DJ1yellow 2wo
1
Parkinson’s Disease and Age Dependent Deficits in Striatal Tyrosine Hydroxylase (TH) and Spontaneous Motor Activity in DJ-1 Knockout (KO) Mice 2 Shantell Nolen, 1 Farzad Mortazavi, 1 Stefano Patassini, 1 Marie-Francoise Chesselet 1 UCLA Department of Neurology, 2 University of California, Berkeley ABSTRACT INTRODUCTION RESULTS Parkinson’s Disease (PD) is a neurodegenerative disorder characterized by multiple motor dysfunctions as a result of significant loss of dopaminergic neurons in the substantia nigra. Furthermore, aging has been recognized as a major risk factor for developing PD. Mutations in the gene coding for the DJ-1 protein has been linked to autosomal-recessive early onset PD. Several studies have implicated DJ-1 protein to have antioxidant and anti-apoptotic properties. In this way, mice lacking the DJ-1 gene, may be more susceptible to oxidative stress and mitochondrion stress, processes involved in PD. Interestingly, previous studies investigating DJ-1 knockout (KO) mice, have reported slight dysfunctions in the nigrastriatal pathway such as increased dopamine transport (DAT) in the striatum. However, these studies have not looked at aging as a factor involved in dopaminergic dysfunction in these mice. For the current study, we are investigating age dependent deficits in DJ-1 KO mice at 12 months and 18 months; spontaneous motor activity is assessed in the cylinder prior to transcardiac perfusion for immunohistochemical analysis and striatal TH content. Spontaneous motor assessment in the cylinder revealed reduced number of rears and hindlimb steps, 2 indices of motor activity in DJ-1 KO at 18 months of age. TH fiber density showed an overall reduction in TH for DJ-1 KO from 12 months to 18 months and an increase in WT 12 months to 18 months. Differences were primarily in the rostral and medial striatum. METHODS CONCLUSION REFERENCES ACKNOWLEDGEMENTS Spontaneous Motor Assessment: Histopathological Analysis: • The mice were individually placed in a cylinder and videotaped for 3 min. Spontaneous motor behavior was assesed by recording their grooming time and their total # of rears, frontlimb (fl) steps, and hindlimb (hl) steps by a blind investigator to the genotype or the age of the mice. The results were then grouped into 1 of 4 categories, WT 12 months, KO 12 months, WT 18 months, and KO 18 months. 2 x 2 ANOVA, (p< 0.05) was used to assess statistical differences between groups. Tyrosine L-Dopa Dopamine • Mice were anesthetized with pentobarbital (100 mg/ kg i.p.) and intracardially perfused with 0.1 M PBS followed by ice cold 4% paraformaldehyde (PFA). Brains were quickly removed, postfixed for 2 h in 4% PFA, then cryoprotected in 30% sucrose in 0.1 M PBS, and frozen on powdered dry ice and stored at 80°C. Forty micrometer free-floating coronal sections were collected for immunohistochemical analysis. Immunohistochemistry: Spontaneous Motor Assessment: WT 12 months N = 3, KO 12 months N = 5 WT 18 months N = 6, KO 18 months N = 4 Figure 1. * KO 18 months compared to WT 18 months. # WT 18 months com- pared to WT and KO 12 months. No Main effect of Genotype. Main effect of Age. Genotype x Age interaction. When compared at 12 and 18 months, the 18 months KO have significant reduction in number of rears. Figure 2. No main effect of Geno- type. No main effect of Age. No Gen- otype x Age interaction. Figure 3. No main effect of Geno- type. No main effect of Age. No Genotype x Age interaction. Figure 4. * KO 18 months compared to WT 18 months. # WT 18 months com- pared to WT and KO 12 months.Main effect of Genotype. No main effect of Age. No Genotype x Age interaction. Overall KO animals have reduced HL Steps compared to WT but this effect is not age dependent. Figure 5. # WT 18 months compared to WT 12 months and KO 18 months. * KO 18 months compared to WT 18 months. No main effect of Genotype. No main effect of Age. Genotype x Age interaction. WT 18 months is significantly higher than WT 12 months and KO 18 months. (2 x 2 ANOVA) WT 12 months N = 3, KO 12 months N = 5 WT 18 months N = 6, KO 18 months N = 5 WT 12 months N = 9, KO 12 months N = 15 