- Email: [email protected]
Neurons in Alzheimer disease emerge from senescence
Mechanisms of Ageing and Development 123 (2001) 3 – 9 www.elsevier.com/locate/mechagedev
Neurons in Alzheimer disease emerge from senescence Arun K. Raina a, Patricia Pardo b, Catherine A. Rottkamp a, Xiongwei Zhu a, Olivia M. Pereira-Smith b, Mark A. Smith a,* a
Institute of Pathology, Case Western Reser6e Uni6ersity, 2085 Adelbert Road, Cle6eland, OH 44106, USA b Roy M. and Phyllis Gough Huffington Center on Aging, Departments of Virology and Microbiology, Molecular and Cellular Biology and Medicine, Baylor College of Medicine, Houston, TX, USA
Abstract A number of cell cycle markers are associated with the selective neuronal pathology found in Alzheimer disease. However, the significance of such cell cycle markers is clouded by duplicity of function in that many such proteins are also involved in apoptosis and/or DNA repair following oxidative damage. To clarify whether or not neurons in Alzheimer disease do in fact emerge from a quiescent status, with subsequent entry into the G1 phase of the cell cycle, in this study we focused on a family of MORF4-related proteins that are associated with emergence from senescence. Our results show that many neurons in vulnerable regions of Alzheimer disease brain, but not in control brain, have increased MORF4-related proteins indicating re-entry into the cell cycle. Immunoblot analysis showed a specific disease-related increase in a 52 kDa protein that is likely the human homologue of the MORF4-related transcription factor. The novel localization of such a transcriptional activating protein to selectively vulnerable neurons in Alzheimer disease provides compelling evidence for mitotic re-entry as part of the pathogenesis of neuronal dysfunction and death in Alzheimer disease. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Alzheimer disease; Cell cycle; Pathogenesis
* Corresponding author. Tel.: +1-216-368-3670; fax: 1 + 216-368-8964. E-mail address: [email protected] (M.A. Smith). 0047-6374/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 3 3 - 5
4
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
1. Introduction Alzheimer disease (AD), an insidious, progressive dementing disorder is characterized by selective primary neuronal loss in the hippocampal and cerebral cortices that correlates clinically with dementia (reviewed in Smith, 1998). While the mechanisms involved in selective neurodegeneration are largely unknown, there is accumulating evidence that these susceptible neuronal populations, which are postmitotic and thus inherently restricted in their mitotic competence, may exit G0 and re-enter the G1 phase of the cell cycle during the progression of AD (reviewed in Raina et al., 2000). The mitotic phenotype within the pyramidal neurons in AD includes the ectopic presence of various cyclins, cyclin dependent kinases (Arendt et al., 1995, 1996; McShea et al., 1997; Busser et al., 1998; Zhu et al., 2000) and cyclin inhibitors (McShea et al., 1997; Nagy et al., 1998). Taken together, these findings suggest that neurons vulnerable to degeneration, morphologically resemble cells that are cycling, rather than terminally differentiated non-dividing cells (Vincent et al., 1996; McShea et al., 1997, 1999a; Raina et al., 1999a,b, 2000; Zhu et al., 1999; Gerst et al., 2000). However, many cell cycle-related proteins, including those found ectopically expressed in AD, are also involved in apoptosis, trophic-deprivation and DNA repair (Elledge, 1996; Stefanis et al., 1996; Park et al., 1997, 1998, 2000; Padmanabhan et al., 1999). In this study to further test the hypothesis that emergence from G0 and subsequent entry into the G1 phase of the cell cycle is a fundamental characteristic of disease pathogenesis, we speculated that other novel marker(s) of entry into the G1 phase should also be present in this vulnerable neuronal population in AD. The members of the MORF4-related protein family are unique markers of emergence out of quiescence and progression into the G1 phase of the cell cycle such that expression is decreased in quiescent and senescent non-dividing normal human cells and protein levels increase when quiescent cells are stimulated to re-enter the cell cycle (Bertram et al., 1999). Importantly, aside from a role in cell cycle, no other functions are known. We therefore initiated this study to determine whether MORF4-related proteins, novel markers of cell cycle progression, are associated with the neuronal pathophysiology of AD.
