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Preservation of DNA integrity and neuronal degeneration
Brain Research Reviews 48 (2005) 347 – 351 www.elsevier.com/locate/brainresrev
Review
Preservation of DNA integrity and neuronal degeneration Simona Francisconi, Mara Codenotti, Giulia Ferrari-Toninelli, Daniela Uberti, Maurizio Memo* Department of Biomedical Sciences and Biotechnologies, University of Brescia, Medical School, Viale Europa 11, 25124 Brescia, Italy Accepted 9 December 2004 Available online 8 February 2005
Abstract The mismatch repair system (MMR) is an important member of the DNA checkpoint, that includes a number of protein deputed to control genomic stability through cell cycle arrest, DNA repair, and apoptosis. Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of MSH2, perhaps the most relevant component of the MMR system. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: MSH2; DNA damage; Alzheimer’s disease; Oxidative stress; DNA repair; Neurodegeneration
Contents 1. Introduction. . . . . . . . . . . . . 2. Contribution of the mismatch repair 3. Concluding remarks . . . . . . . . Acknowledgments . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
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1. Introduction DNA repair is one of the most essential system for saving the inherited nucleotide sequence of genomic DNA over time. Cells engage very efficient mechanisms involving DNA surveillance/repair proteins so as to maintain inherited nucleotide sequence of genomic DNA over time. After DNA damage occurring during duplication or after genotoxic stimuli, cells activate intracellular pathways which are able to recognize the damage, arrest cell cycle, recruit DNA repair factors, and finally repair the damage. This relevant process is finalized to prevent the generation and the persistence of * Corresponding author. Fax: +39 030 3717409. E-mail address: [email protected] (M. Memo). 0165-0173/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2004.12.023
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impaired cells which may ultimately be detrimental to the organism. If damage repair fails, the same molecular participants trigger elimination of the target cell by apoptosis. It is well recognized that mutation of genes related with DNA damage repair are associated with specific cancerprone syndromes. Interestingly, as recently pointed out by Rolig and McKinnon [19], many human pathological conditions with genetic defects in DNA damage responses are also characterized by neurological deficits. These neurological deficits can manifest themselves during many stages of development, suggesting an important role for DNA repair during the development and maintenance of the brain. However, only little is known about the role of DNA damage sensors and repair factors in terminally differentiated, not proliferating cells, like neurons.
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S. Francisconi et al. / Brain Research Reviews 48 (2005) 347–351
Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of a component of the DNA repair system, the protein MSH2. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes.
2. Contribution of the mismatch repair system to neuronal degeneration The mismatch repair (MMR) system is an important member of the DNA checkpoint, that includes a number of proteins involved in the control of genomic stability through cell cycle arrest, DNA repair, and apoptosis. Repair of damaged genes is the prominent role of this system, that is shared with the other major repair systems, such as the Nucleotide Excision Repair (NER) and the Base Excision Repair (BER). It is well established that mutation of at least one of the MMR genes is associated with the development of non-polyposis colon–rectal cancer [12]. Several studies have led to a biochemical model for postreplication mismatch repair in E. coli. Initiation of an MMR event occurs when MutS recognizes and binds mispaired nucleotides that result from polymerase misincorporation errors [20]. In eukaryotics, there are at least seven MutS homologues (MSH1 to MSH7) and five MutL homologues designated MLH1, MLH2, MLH3, PMS1 and PMS2. With the exception of the homodimer MSH1– MSH1, involved in mitochondrial genomic stability in yeast and plant, the eukaryotic MutS and MutL homologues typically form heterodimers. MSH heterodimers are specialized for different but overlapping classes of mismatches: in mammals, the most important dimers are MSH2–MSH6 (MutSa) heterodimers, that generally recognize base mispairs (e.g., G/T, A/C) and short insertion–deletion loops (IDLs), and MSH2–MSH3 heterodimers (MutSh), that bind to IDLs [1]. An oversimplified scheme of MMR gene interaction and function is illustrated in Fig. 1. MutS proteins interact also with other DNA lesions: small
Fig. 1. Simplified scheme of MMR proteins interaction and function.
