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LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors.

Hinkle KM, Yue M, Behrouz B, Dächsel JC, Lincoln SJ, Bowles EE, Beevers JE, Dugger B, Winner B, Prots I, Kent CB, Nishioka K, Lin WL, Dickson DW, Janus CJ, Farrer MJ, Melrose HL - Mol Neurodegener (2012)

Bottom Line: Mutations in the LRRK2 gene are the most common cause of genetic Parkinson's disease.We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice.Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, Florida 32224, USA.

ABSTRACT
Mutations in the LRRK2 gene are the most common cause of genetic Parkinson's disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

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Generation and expression characterization ofLRRK2KO mice.(a) Schematic diagram (courtesy of Ozgene PLC) showing targeted locus. Exon 41 was flanked with LoxP sites to allow deletion with Cre recombinase. PKG-Neo-pA-SD-IS is Ozgene’s standard selection cassette and was inserted downstream of exon 41. The PKG-neo cassette was also flanked by FRT sites to allow FLPe recombinase deletion. The targeting vector was constructed from three fragments, the 5’homology arm, the 3’ homology arm and the lox P arm, which were generated by PCR. Splicing of exon 40 to 42 causes a frame shift mutation, with the introduction of an early stop codon (TGA). (b) Northern blot hybridized with a probe to LRRK2 exon 24–27 showing absence of transcript in KO and diminished transcript in HET. A histone probe was used as loading control. (c) Immunoblot with LRRK2 antibody 1182E, raised to amino acids 841–960 showing the absence of LRRK2 protein in KO and diminished signal in HET. GAPDH was used as a loading control. (d) Immunohistochemistry with MJFF2 c41-2 antibody showing WT and KO brain sections at the level of the striatum. Specific signal is seen in the WT compared to KO. Rabbit IgG was used as an isotype control. Boxes depict enlarged images to the right. Scale bar 50 microns.
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Figure 1: Generation and expression characterization ofLRRK2KO mice.(a) Schematic diagram (courtesy of Ozgene PLC) showing targeted locus. Exon 41 was flanked with LoxP sites to allow deletion with Cre recombinase. PKG-Neo-pA-SD-IS is Ozgene’s standard selection cassette and was inserted downstream of exon 41. The PKG-neo cassette was also flanked by FRT sites to allow FLPe recombinase deletion. The targeting vector was constructed from three fragments, the 5’homology arm, the 3’ homology arm and the lox P arm, which were generated by PCR. Splicing of exon 40 to 42 causes a frame shift mutation, with the introduction of an early stop codon (TGA). (b) Northern blot hybridized with a probe to LRRK2 exon 24–27 showing absence of transcript in KO and diminished transcript in HET. A histone probe was used as loading control. (c) Immunoblot with LRRK2 antibody 1182E, raised to amino acids 841–960 showing the absence of LRRK2 protein in KO and diminished signal in HET. GAPDH was used as a loading control. (d) Immunohistochemistry with MJFF2 c41-2 antibody showing WT and KO brain sections at the level of the striatum. Specific signal is seen in the WT compared to KO. Rabbit IgG was used as an isotype control. Boxes depict enlarged images to the right. Scale bar 50 microns.

Mentions: The targeting strategy for generation of LRRK2 knockout (KO) mice is shown in Figure 1A. Homozygous mice received from Ozgene PLC were bred to Jackson C57BL6/J mice and subsequent heterozygous offspring were bred together to obtain wild type (WT), heterozygous (HET) and KO animals. Northern blotting analysis with a probe designed to LRRK2 exon 24–27 confirmed the absence of the ~9 kb LRRK2 mRNA transcript in KO mice and a reduced transcript signal in the HET mice (Figure 1B). Similarly, immunoblotting confirmed absence of LRRK2 protein band in the KO mice and a diminished signal in HET mice (Figure 1C). Immunohistochemistry also revealed specific signal in the WT compared to KO (Figure 1D).


LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors.

Hinkle KM, Yue M, Behrouz B, Dächsel JC, Lincoln SJ, Bowles EE, Beevers JE, Dugger B, Winner B, Prots I, Kent CB, Nishioka K, Lin WL, Dickson DW, Janus CJ, Farrer MJ, Melrose HL - Mol Neurodegener (2012)

Generation and expression characterization ofLRRK2KO mice.(a) Schematic diagram (courtesy of Ozgene PLC) showing targeted locus. Exon 41 was flanked with LoxP sites to allow deletion with Cre recombinase. PKG-Neo-pA-SD-IS is Ozgene’s standard selection cassette and was inserted downstream of exon 41. The PKG-neo cassette was also flanked by FRT sites to allow FLPe recombinase deletion. The targeting vector was constructed from three fragments, the 5’homology arm, the 3’ homology arm and the lox P arm, which were generated by PCR. Splicing of exon 40 to 42 causes a frame shift mutation, with the introduction of an early stop codon (TGA). (b) Northern blot hybridized with a probe to LRRK2 exon 24–27 showing absence of transcript in KO and diminished transcript in HET. A histone probe was used as loading control. (c) Immunoblot with LRRK2 antibody 1182E, raised to amino acids 841–960 showing the absence of LRRK2 protein in KO and diminished signal in HET. GAPDH was used as a loading control. (d) Immunohistochemistry with MJFF2 c41-2 antibody showing WT and KO brain sections at the level of the striatum. Specific signal is seen in the WT compared to KO. Rabbit IgG was used as an isotype control. Boxes depict enlarged images to the right. Scale bar 50 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Generation and expression characterization ofLRRK2KO mice.(a) Schematic diagram (courtesy of Ozgene PLC) showing targeted locus. Exon 41 was flanked with LoxP sites to allow deletion with Cre recombinase. PKG-Neo-pA-SD-IS is Ozgene’s standard selection cassette and was inserted downstream of exon 41. The PKG-neo cassette was also flanked by FRT sites to allow FLPe recombinase deletion. The targeting vector was constructed from three fragments, the 5’homology arm, the 3’ homology arm and the lox P arm, which were generated by PCR. Splicing of exon 40 to 42 causes a frame shift mutation, with the introduction of an early stop codon (TGA). (b) Northern blot hybridized with a probe to LRRK2 exon 24–27 showing absence of transcript in KO and diminished transcript in HET. A histone probe was used as loading control. (c) Immunoblot with LRRK2 antibody 1182E, raised to amino acids 841–960 showing the absence of LRRK2 protein in KO and diminished signal in HET. GAPDH was used as a loading control. (d) Immunohistochemistry with MJFF2 c41-2 antibody showing WT and KO brain sections at the level of the striatum. Specific signal is seen in the WT compared to KO. Rabbit IgG was used as an isotype control. Boxes depict enlarged images to the right. Scale bar 50 microns.
Mentions: The targeting strategy for generation of LRRK2 knockout (KO) mice is shown in Figure 1A. Homozygous mice received from Ozgene PLC were bred to Jackson C57BL6/J mice and subsequent heterozygous offspring were bred together to obtain wild type (WT), heterozygous (HET) and KO animals. Northern blotting analysis with a probe designed to LRRK2 exon 24–27 confirmed the absence of the ~9 kb LRRK2 mRNA transcript in KO mice and a reduced transcript signal in the HET mice (Figure 1B). Similarly, immunoblotting confirmed absence of LRRK2 protein band in the KO mice and a diminished signal in HET mice (Figure 1C). Immunohistochemistry also revealed specific signal in the WT compared to KO (Figure 1D).

Bottom Line: Mutations in the LRRK2 gene are the most common cause of genetic Parkinson's disease.We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice.Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, Florida 32224, USA.

ABSTRACT
Mutations in the LRRK2 gene are the most common cause of genetic Parkinson's disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

Show MeSH
Related in: MedlinePlus