WT 18 months N = 18, KO 18 months N = 15 Figure 7. No statistical differences ob- served at this level. (2 x 2 ANOVA) Figure 8. * KO 18 months compared to WT 12 months and KO 12 months. # WT 18 months compared to WT 12 and KO 12 months. No main effect of Geno- type. No main effect of Age. Genotype x Age interaction. At 18 months there is a significant increase for WT mice and a significant decrease for KO mice. (2 x 2 ANOVA) For the following 4 graphs • PD is a progressive neurodegenerative disease effecting 1% of the population over 65 years old. • Exact etiology is unknown but evidence supports that mitochondrial dysfunction, protesome inhibition, and œ – synuclein aggregation play a role in PD. • In addition to environmental factors, the pathology of familial PD has been linked to mutations including œ – synuclein, Parkin, DJ-1, Pink-1, and LRRK2. • DJ-1 linked to autosomal-recessive early onset PD • DJ-1 has neuroprotective capabilities in response too oxidative stress, oxidation/ reduction sensor, chaperone, and/or involved in proteasomal degradation. Rostral Medial Caudal Tyrosine Hydroxylase (TH): • Sections were washed in 0.1 M PBS (pH=7.4) and incubated in 0.3% hydrogen peroxide for 15 minutes in order to block endogenous peroxidases. Sections were washed in PBS and incubated for 1 hr in 10% normal goat serum/0.3% triton X-100 in PBS followed by incubation overnight at 4°C in the primary antibody with 2% normal goat serum: rabbit anti-tyrosine hydroxylase (1/600, Pel Freeze, Rogers, Arkansas). After washing in PBS, sections were incubated with the corresponding biotynilated secondary antibody, either goat anti-rabbit IgG (1/600, Vector, Laboratories, Burlingame, CA) or goat anti-rat IgG (1/600, Vector Laboratories, Burlingame, CA) at room temperature for 2 hrs. The ABC elite kit (Vector Laboratories, Burlingame, CA) was used for the avidin- biotin complex, and the reaction was visualized by 3,3”-diaminobenzidine tetrachloride (DAB, Sigma Chemical). TH-IR fibers in the striatum were measured in three slices from each animal: rostral striatum (Bregma 1.1mm) and medial striatum (Bregma 0.14mm) and caudal striatum (Bregma 0.22 mm). Optical density measurements were carried out using ImageJ software, version 1.38x (NIH, USA, HTTP://rsb.info.nih.gov/ij). Spontaneous Motor Activity • Motor deficits observed at 18 months in KO, in both the number of rears and HL steps, compared to WT 18 months. Immunohistochemistry • Rostral striatum: TH reduction in KO 18 months but increase in WT 18 months • Medial striatum: same as Rostral • Overall striatum: KO mice had decrease in TH from 12 months to 18 months, WT mice had increase in TH from 12 months to 18 months • KO 18 months have reduction in striatal TH, whereas in WT mice at 18 months, an increase in striatal TH compared to the 12 month old mice. • The reduction in striatal TH at 18 months in KO mice correlates with the motor deficits observed in these mice. • An increase in TH in WT mice is most likely due to compensatory mechanisms, which is not observed in the DJ1 KO mice. • The current results indicate the succeptibility of the nigrostriatal dopaminergic system to DJ1 mutations and that the processe of aging may exacerbate both motor and pathological processes involved in PD. WT 12 months N = 3, KO 12 months N = 5 WT 18 months N = 6, KO 18 months N = 5 Figure 6. # KO 12 months compared to WT 12 months. * KO 18 months compared to KO 12 months. No main effect of Genotype. No main effect of Age. No Genotype x Age interaction. WT 12 months is significantly higher than KO 12 months. KO 18 months is significantly lower than KO 12 months. (2 x 2 ANOVA) 0 5 10 15 20 25 30 35 40 45 12 months 18 months !"#sit' o* +,-.osi/0" 1i2"rs Rostral Striatum WT KO 0 10 20 30 40 50 60 12 Months 18 Months !e#$it' )* +,-.)$i/0e 1i2er$ Medial Striatum WT KO * # 0 5 10 15 20 25 30 35 40 45 12 Months 18 Months !"#$it' )* +,-.)$i/0" 1i2"r$ Caudal Striatum WT KO 0 5 10 15 20 25 30 35 40 45 12 Months 18 Months !e#$it' )* +,-.)