alteration such as O 6-methylguanine, 8-oxoguanine and thymine glycol, and major ultraviolet light photoproducts such as Cyclobutane pyrimidine dimers (CPDs) and 1,2 intrastrand G–G intrastrands cross-links produced by cysplatin [6]. A protein named Proliferating Cell Nuclear Antigen (PCNA) has recently been added to the list of members of the MMR system; this protein was originally thought to interact with DNA polymerases and increase their activity. Recently, it was found that yeast MSH3 and MSH6 contain N-terminal sequence motifs characteristics of proteins that bind to PCNA [7] and that human PCNA co-immunoprecipitates in a complex containing MLH1 and PMS2 [13]. These evidences suggest that PCNA has also been implicated in MMR before the DNA synthesis step, directly in mispair recognition, although the biochemical basis for this additional role of PCNA remains unclear [22].DNA repair is not the only function of MMR system; it is likely part of the DNA checkpoint system involved in choosing cell fate. It has recently been shown that key proteins involved in DNA repair could also play additional roles, in excess of DNA damage, and become pro-apoptotic proteins. Proteins of MMR system, i.e., MSH2 and MSH6, appear to play this dual role [4,15]. In this line, hMutSa and hMutLh increase the phosphorylation of p53 in response to DNA methylation damage [11]. Human epithelial and mouse embryo fibroblast cell lines lacking MLH1 protein are more resistant to hydrogen peroxide, likely via the dysregulation of apoptosis. In fact, MLH1 proficient cells treated with hydrogen peroxide undergo apoptotic death that involves mitochondrial permeability and caspase 3 activation [14]. Mouse embryonic stem cells and mouse embryo fibroblast cell lines derived from MSH2 defective mice are more resistant to low-level radiations [10], methylating agents [16], and to hydrogen peroxide treatment [8]. Very little is known about the possible role of the MMR system in the CNS. Marietta et al. [17] demonstrated that proteins of MutSa complex are expressed in developing and adult rat brain tissues suggesting that adult brain cells have the capacity to carry out DNA mismatch repair. Nuclear extracts from adult rat brain neurons were also found to be able to repair DNA mismatches [5]. MMR activity in post-mitotic cells may be necessary to correct mismatches occurring during repair of spontaneous deamination of 5-methylcytosine or 8-OH-deoxyguanosine induced by oxidative stress and not corrected by polymerase beta that is an error prone enzyme for the loss of proofreading activity. In this contest, we further explored the expression of MMR proteins in neurons by in vivo and in vitro studies. The distribution of MSH2, one of the key proteins involved in recognition of damaged DNA, was evaluated by immunohistochemistry in the adult rat CNS. MSH2 was expressed in several areas of the brain including hippocampus, cerebellum, cortex, striatum, substantia nigra, and in spinal cord. MSH2 was detected in nuclei of neuronal cells and not in glial cells [2]. We also studied
S. Francisconi et al. / Brain Research Reviews 48 (2005) 347–351
the involvement of MSH2 in neuronal degeneration caused by systemic administration of kainic acid in rats. This is a well-known experimental paradigm of excitotoxicity, characterized by specific cell loss in CA3/CA4 hippocampal subfields. Kainate injection resulted in a dosedependent increase of MSH2 expression specifically in the pyramidal cells of CA3/CA4 subfield. MSH2 increased expression could be interpreted as a part of a pro-apoptotic signaling. This is in line with previous data showing overexpression of MSH2 and MLH1 proteins in cells undergoing apoptosis [24]. In this regard, we also found an overexpression of both MSH2 and MSH6 proteins in SH-SY5Y neuronal cells exposed to doxorubicine [3]. We further explored the possible involvement of MMR in the repair of base oxidation induced by H2O2 in our
349
model of neuronal cells. SH-SY5Y neuroblastoma cells were differentiated with retinoic acid and then treated with a H2O2 pulse. This lesion caused a marked oxidative DNA damage, as detected by 8-OH-deoxyguanosine immunoreactivity, within 15 min and lasted until 2 h after the H2O2 pulse (Fig. 2A). Interestingly, in the same time frame, H2O2 induces also MSH2 nuclear translocation (Figs. 2B and C) [21]. Additional evidence for MMR activity in neurons was obtained in functional studies from both undifferentiated and differentiated Ntera2 extracts [9]. To more specifically address the question of whether MSH2 is involved in an apoptotic program, we analyzed apoptosis induced by H2O2 in isolated hippocampal neurons from MSH2 heterozygous mice. Using comet assay to
Fig. 2. Generation of DNA oxidative damage and MSH2 protein activation in SH-SY5Y neuronal cell line treated with H2O2. SH-SY5Y human neuroblastoma cells were treated with retinoic acid for 1 week to acquire a neuronal phenotype. When differentiated, cells were exposed to 2.5 mM H2O2 for 20 min. Immunocytochemistry with anti-8OH-dG antibody (A) was performed at different times as indicated on the top-right of each individual micrograph. Arrows indicate the nuclear localization of the signal. Some positive spots were also present in extranuclear, possibly mitochondria, subcellular regions. MSH2 expression was studied at different times after the H2O2 pulse by Western blot analysis (B) using protein extracts from the cytosol or nuclear fraction, as indicated. Immunocytochemistry (C) was performed in cells at 2 h after the H2O2 pulse. Both Western blot and immunocytochemical analysis were performed using the same anti-MSH2 antibody.