2. Materials and methods
2.1. Tissue section preparation Hippocampal tissue samples were obtained postmortem from patients (n= 8, ages 68– 95) with histopathologically-confirmed AD, as well as from non-AD young and aged-matched controls (n =8, ages 63– 85). All clinical and pathological diagnoses were according to standardized criteria (Khachaturian, 1985; Mirra et al., 1991). From the clinical reports available to us, we found no obvious differences in agonal status or other potential confounders between the groups. Tissue was fixed in methacarn (methanol:chloroform:acetic acid in a 6:3:1 v/v/v) or buffered forma-
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
5
lin for 16 h at 4 °C. Tissue was subsequently dehydrated through graded ethanol and xylene solutions and embedded in paraffin. Microtome sections 6 mm thick were prepared and placed on silane-coated slides (Sigma, St. Louis, MO).
2.2. Immunocytochemistry Immunocytochemistry with an anti-chromodomain region of the MORF4 family of proteins peptide antibody (Bertram et al., 1999) was performed essentially as previously described (Sternberger, 1986; Smith et al., 1994). Briefly, following immersion in xylene and hydration through graded ethanol solutions, endogenous peroxidase activity was eliminated by incubation of the sections of 3% hydrogen peroxidase for 30 min. To reduce non-specific binding, sections were incubated for 30 min at room temperature in 10% normal goat serum (NGS) in Tris-buffered saline (TBS; 50 mM Tris– HCl, 150 mM NaCl, pH 7.6). After rinsing briefly with 1% NGS, sections were incubated overnight in either MORF4 antisera (1/100) or a mouse monoclonal antibody, AT8, to altered cytoskeletal t protein (Smith et al., 1994). The sections were then incubated in either goat anti-rabbit or goat antimouse antisera (ICN Biomedicals, Costa Mesa, CA), followed by species-specific peroxidase anti-peroxidase complex (Sternberger Monoclonals Inc., Lutherville, MD and ICN Biomedicals, Costa Mesa, CA). Antibodies were localized using 3,3 diaminobenzidine (DAB) as a chromogen (DAKO Corporation, Carpinteria, CA). To ensure the specificity of the reactivity observed, immunostaining was also performed in parallel using antibody that had been pre-incubated with the purified immunizing peptide (50 mg/ml) (Bertram et al., 1999).
2.3. Immunoblot analysis Tissue samples were solubilized in RIPA buffer, protein concentration determined and 20 mg protein loaded on SDS PAGE gels. The proteins were transferred to membranes and probed with 1:1000 of our primary antibody and 1:2000 secondary rabbit polyclonal antibody and visualized with ECL according to the manufacturer’s instructions (Santa Cruz, Santa Cruz, CA).
3. Results The antisera made against the MORF4 family chromodomain region specifically labels large pyramidal neurons in AD cases, but not age-matched controls (Fig. 1). There was significant overlap of the immunostaining profiles of MORF4-related protein and phosphorylated t in neurofibrillary pathology assessed with the antibody AT8, which recognizes t protein only when serine 202 and threonine 205 are phosphorylated. In marked contrast, age-matched normal controls did not show any immunoreactivity. The specificity of this select immunolocalization of MORF4related protein was confirmed by the almost complete abolition of immunostaining using antibody pre-incubated with the immunizing peptide.
6
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
Fig. 1. Antisera to the chromodomain region of MORF4-related proteins specifically recognize intraneuronal neurofibrillary pathology in cases of AD (A), whereas, conversely, there is little specific localization in control cases.
Immunoblot analyses of homogenates of AD and control cortex reveals a single 52 kDa band in 3/3 AD cases and 1/3 control cases recognized by the antibody to MORF4 (Fig. 2). We determined that these bands were specifically eliminated in immunoblot analyses performed with the antibody pre-incubated with the peptide used to obtain the antiserum, indicating that the antisera is specific to the chromodomain region of this family of proteins. Recently, two chromodomain proteins with molecular weights corresponding to about 52 kDa and with homology to the peptide region against which our antibody was raised (Fig. 3) were cloned, namely MOF (Neal et al., 2000; Eisen et al., 2001) and an alternatively spliced form of MSL3L1 (Prakash et al., 1999). MOF is a histone acetyl transferase (HAT) and MSL3L1 was cloned during studies to identify the microopthalmia gene.