$i/ve 1i2er$ Overall Striatum WT KO 0 5 10 15 20 25 12 Months 18 Months Seconds of Grooming Grooming WT KO 0 10 20 30 40 50 60 70 80 12 Months 18 Months Number of HL Steps HL Steps WT KO 0 10 20 30 40 50 60 70 80 90 100 12 Months 18 Months Number of FL Steps FL Steps WT KO # * 0 2 4 6 8 10 12 14 16 18 20 12 Months 18 Months Number of Rears Rearing WT KO # * TH Optical Density (OD) measurements: WT 12 months N = 3, KO 12 months N = 5 WT 18 months N = 6, KO 18 months N = 5 # * # * Thank you to the UCLA Alliance for Graduate Education and the Professoriate (AGEP) sponsored by the Na- tional Science Foundation (NSF) grant, for the opportunity to be apart of the program. Thank you to everyone in the Chesselet lab and Dr. Marie-Francoise Chesselet for allowing me to be apart of the lab this summer. A spe- cial thanks to Dr. Farzad Mortazavi for being my mentor for the summer and letting me take part in his research. 1. Liu, Fang, Jamie L. Nguyen, John D. Hullerman, Li Li, and Jean-Christophe Rochet. “Mechanisms of DJ-1 neuroprotection in a cellular model of Parkinson’s disease.” Journal of Neurochemistry 105.6 (2008): 2435-453. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/18331584?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum>. 2. Manning-Bog, Amy B., W. M. Caudle, Xiomara A. Perez, Stephen H. Reaney, Ronald Palwtzki, Martha Z. Isla, Vivian P. Chou, Alison L. McCormack, Gary W. Miller, J. W. Langston, Charles R. Gerfen, and Donato A. DiMonte. “Increased vulnerability of nigrostriatal terminals in DJ-1-deficient mice is mediated by the dopamine transporter.” Neurobiology of Disease 27 (2007): 141-50. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/sites/entrez>. 3. Savitt, Joseph M., Valina L. Dawson, and Ted M. Dawson. “Diagnosis and treatment of Parkinson disease: molecules to medicine.” The Journal of Clinical Investigation 116.7 (2006): 1744-754. Pubmed. Web. 1 July 2009. <http://www.ncbi. nlm.nih.gov/pubmed/16823471?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_ DefaultReportPanel.Pubmed_RVDocSum>. 4. Yang, Wonsuk, Linan Chen, Yunmin Ding, Xiaoxi Zhuang, and Un J. Kang. “Paraquat induces dopaminergic dysfunction and proteasome impairment in DJ-1-deficient mice.” Human Molecular Genetics 16.23 (2007): 2900-910. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/sites/entrez>.
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Transcript of DJ1yellow 2wo
Parkinson’s Disease and Age Dependent Deficits in Striatal Tyrosine Hydroxylase (TH) and Spontaneous Motor Activity in DJ-1 Knockout (KO) Mice
2Shantell Nolen, 1Farzad Mortazavi, 1Stefano Patassini, 1Marie-Francoise Chesselet1UCLA Department of Neurology, 2University of California, Berkeley
ABSTRACT
INTRODUCTION
RESULTS
Parkinson’s Disease (PD) is a neurodegenerative disorder characterized by multiple motor dysfunctions as a result of significant loss of dopaminergic neurons in the substantia nigra. Furthermore, aging has been recognized as a major risk factor for developing PD. Mutations in the gene coding for the DJ-1 protein has been linked to autosomal-recessive early onset PD. Several studies have implicated DJ-1 protein to have antioxidant and anti-apoptotic properties. In this way, mice lacking the DJ-1 gene, may be more susceptible to oxidative stress and mitochondrion stress, processes involved in PD. Interestingly, previous studies investigating DJ-1 knockout (KO) mice, have reported slight dysfunctions in the nigrastriatal pathway such as increased dopamine transport (DAT) in the striatum. However, these studies have not looked at aging as a factor involved in dopaminergic dysfunction in these mice. For the current study, we are investigating age dependent deficits in DJ-1 KO mice at 12 months and 18 months; spontaneous motor activity is assessed in the cylinder prior to transcardiac perfusion for immunohistochemical analysis and striatal TH content. Spontaneous motor assessment in the cylinder revealed reduced number of rears and hindlimb steps, 2 indices of motor activity in DJ-1 KO at 18 months of age. TH fiber density showed an overall reduction in TH for DJ-1 KO from 12 months to 18 months and an increase in WT 12 months to 18 months. Differences were primarily in the rostral and medial striatum.