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detect DNA degradation, we found that hippocampal neurons from MSH2 heterozygous mice were 3-fold more resistant to H2O2-induced apoptosis than hippocampal neurons from wild-type mice (data not shown). Thus, neurons lacking at least one component of the MMR system, namely, MSH2, were unable to activate an apoptotic program. The relevance of these observations in neurodegenerative diseases definitely requires further investigations. Consistent with this putative role are the data from Wheeler et al. [23] who show, by studying the CAG tract instability in an animal model of Huntington’s disease, that lack of MSH2 delays the timing of mutant protein accumulation and disease onset.
3. Concluding remarks Historically, one of the most well-known connections between abnormalities of the DNA damage response and neurodegeneration has been the human syndrome of ataxia telangiectasia. However, other syndromes associated with defective DNA damage response also include neurological symptoms as a primary feature of their phenotypes [19]. This argues that defects in the repair of, or response to, DNA damage impact significantly on brain function. We speculate that the list of neuropathologies associated with an impairment of the DNA damage repair system could be even larger including chronic and progressive neurodegenerative diseases like Alzheimer’s disease (AD). In this regard, quantitative and histopathological markers of oxidative DNA damage have been found in the brain of AD patients [18] suggesting that the neuronal machinery devoted to restore damaged DNA might be impaired also in AD brain. Future studies focusing on these and other aspects of h-amyloid neurotoxicity will be important for the molecular description of specific DNA processing and repair mechanisms in brain as well as to define whether cancer and neurodegeneration share common genetic risk factors for the development and progression of the underlying diseases.In this regard, DNA repair genes are good candidate for such a dual role. In fact, the activity of the DNA repair systems in the brain decreases as a function of age so that the capability to recognize and repair various types of DNA damage is reduced in aging brain. One of the most intriguing aspects of this topic is whether or not neuronal cells lacking DNA repair factors are able to survive after DNA damage. One possibility is that they do survive and acquire a novel phenotype, that is a neuron with altered DNA. This could be relevant or not. If DNA damage is located in a region not important for transcription or in a coding region of a protein which is not important for cell function, they may survive without showing significant impairment. On the other hand, DNA damage may be located in crucial sites along the DNA filament, thus altering transcription of proteins deeply involved in cell function. The decrease efficiency of the DNA repair
systems occurring in aging brain may increase the risk to generate and accumulate gene mutations within individual neurons and, as consequence, develop cells with functional alterations that might be defined as bdysfunctional neurons Q. This view takes into consideration at least two features of many neurodegenerative diseases: anatomy, i.e., cell specificity, and timing, i.e., years for the clinical manifestation of the disease.
Acknowledgments This work was supported by the Italian MIUR, PRIN 2004 and an educational grant from Newron Pharmaceuticals, Italy. S.F. is on leave of absence from Newron Pharmaceuticals, Italy.
