TrkB expression is found in nigral DA neurons [17], but also in other neuronal populations in the entire ventral midbrain (unpublished data). The conditional trkBlx allele has previously been used for region-specific removal of TrkB in several studies [25,28], indicating that this locus can efficiently be modified by Cre recombination. We were unable to visualize loss of TrkB by immunostaining and Western blotting in DAT-TrkBlx/lx mice, because the TH-positive subpopulation expresses low amounts of TrkB and is a minor population within the TrkB expression domain. However, we detected the recombined TrkBlx allele by PCR specifically in SNpc, but not in striatum, of DAT-TrkBlx/lx mice (Figure S1A). In addition, using laser capture microdissection combined with single-cell RT-PCR, we found a 65% decrease of TrkB mRNA–positive DA neurons in the SNpc in DAT-TrkBlx/lx mice compared to controls (Figure S1B–S1D).

Bottom Line: Support of ageing neurons by endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration.We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation.These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max-Planck Institute of Neurobiology, Martinsried, Germany. ekramer@neuro.mpg.de

ABSTRACT
Support of ageing neurons by endogenous neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) may determine whether the neurons resist or succumb to neurodegeneration. GDNF has been tested in clinical trials for the treatment of Parkinson disease (PD), a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. BDNF modulates nigrostriatal functions and rescues DA neurons in PD animal models. The physiological roles of GDNF and BDNF signaling in the adult nigrostriatal DA system are unknown. We generated mice with regionally selective ablations of the genes encoding the receptors for GDNF (Ret) and BDNF (TrkB). We find that Ret, but not TrkB, ablation causes progressive and adult-onset loss of DA neurons specifically in the substantia nigra pars compacta, degeneration of DA nerve terminals in striatum, and pronounced glial activation. These findings establish Ret as a critical regulator of long-term maintenance of the nigrostriatal DA system and suggest conditional Ret mutants as useful tools for gaining insights into the molecular mechanisms involved in the development of PD.

Show MeSH
Related in: MedlinePlus