METHODS
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
Spontaneous Motor Assessment:
Histopathological Analysis:
• The mice were individually placed in a cylinder and videotaped for 3 min. Spontaneous motor behavior was assesed by recording their grooming time and their total # of rears, frontlimb (fl) steps, and hindlimb (hl) steps by a blind investigator to the genotype or the age of the mice. The results were then grouped into 1 of 4 categories, WT 12 months, KO 12 months, WT 18 months, and KO 18 months. 2 x 2 ANOVA, (p< 0.05) was used to assess statistical differences between groups.
Tyrosine L-Dopa Dopamine
• Mice were anesthetized with pentobarbital (100 mg/ kg i.p.) and intracardially perfused with 0.1 M PBS followed by ice cold 4% paraformaldehyde (PFA). Brains were quickly removed, postfixed for 2 h in 4% PFA, then cryoprotected in 30% sucrose in 0.1 M PBS, and frozen on powdered dry ice and stored at 80°C. Forty micrometer free-floating coronal sections were collected for immunohistochemical analysis.
Immunohistochemistry:
Spontaneous Motor Assessment:WT 12 months N = 3, KO 12 months N = 5WT 18 months N = 6, KO 18 months N = 4
Figure 1. * KO 18 months compared to WT 18 months. # WT 18 months com-pared to WT and KO 12 months. No Main effect of Genotype. Main effect of Age. Genotype x Age interaction. When compared at 12 and 18 months, the 18 months KO have significant reduction in number of rears.
Figure 2. No main effect of Geno-type. No main effect of Age. No Gen-otype x Age interaction.
Figure 3. No main effect of Geno-type. No main effect of Age. No Genotype x Age interaction.
Figure 4. * KO 18 months compared to WT 18 months. # WT 18 months com-pared to WT and KO 12 months.Main effect of Genotype. No main effect of Age. No Genotype x Age interaction. Overall KO animals have reduced HL Steps compared to WT but this effect is not age dependent.
Figure 5. # WT 18 months compared to WT 12 months and KO 18 months. * KO 18 months compared to WT 18 months. No main effect of Genotype. No main effect of Age. Genotype x Age interaction. WT 18 months is significantly higher than WT 12 months and KO 18 months. (2 x 2 ANOVA)
WT 12 months N = 3, KO 12 months N = 5WT 18 months N = 6, KO 18 months N = 5
WT 12 months N = 9, KO 12 months N = 15WT 18 months N = 18, KO 18 months N = 15
Figure 7. No statistical differences ob-served at this level. (2 x 2 ANOVA)
Figure 8. * KO 18 months compared to WT 12 months and KO 12 months. # WT 18 months compared to WT 12 and KO 12 months. No main effect of Geno-type. No main effect of Age. Genotype x Age interaction. At 18 months there is a significant increase for WT mice and a significant decrease for KO mice. (2 x 2 ANOVA)
For the following 4 graphs
•PD is a progressive neurodegenerative disease effecting 1% of the population over 65 years old.
•Exact etiology is unknown but evidence supports that mitochondrial dysfunction, protesome inhibition, and œ – synuclein aggregation play a role in PD.
•In addition to environmental factors, the pathology of familial PD has been linked to mutations including œ – synuclein, Parkin, DJ-1, Pink-1, and LRRK2.
•DJ-1 linked to autosomal-recessive early onset PD •DJ-1 has neuroprotective capabilities in response too oxidative stress, oxidation/
reduction sensor, chaperone, and/or involved in proteasomal degradation.
