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Increased DJ-1 expression under oxidative stress and in Alzheimer's disease brains.

Baulac S, Lu H, Strahle J, Yang T, Goldberg MS, Shen J, Schlossmacher MG, Lemere CA, Lu Q, Xia W - Mol Neurodegener (2009)

Bottom Line: We found that DJ-1 was expressed early during zebrafish development and throughout adulthood.While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos.Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Harvard University, Boston, MA 02115, USA. wxia@rics.bwh.harvard.edu.

ABSTRACT
Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

No MeSH data available.


Related in: MedlinePlus

Knockdown of DJ-1 in zebrafish embryos. A. Abundant DJ-1 expression in human brain lysate from a control case, as detected by Western Blot (WB) with KAM-SA100 and anti-DJ-1-N antibodies, but not when anti-DJ-1-N was pre-absorbed with the synthetic DJ-1 peptide. Specificity of anti-DJ-1-N antibody was confirmed by detecting DJ-1 in wild type mouse brain lysate but not in the DJ-1 knockout mouse brain lysate. A minor cross-reacting band at ~70 kDa was detected in extracts of both wild type and DJ-1 knockout mouse brain, but not in human brain extracts. B. Zebrafish embryos at different developmental stages and brains of adult zebrafish were lysed for Western blotting with antibody E2.19. High levels of DJ-1 protein were found in embryos at all developmental stages and in adult brains. C. Extracts taken from individual embryos at 24 hpf were run on WB with antibody E2.19 (bottom panel). The same blot was re-probed with antibody against α-tubulin (top panel). DJ-1 protein levels were dramatically reduced in DJ-1 KD embryos compared to control embryos.
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Figure 1: Knockdown of DJ-1 in zebrafish embryos. A. Abundant DJ-1 expression in human brain lysate from a control case, as detected by Western Blot (WB) with KAM-SA100 and anti-DJ-1-N antibodies, but not when anti-DJ-1-N was pre-absorbed with the synthetic DJ-1 peptide. Specificity of anti-DJ-1-N antibody was confirmed by detecting DJ-1 in wild type mouse brain lysate but not in the DJ-1 knockout mouse brain lysate. A minor cross-reacting band at ~70 kDa was detected in extracts of both wild type and DJ-1 knockout mouse brain, but not in human brain extracts. B. Zebrafish embryos at different developmental stages and brains of adult zebrafish were lysed for Western blotting with antibody E2.19. High levels of DJ-1 protein were found in embryos at all developmental stages and in adult brains. C. Extracts taken from individual embryos at 24 hpf were run on WB with antibody E2.19 (bottom panel). The same blot was re-probed with antibody against α-tubulin (top panel). DJ-1 protein levels were dramatically reduced in DJ-1 KD embryos compared to control embryos.

Mentions: We generated an affinity purified polyclonal antibody DJ-1-N against the N-terminus of DJ-1 that recognizes a single band at 23 kDa corresponding to endogenous DJ-1 from human cortex homogenate; this band was absent when the antibody was preabsorbed with its cognate peptide (Fig. 1A). The same band was recognized by a commercially available monoclonal antibody (KAM-SA100) raised against full-length human recombinant DJ-1 (Fig. 1A). We further tested the specificity of this antibody by Western blotting of brain homogenate from a wild type or DJ-1 knockout mouse. Using the antibody DJ-1-N we detected the corresponding 23 kDa band in wild type but not DJ-1 knockout mouse brain lysates (Fig. 1A). We then prepared zebrafish embryos at different developmental stages as well as brain lysates from adult zebrafish for Western blotting using another commercially available monoclonal antibody E2.19 (Fig. 1B). Staining of zebrafish lysates with DJ-1-N showed the same results (data not shown). We found that DJ-1 was expressed early in embryonic development and persisted into adulthood (Fig. 1B).


Increased DJ-1 expression under oxidative stress and in Alzheimer's disease brains.

Baulac S, Lu H, Strahle J, Yang T, Goldberg MS, Shen J, Schlossmacher MG, Lemere CA, Lu Q, Xia W - Mol Neurodegener (2009)

Knockdown of DJ-1 in zebrafish embryos. A. Abundant DJ-1 expression in human brain lysate from a control case, as detected by Western Blot (WB) with KAM-SA100 and anti-DJ-1-N antibodies, but not when anti-DJ-1-N was pre-absorbed with the synthetic DJ-1 peptide. Specificity of anti-DJ-1-N antibody was confirmed by detecting DJ-1 in wild type mouse brain lysate but not in the DJ-1 knockout mouse brain lysate. A minor cross-reacting band at ~70 kDa was detected in extracts of both wild type and DJ-1 knockout mouse brain, but not in human brain extracts. B. Zebrafish embryos at different developmental stages and brains of adult zebrafish were lysed for Western blotting with antibody E2.19. High levels of DJ-1 protein were found in embryos at all developmental stages and in adult brains. C. Extracts taken from individual embryos at 24 hpf were run on WB with antibody E2.19 (bottom panel). The same blot was re-probed with antibody against α-tubulin (top panel). DJ-1 protein levels were dramatically reduced in DJ-1 KD embryos compared to control embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654450&req=5

Figure 1: Knockdown of DJ-1 in zebrafish embryos. A. Abundant DJ-1 expression in human brain lysate from a control case, as detected by Western Blot (WB) with KAM-SA100 and anti-DJ-1-N antibodies, but not when anti-DJ-1-N was pre-absorbed with the synthetic DJ-1 peptide. Specificity of anti-DJ-1-N antibody was confirmed by detecting DJ-1 in wild type mouse brain lysate but not in the DJ-1 knockout mouse brain lysate. A minor cross-reacting band at ~70 kDa was detected in extracts of both wild type and DJ-1 knockout mouse brain, but not in human brain extracts. B. Zebrafish embryos at different developmental stages and brains of adult zebrafish were lysed for Western blotting with antibody E2.19. High levels of DJ-1 protein were found in embryos at all developmental stages and in adult brains. C. Extracts taken from individual embryos at 24 hpf were run on WB with antibody E2.19 (bottom panel). The same blot was re-probed with antibody against α-tubulin (top panel). DJ-1 protein levels were dramatically reduced in DJ-1 KD embryos compared to control embryos.
Mentions: We generated an affinity purified polyclonal antibody DJ-1-N against the N-terminus of DJ-1 that recognizes a single band at 23 kDa corresponding to endogenous DJ-1 from human cortex homogenate; this band was absent when the antibody was preabsorbed with its cognate peptide (Fig. 1A). The same band was recognized by a commercially available monoclonal antibody (KAM-SA100) raised against full-length human recombinant DJ-1 (Fig. 1A). We further tested the specificity of this antibody by Western blotting of brain homogenate from a wild type or DJ-1 knockout mouse. Using the antibody DJ-1-N we detected the corresponding 23 kDa band in wild type but not DJ-1 knockout mouse brain lysates (Fig. 1A). We then prepared zebrafish embryos at different developmental stages as well as brain lysates from adult zebrafish for Western blotting using another commercially available monoclonal antibody E2.19 (Fig. 1B). Staining of zebrafish lysates with DJ-1-N showed the same results (data not shown). We found that DJ-1 was expressed early in embryonic development and persisted into adulthood (Fig. 1B).

Bottom Line: We found that DJ-1 was expressed early during zebrafish development and throughout adulthood.While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos.Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Harvard University, Boston, MA 02115, USA. wxia@rics.bwh.harvard.edu.

ABSTRACT
Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.

No MeSH data available.


Related in: MedlinePlus