References [1] D.J. Allen, M. Makhov, M. Grilley, J. Taylor, R. Thresher, P. Modrich, D.J. Griffith, MutS mediates heteroduplex loop formation by a translocation mechanism, EMBO J. 16 (1997) 4467 – 4476. [2] M. Belloni, D. Uberti, C. Rizzini, G. Ferrari-Toninelli, P. Rizzonelli, J. Jiricny, P. Spano, M. Memo, Distribution and kainate-mediated induction of the DNA mismatch repair protein MSH2 in rat brain, Neuroscience 94 (1999) 1323 – 1331. [3] M. Belloni, D. Uberti, C. Rizzini, J. Jiricny, M. Memo, Induction of two DNA mismatch repair proteins, MSH2 and MSH6, in differentiated human neuroblastoma SH-SY5Y cells exposed to doxorubicin, J. Neurochem. 72 (1999) 974 – 979. [4] C. Bernstein, H. Bernstein, C.M. Payne, H. Garewal, DNA repair/proapoptotic dual role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis, Mutat. Res. 511 (2002) 145 – 178. [5] P.J. Brooks, C. Marietta, D. Goldman, DNA mismatch repair and DNA methylation in adult brain neurons, J. Neurosci. 16 (1996) 939 – 945. [6] M. Christmann, B. Kaina, Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents, J.B.C 275 (2000) 36256 – 36262. [7] A.B. Clark, F. Valle, K. Drotschmann, R.K. Gary, T.A. Kunkel, Functional interaction of proliferating cell nuclear antigen with MSH2–MSH6 and MSH2 and MSH3 complexes, J. Biol. Chem. 275 (2000) 36498 – 36501. [8] C. Colussi, E. Parlanti, P. Degan, G. Aquilina, D. Barnes, P. Macpherson, P. Karran, M. Crescenzi, E. Dogliotti, M. Bignami, The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool, Curr. Biol. 12 (2002) 912 – 918. [9] P. David, DNA replication and postreplication mismatch repair in cellfree extracts from cultured human neuroblastoma and fibroblast cells, J. Neurosci. 17 (1997) 8711 – 8720. [10] T.L. DeWeese, J.M. Shipman, N.A. Larrier, N.M. Buckley, L.R. Kidd, J.D. Groopman, R.G. Cutler, H. Te Riele, W.G. Nelson, Mouse embrionic stem cells carrying one or two defective MSH2 alleles respond abnormally to oxidative stress inflicted by low-level radiation, Proc. Natl. Acad. Sci. 95 (1998) 11915 – 11920. [11] D.R. Duckett, S.M. Bronstein, Y. Taya, P. Modrich, hMutSa and hMutLa-dependent phosphorylation of p53 in response to DNA methylator damage, Proc. Natl. Acad. Sci. 96 (1999) 12384 – 12388. [12] R. Fishel, The selection for mismatch repair defects in hereditary nonpolyposis colorectal cancer: revising the mutator hypothesis, Cancer Res. 61 (2001) 7369 – 7374.
S. Francisconi et al. / Brain Research Reviews 48 (2005) 347–351 [13] L. Gu, Y. Hong, S. McCulloch, H. Watanabe, G. Li, ATPdependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair, Nucleic Acids Res. 26 (1998) 1173 – 1178. [14] R.A. Hardman, C.A. Afshari, J.C. Barrett, Involvement of mammalian MLH1 in the apoptotic response to peroxide-induced oxidative stress, Cancer Res. 61 (2001) 1392 – 1397. [15] M.J. Hickman, L.D. Samson, Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents, Proc. Natl. Acad. Sci. 96 (1999) 10764 – 10769. [16] O. Humbert, S. Fiumicino, G. Aquilina, P. Branch, S. Oda, A. Zijno, P. Karran, M. Bignami, Mismatch repair and differential sensitivity of mouse and human cells to methylating agents, Carcinogenesis 20 (1999) 205 – 214. [17] C. Marietta, F. Palombo, P. Gallinari, J. Jiricny, P.J. Brooks, Expression of long-patch and short-patch DNA mismatch repair proteins in the embryonic and adult mammalian brain, Mol. Brain Res. 53 (1998) 317 – 320. [18] D. Pratico, N. Delanty, Oxidative injury in diseases of the central
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nervous system: focus on Alzheimer’s disease, Am. J. Med. 109 (2000) 577 – 585. R.L. Rolig, P.J. McKinnon, Linking DNA damage and neurodegeneration, Trends Neurosci. 23 (2000) 417 – 424. S.S. Su, P. Modrich, Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs, Proc. Natl. Acad. Sci. 83 (1986) 5057 – 5061. D. Uberti, G. Ferrari-Toninelli, M. Memo, Involvement of DNA damage and repair systems in neurodegenerative process, Toxicol. Lett. 139 (2003) 99 – 105. A. Umar, A.B. Buermeyer, J.A. Simon, D.C. Thomas, A.B. Clark, R.M. Liskay, T.A. Kunkel, Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis, Cell 87 (1996) 65 – 73. V.C. Wheeler, L.A. Lebel, V. Vrbanac, A. Teed, H. te Riele, M.E. MacDonald, Mismatch repair gene Msh2 modifies the timing of early disease in HdhQ111 striatum, Hum. Mol. Genet. 12 (2003) 273 – 281. H. Zhang, B. Richards, T. Wilson, M. Lloyd, A. Cranston, A. Thorburn, R. Fishel, M. Meuth, Apoptosis induced by overexpression of hMSH2 or hMLH1, Cancer Res. 59 (1999) 3021 – 3027.