Fig. 2. Western analysis of AD (lanes 1 –3) and normal samples (lanes 4 – 6). HeLa cells transfected with MRG 15 driven by the CMV promoter demonstrate the position of the MRG 15 protein at 37 kDa (lanes 7, 8) and the cross reactivity of the antibody with a protein about 52 kDa (lane 8).
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
7
Fig. 3. Amino acid alignment of the chromodomain region of human MRG15, MSL3L1 and MOF. The chromodomain regions of MRG15 (GeneBank Accession Number AF100615), the two splicing variants of MSL3L1 (GeneBank Accession Number AF117065) and MOF proteins were aligned using Clustal analysis. The sequence of the peptide used to obtain the antibody is underlined. Identical residues are shown in bold and similar residues in italics.
4. Discussion Primary neurons in the normal adult brain are generally viewed as being quiescent. However, multiple lines of evidence implicate emergence from this quiescent state, and subsequent attempted re-entry into the cell division cycle, as a proximal pathophysiology in AD (reviewed in Raina et al., 2000). While, thus far, there is no evidence that successful nuclear division occurs in these neurons, proteins associated with exit from the cell cycle are apparently upregulated (Arendt et al., 1998; McShea et al., 1999a,b). These include members of the cell cycle engine, namely CDKs, along with their cognate cyclins in vulnerable neurons (reviewed in Raina et al. 1999a, 2000) and indeed, these neurons in AD are morphologically similar to cells that are cycling, rather than terminally differentiated non-dividing cells (Raina et al., 2000). In such a scenario, one would predict that novel markers, whose presence is obligatory in the G1 phase of the cell cycle, would also be seen in the vulnerable neurons of AD. In this study, we found that a 52 kDa protein MORF4-related cell cycle protein is associated with intraneuronal neurofibrillary pathology in select neuronal population that are vulnerable in AD, while all age matched controls have no immunostaining. Immunoblot analysis confirmed these findings although one of three control individuals did express detectable levels of the protein, which may be an indicator of pathology that might arise in the future. Two such proteins, 52 kD in size, have recently been cloned. One gene, MOF, encodes a HAT and the other being MSL3L1, which was cloned during studies localizing the microopthalmia gene. These, together with MRG15, all encode chromodomain regions. Chromodomains are known to be involved in protein–
8
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
protein interactions and form complexes that remodel chromatin, making transcription factors accessible to gene promoters. Such complexes often contain HATs. The best-studied chromodomain protein is Drosophila msl3 (male specific lethal) that is involved in dosage compensation and up-regulation of transcription on the X chromosome in males. The chromodomain regions of msl3, MRG15, MOF, and MSL3L1 are very similar and the prediction is that these proteins will be present in transcriptional activating complexes that most likely will include a HAT activity. The presence of a positive transcriptional controlling protein in association with intraneuronal neurofibrillary pathology in vulnerable neurons of AD is consistent with the many changes in gene expression that occur and adds credence to the role of proximal mitotic mechanisms in AD. This return to mitotic competence in AD can have profound implications for both screening and therapeutics to modify the nature history of this disease.
Acknowledgements This work was supported by grants from the National Institutes of Health (R01 NS38648 to M.A.S. and R37 AG05333 to O.M.P.S.) and the Alzheimer’s Association (Stephanie B. Overstreet Scholars, IIRG-00-2163, to M.A.S.).