Rostral Medial Caudal
Tyrosine Hydroxylase (TH):• Sections were washed in 0.1 M PBS (pH=7.4) and incubated in 0.3%
hydrogen peroxide for 15 minutes in order to block endogenous peroxidases. Sections were washed in PBS and incubated for 1 hr in 10% normal goat serum/0.3% triton X-100 in PBS followed by incubation overnight at 4°C in the primary antibody with 2% normal goat serum: rabbit anti-tyrosine hydroxylase (1/600, Pel Freeze, Rogers, Arkansas). After washing in PBS, sections were incubated with the corresponding biotynilated secondary antibody, either goat anti-rabbit IgG (1/600, Vector, Laboratories, Burlingame, CA) or goat anti-rat IgG (1/600, Vector Laboratories, Burlingame, CA) at room temperature for 2 hrs. The ABC elite kit (Vector Laboratories, Burlingame, CA) was used for the avidin-biotin complex, and the reaction was visualized by 3,3”-diaminobenzidine tetrachloride (DAB, Sigma Chemical). TH-IR fibers in the striatum were measured in three slices from each animal: rostral striatum (Bregma 1.1mm) and medial striatum (Bregma 0.14mm) and caudal striatum (Bregma 0.22 mm). Optical density measurements were carried out using ImageJ software, version 1.38x (NIH, USA, HTTP://rsb.info.nih.gov/ij).
Spontaneous Motor Activity•Motor deficits observed at 18 months in KO, in both the number of rears and HL steps,
compared to WT 18 months. Immunohistochemistry•Rostral striatum: TH reduction in KO 18 months but increase in WT 18 months•Medial striatum: same as Rostral•Overall striatum: KO mice had decrease in TH from 12 months to 18 months, WT mice
had increase in TH from 12 months to 18 months•KO 18 months have reduction in striatal TH, whereas in WT mice at 18 months, an
increase in striatal TH compared to the 12 month old mice. •The reduction in striatal TH at 18 months in KO mice correlates with the motor deficits
observed in these mice.•An increase in TH in WT mice is most likely due to compensatory mechanisms, which is
not observed in the DJ1 KO mice.• The current results indicate the succeptibility of the nigrostriatal dopaminergic system to
DJ1 mutations and that the processe of aging may exacerbate both motor and pathological processes involved in PD.
WT 12 months N = 3, KO 12 months N = 5WT 18 months N = 6, KO 18 months N = 5
Figure 6. # KO 12 months compared to WT 12 months. * KO 18 months compared to KO 12 months. No main effect of Genotype. No main effect of Age. No Genotype x Age interaction. WT 12 months is significantly higher than KO 12 months. KO 18 months is significantly lower than KO 12 months. (2 x 2 ANOVA)
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Thank you to the UCLA Alliance for Graduate Education and the Professoriate (AGEP) sponsored by the Na-tional Science Foundation (NSF) grant, for the opportunity to be apart of the program. Thank you to everyone in the Chesselet lab and Dr. Marie-Francoise Chesselet for allowing me to be apart of the lab this summer. A spe-cial thanks to Dr. Farzad Mortazavi for being my mentor for the summer and letting me take part in his research.
1. Liu, Fang, Jamie L. Nguyen, John D. Hullerman, Li Li, and Jean-Christophe Rochet. “Mechanisms of DJ-1 neuroprotection in a cellular model of Parkinson’s disease.” Journal of Neurochemistry 105.6 (2008): 2435-453. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/18331584?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum>.
2. Manning-Bog, Amy B., W. M. Caudle, Xiomara A. Perez, Stephen H. Reaney, Ronald Palwtzki, Martha Z. Isla, Vivian P. Chou, Alison L. McCormack, Gary W. Miller, J. W. Langston, Charles R. Gerfen, and Donato A. DiMonte. “Increased vulnerability of nigrostriatal terminals in DJ-1-deficient mice is mediated by the dopamine transporter.” Neurobiology of Disease 27 (2007): 141-50. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/sites/entrez>.
3. Savitt, Joseph M., Valina L. Dawson, and Ted M. Dawson. “Diagnosis and treatment of Parkinson disease: molecules to medicine.” The Journal of Clinical Investigation 116.7 (2006): 1744-754. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/pubmed/16823471?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum>.
4. Yang, Wonsuk, Linan Chen, Yunmin Ding, Xiaoxi Zhuang, and Un J. Kang. “Paraquat induces dopaminergic dysfunction and proteasome impairment in DJ-1-deficient mice.” Human Molecular Genetics 16.23 (2007): 2900-910. Pubmed. Web. 1 July 2009. <http://www.ncbi.nlm.nih.gov/sites/entrez>.