Review
Preservation of DNA integrity and neuronal degeneration Simona Francisconi, Mara Codenotti, Giulia Ferrari-Toninelli, Daniela Uberti, Maurizio Memo* Department of Biomedical Sciences and Biotechnologies, University of Brescia, Medical School, Viale Europa 11, 25124 Brescia, Italy Accepted 9 December 2004 Available online 8 February 2005
Abstract The mismatch repair system (MMR) is an important member of the DNA checkpoint, that includes a number of protein deputed to control genomic stability through cell cycle arrest, DNA repair, and apoptosis. Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of MSH2, perhaps the most relevant component of the MMR system. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: MSH2; DNA damage; Alzheimer’s disease; Oxidative stress; DNA repair; Neurodegeneration
Contents 1. Introduction. . . . . . . . . . . . . 2. Contribution of the mismatch repair 3. Concluding remarks . . . . . . . . Acknowledgments . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
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1. Introduction DNA repair is one of the most essential system for saving the inherited nucleotide sequence of genomic DNA over time. Cells engage very efficient mechanisms involving DNA surveillance/repair proteins so as to maintain inherited nucleotide sequence of genomic DNA over time. After DNA damage occurring during duplication or after genotoxic stimuli, cells activate intracellular pathways which are able to recognize the damage, arrest cell cycle, recruit DNA repair factors, and finally repair the damage. This relevant process is finalized to prevent the generation and the persistence of * Corresponding author. Fax: +39 030 3717409. E-mail address: [email protected] (M. Memo). 0165-0173/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2004.12.023
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impaired cells which may ultimately be detrimental to the organism. If damage repair fails, the same molecular participants trigger elimination of the target cell by apoptosis. It is well recognized that mutation of genes related with DNA damage repair are associated with specific cancerprone syndromes. Interestingly, as recently pointed out by Rolig and McKinnon [19], many human pathological conditions with genetic defects in DNA damage responses are also characterized by neurological deficits. These neurological deficits can manifest themselves during many stages of development, suggesting an important role for DNA repair during the development and maintenance of the brain. However, only little is known about the role of DNA damage sensors and repair factors in terminally differentiated, not proliferating cells, like neurons.
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S. Francisconi et al. / Brain Research Reviews 48 (2005) 347–351
Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of a component of the DNA repair system, the protein MSH2. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes.
2. Contribution of the mismatch repair system to neuronal degeneration The mismatch repair (MMR) system is an important member of the DNA checkpoint, that includes a number of proteins involved in the control of genomic stability through cell cycle arrest, DNA repair, and apoptosis. Repair of damaged genes is the prominent role of this system, that is shared with the other major repair systems, such as the Nucleotide Excision Repair (NER) and the Base Excision Repair (BER). It is well established that mutation of at least one of the MMR genes is associated with the development of non-polyposis colon–rectal cancer [12]. Several studies have led to a biochemical model for postreplication mismatch repair in E. coli. Initiation of an MMR event occurs when MutS recognizes and binds mispaired nucleotides that result from polymerase misincorporation errors [20]. In eukaryotics, there are at least seven MutS homologues (MSH1 to MSH7) and five MutL homologues designated MLH1, MLH2, MLH3, PMS1 and PMS2. With the exception of the homodimer MSH1– MSH1, involved in mitochondrial genomic stability in yeast and plant, the eukaryotic MutS and MutL homologues typically form heterodimers. MSH heterodimers are specialized for different but overlapping classes of mismatches: in mammals, the most important dimers are MSH2–MSH6 (MutSa) heterodimers, that generally recognize base mispairs (e.g., G/T, A/C) and short insertion–deletion loops (IDLs), and MSH2–MSH3 heterodimers (MutSh), that bind to IDLs [1]. An oversimplified scheme of MMR gene interaction and function is illustrated in Fig. 1. MutS proteins interact also with other DNA lesions: small
Fig. 1. Simplified scheme of MMR proteins interaction and function.