References Arendt, T., Holzer, M., Grossmann, A., Zedlick, D., Bruckner, M.K., 1995. Increased expression and subcellular translocation of the mitogen activated protein kinase and mitogen-activated protein kinase in Alzheimer’s disease. Neuroscience 68, 5 – 18. Arendt, T., Rodel, L., Gartner, U., Holzer, M., 1996. Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer’s disease. Neuroreport 7, 3047 – 3049. Arendt, T., Holzer, M., Gartner, U., 1998. Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer’s disease. J. Neural Transm. 105, 949 – 960. Bertram, M.J., Berube, N.G., Hang-Swanson, X., Ran, Q., Leung, J.K., Bryce, S., Spurgers, K., Bick, R.J., Baldini, A., Ning, Y., Clark, L.J., Parkinson, E.K., Barrett, J.C., Smith, J.R., Pereira-Smith, O.M., 1999. Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol. Cell. Biol. 19, 1479 – 1485. Busser, J., Geldmacher, D.S., Herrup, K., 1998. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer’s disease brain. J. Neurosci. 18, 2801 – 2807. Eisen, A., Utley, R.T., Nourani, A., Allard, S., Schmidt, P., Lane, W.S., Lucchesi, J.C., Coˆ te´ , J., 2001. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation. J. Biol. Chem. 276, 3484 – 3491. Elledge, S.J., 1996. Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664 – 1672. Gerst, J.L., Raina, A.K., Pirim, I., McShea, A., Harris, P.L.R., Siedlak, S.L., Takeda, A., Petersen, R.B., Smith, M.A., 2000. Altered cell-matrix associated ADAM in Alzheimer disease. J. Neurosci. Res. 59, 680 –684. Khachaturian, Z.S., 1985. Diagnosis of Alzheimer’s disease. Arch. Neurol. 42, 1097 – 1105. McShea, A., Harris, P.L.R., Webster, K.R., Wahl, A.F., Smith, M.A., 1997. Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer’s disease. Am. J. Pathol. 150, 1933 – 1939. McShea, A., Wahl, A.F., Smith, M.A., 1999a. Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med. Hypotheses 52, 525 – 527.
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
9
McShea, A., Zelasko, D.A., Gerst, J.L., Smith, M.A., 1999b. Signal transduction abnormalities in Alzheimer’s disease: evidence of a pathogenic stimuli. Brain Res. 815, 237 – 242. Mirra, S.S., Heyman, A., McKeel, D., Sumi, S.M., Crain, B.J., Brownlee, L.M., Vogel, F.S., Hughes, J.P., van, B.G., Berg, L., 1991. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology 41, 479 – 486. Nagy, Z., Esiri, M.M., Smith, A.D., 1998. The cell division cycle and the pathophysiology of Alzheimer’s disease. Neuroscience 87, 731 – 739. Neal, K.C., Pannuti, A., Smith, E.R., Lucchesi, J.C., 2000. A new human member of the MYST family of histone acetyl transferases with high sequence similarity to Drosophila MOF. Biochim. Biophys. Acta 1490, 170 –174. Padmanabhan, J., Park, D.S., Greene, L.A., Shelanski, M.L., 1999. Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis. J. Neurosci. 19, 8747 – 8756. Park, D.S., Levine, B., Ferrari, G., Greene, L.A., 1997. Cyclin dependent kinase inhibitors and dominant negative cyclin dependent kinase 4 and 6 promote survival of NGF-deprived sympathetic neurons. J. Neurosci. 17, 8975 –8983. Park, D.S., Morris, E.J., Padmanabhan, J., Shelanski, M.L., Geller, H.M., Greene, L.A., 1998. Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents. J. Cell Biol. 143, 457 –467. Park, D.S., Obeidat, A., Giovanni, A., Greene, L.A., 2000. Cell cycle regulators in neuronal death evoked by excitotoxic stress: implications for neurodegeneration and its treatment. Neurobiol. Aging 21, 771 – 781. Prakash, S.K., Van den Veyver, I.B., Franco, B., Volta, M., Ballabio, A., Zoghbi, H.Y., 1999. Characterization of a novel chromo domain gene in Xp22.3 with homology to Drosophila msl-3. Genomics 59, 77 –84. Raina, A.K., Monteiro, M.J., McShea, A., Smith, M.A., 1999a. The role of cell cycle-mediated events in Alzheimer’s disease. Int. J. Exp. Pathol. 80, 71 – 76. Raina, A.K., Takeda, A., Smith, M.A., 1999b. Mitotic neurons: a dogma succumbs. Exp. Neurol. 159, 248–249. Raina, A.K., Zhu, X., Rottkamp, C.A., Monteiro, M., Takeda, A., Smith, M.A., 2000. Cyclin’ toward dementia: cell cycle abnormalities and abortive oncogenesis in Alzheimer disease. J. Neurosci. Res. 61, 128 –133. Smith, M.A., Taneda, S., Richey, P.L., Miyata, S., Yan, S.-D., Stern, D., Sayre, L.M., Monnier, V.M., Perry, G., 1994. Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc. Natl. Acad. Sci. USA 91, 5710 – 5714. Smith, M.A., 1998. Alzheimer disease. In: Bradley, R.J., Harris, R.A. (Eds.), International Review of Neurobiology, Vol. 42, Academic Press, Inc., San Diego, pp. 1 – 54. Stefanis, L., Park, D.S., Yan, C.Y., Farinelli, S.E., Troy, C.M., Shelanski, M.L., Greene, L.A., 1996. Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271, 30663 –30671. Sternberger, L.A., 1986. Immunocytochemistry, 3rd ed. Wiley, New York. Vincent, I., Rosado, M., Davies, P., 1996. Mitotic mechanisms in Alzheimer’s disease? J. Cell Biol. 132, 413–425. Zhu, X., Raina, A.K., Smith, M.A., 1999. Cell cycle events in neurons: proliferation or death? Am. J. Pathol. 155, 327 –329. Zhu, X., Raina, A.K., Boux, H., Simmons, Z.L., Takeda, A., Smith, M.A., 2000. Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int. J. Dev. Neurosci. 18, 433–437.