alteration such as O 6-methylguanine, 8-oxoguanine and thymine glycol, and major ultraviolet light photoproducts such as Cyclobutane pyrimidine dimers (CPDs) and 1,2 intrastrand G–G intrastrands cross-links produced by cysplatin [6]. A protein named Proliferating Cell Nuclear Antigen (PCNA) has recently been added to the list of members of the MMR system; this protein was originally thought to interact with DNA polymerases and increase their activity. Recently, it was found that yeast MSH3 and MSH6 contain N-terminal sequence motifs characteristics of proteins that bind to PCNA [7] and that human PCNA co-immunoprecipitates in a complex containing MLH1 and PMS2 [13]. These evidences suggest that PCNA has also been implicated in MMR before the DNA synthesis step, directly in mispair recognition, although the biochemical basis for this additional role of PCNA remains unclear [22].DNA repair is not the only function of MMR system; it is likely part of the DNA checkpoint system involved in choosing cell fate. It has recently been shown that key proteins involved in DNA repair could also play additional roles, in excess of DNA damage, and become pro-apoptotic proteins. Proteins of MMR system, i.e., MSH2 and MSH6, appear to play this dual role [4,15]. In this line, hMutSa and hMutLh increase the phosphorylation of p53 in response to DNA methylation damage [11]. Human epithelial and mouse embryo fibroblast cell lines lacking MLH1 protein are more resistant to hydrogen peroxide, likely via the dysregulation of apoptosis. In fact, MLH1 proficient cells treated with hydrogen peroxide undergo apoptotic death that involves mitochondrial permeability and caspase 3 activation [14]. Mouse embryonic stem cells and mouse embryo fibroblast cell lines derived from MSH2 defective mice are more resistant to low-level radiations [10], methylating agents [16], and to hydrogen peroxide treatment [8]. Very little is known about the possible role of the MMR system in the CNS. Marietta et al. [17] demonstrated that proteins of MutSa complex are expressed in developing and adult rat brain tissues suggesting that adult brain cells have the capacity to carry out DNA mismatch repair. Nuclear extracts from adult rat brain neurons were also found to be able to repair DNA mismatches [5]. MMR activity in post-mitotic cells may be necessary to correct mismatches occurring during repair of spontaneous deamination of 5-methylcytosine or 8-OH-deoxyguanosine induced by oxidative stress and not corrected by polymerase beta that is an error prone enzyme for the loss of proofreading activity. In this contest, we further explored the expression of MMR proteins in neurons by in vivo and in vitro studies. The distribution of MSH2, one of the key proteins involved in recognition of damaged DNA, was evaluated by immunohistochemistry in the adult rat CNS. MSH2 was expressed in several areas of the brain including hippocampus, cerebellum, cortex, striatum, substantia nigra, and in spinal cord. MSH2 was detected in nuclei of neuronal cells and not in glial cells [2]. We also studied
S. Francisconi et al. / Brain Research Reviews 48 (2005) 347–351
the involvement of MSH2 in neuronal degeneration caused by systemic administration of kainic acid in rats. This is a well-known experimental paradigm of excitotoxicity, characterized by specific cell loss in CA3/CA4 hippocampal subfields. Kainate injection resulted in a dosedependent increase of MSH2 expression specifically in the pyramidal cells of CA3/CA4 subfield. MSH2 increased expression could be interpreted as a part of a pro-apoptotic signaling. This is in line with previous data showing overexpression of MSH2 and MLH1 proteins in cells undergoing apoptosis [24]. In this regard, we also found an overexpression of both MSH2 and MSH6 proteins in SH-SY5Y neuronal cells exposed to doxorubicine [3]. We further explored the possible involvement of MMR in the repair of base oxidation induced by H2O2 in our
349
model of neuronal cells. SH-SY5Y neuroblastoma cells were differentiated with retinoic acid and then treated with a H2O2 pulse. This lesion caused a marked oxidative DNA damage, as detected by 8-OH-deoxyguanosine immunoreactivity, within 15 min and lasted until 2 h after the H2O2 pulse (Fig. 2A). Interestingly, in the same time frame, H2O2 induces also MSH2 nuclear translocation (Figs. 2B and C) [21]. Additional evidence for MMR activity in neurons was obtained in functional studies from both undifferentiated and differentiated Ntera2 extracts [9]. To more specifically address the question of whether MSH2 is involved in an apoptotic program, we analyzed apoptosis induced by H2O2 in isolated hippocampal neurons from MSH2 heterozygous mice. Using comet assay to
Fig. 2. Generation of DNA oxidative damage and MSH2 protein activation in SH-SY5Y neuronal cell line treated with H2O2. SH-SY5Y human neuroblastoma cells were treated with retinoic acid for 1 week to acquire a neuronal phenotype. When differentiated, cells were exposed to 2.5 mM H2O2 for 20 min. Immunocytochemistry with anti-8OH-dG antibody (A) was performed at different times as indicated on the top-right of each individual micrograph. Arrows indicate the nuclear localization of the signal. Some positive spots were also present in extranuclear, possibly mitochondria, subcellular regions. MSH2 expression was studied at different times after the H2O2 pulse by Western blot analysis (B) using protein extracts from the cytosol or nuclear fraction, as indicated. Immunocytochemistry (C) was performed in cells at 2 h after the H2O2 pulse. Both Western blot and immunocytochemical analysis were performed using the same anti-MSH2 antibody.
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detect DNA degradation, we found that hippocampal neurons from MSH2 heterozygous mice were 3-fold more resistant to H2O2-induced apoptosis than hippocampal neurons from wild-type mice (data not shown). Thus, neurons lacking at least one component of the MMR system, namely, MSH2, were unable to activate an apoptotic program. The relevance of these observations in neurodegenerative diseases definitely requires further investigations. Consistent with this putative role are the data from Wheeler et al. [23] who show, by studying the CAG tract instability in an animal model of Huntington’s disease, that lack of MSH2 delays the timing of mutant protein accumulation and disease onset.
3. Concluding remarks Historically, one of the most well-known connections between abnormalities of the DNA damage response and neurodegeneration has been the human syndrome of ataxia telangiectasia. However, other syndromes associated with defective DNA damage response also include neurological symptoms as a primary feature of their phenotypes [19]. This argues that defects in the repair of, or response to, DNA damage impact significantly on brain function. We speculate that the list of neuropathologies associated with an impairment of the DNA damage repair system could be even larger including chronic and progressive neurodegenerative diseases like Alzheimer’s disease (AD). In this regard, quantitative and histopathological markers of oxidative DNA damage have been found in the brain of AD patients [18] suggesting that the neuronal machinery devoted to restore damaged DNA might be impaired also in AD brain. Future studies focusing on these and other aspects of h-amyloid neurotoxicity will be important for the molecular description of specific DNA processing and repair mechanisms in brain as well as to define whether cancer and neurodegeneration share common genetic risk factors for the development and progression of the underlying diseases.In this regard, DNA repair genes are good candidate for such a dual role. In fact, the activity of the DNA repair systems in the brain decreases as a function of age so that the capability to recognize and repair various types of DNA damage is reduced in aging brain. One of the most intriguing aspects of this topic is whether or not neuronal cells lacking DNA repair factors are able to survive after DNA damage. One possibility is that they do survive and acquire a novel phenotype, that is a neuron with altered DNA. This could be relevant or not. If DNA damage is located in a region not important for transcription or in a coding region of a protein which is not important for cell function, they may survive without showing significant impairment. On the other hand, DNA damage may be located in crucial sites along the DNA filament, thus altering transcription of proteins deeply involved in cell function. The decrease efficiency of the DNA repair
systems occurring in aging brain may increase the risk to generate and accumulate gene mutations within individual neurons and, as consequence, develop cells with functional alterations that might be defined as bdysfunctional neurons Q. This view takes into consideration at least two features of many neurodegenerative diseases: anatomy, i.e., cell specificity, and timing, i.e., years for the clinical manifestation of the disease.
Acknowledgments This work was supported by the Italian MIUR, PRIN 2004 and an educational grant from Newron Pharmaceuticals, Italy. S.F. is on leave of absence from Newron Pharmaceuticals, Italy.
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