Neurons in Alzheimer disease emerge from senescence Arun K. Raina a, Patricia Pardo b, Catherine A. Rottkamp a, Xiongwei Zhu a, Olivia M. Pereira-Smith b, Mark A. Smith a,* a
Institute of Pathology, Case Western Reser6e Uni6ersity, 2085 Adelbert Road, Cle6eland, OH 44106, USA b Roy M. and Phyllis Gough Huffington Center on Aging, Departments of Virology and Microbiology, Molecular and Cellular Biology and Medicine, Baylor College of Medicine, Houston, TX, USA
Abstract A number of cell cycle markers are associated with the selective neuronal pathology found in Alzheimer disease. However, the significance of such cell cycle markers is clouded by duplicity of function in that many such proteins are also involved in apoptosis and/or DNA repair following oxidative damage. To clarify whether or not neurons in Alzheimer disease do in fact emerge from a quiescent status, with subsequent entry into the G1 phase of the cell cycle, in this study we focused on a family of MORF4-related proteins that are associated with emergence from senescence. Our results show that many neurons in vulnerable regions of Alzheimer disease brain, but not in control brain, have increased MORF4-related proteins indicating re-entry into the cell cycle. Immunoblot analysis showed a specific disease-related increase in a 52 kDa protein that is likely the human homologue of the MORF4-related transcription factor. The novel localization of such a transcriptional activating protein to selectively vulnerable neurons in Alzheimer disease provides compelling evidence for mitotic re-entry as part of the pathogenesis of neuronal dysfunction and death in Alzheimer disease. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Alzheimer disease; Cell cycle; Pathogenesis
* Corresponding author. Tel.: +1-216-368-3670; fax: 1 + 216-368-8964. E-mail address: [email protected] (M.A. Smith). 0047-6374/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 3 3 3 - 5
4
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
1. Introduction Alzheimer disease (AD), an insidious, progressive dementing disorder is characterized by selective primary neuronal loss in the hippocampal and cerebral cortices that correlates clinically with dementia (reviewed in Smith, 1998). While the mechanisms involved in selective neurodegeneration are largely unknown, there is accumulating evidence that these susceptible neuronal populations, which are postmitotic and thus inherently restricted in their mitotic competence, may exit G0 and re-enter the G1 phase of the cell cycle during the progression of AD (reviewed in Raina et al., 2000). The mitotic phenotype within the pyramidal neurons in AD includes the ectopic presence of various cyclins, cyclin dependent kinases (Arendt et al., 1995, 1996; McShea et al., 1997; Busser et al., 1998; Zhu et al., 2000) and cyclin inhibitors (McShea et al., 1997; Nagy et al., 1998). Taken together, these findings suggest that neurons vulnerable to degeneration, morphologically resemble cells that are cycling, rather than terminally differentiated non-dividing cells (Vincent et al., 1996; McShea et al., 1997, 1999a; Raina et al., 1999a,b, 2000; Zhu et al., 1999; Gerst et al., 2000). However, many cell cycle-related proteins, including those found ectopically expressed in AD, are also involved in apoptosis, trophic-deprivation and DNA repair (Elledge, 1996; Stefanis et al., 1996; Park et al., 1997, 1998, 2000; Padmanabhan et al., 1999). In this study to further test the hypothesis that emergence from G0 and subsequent entry into the G1 phase of the cell cycle is a fundamental characteristic of disease pathogenesis, we speculated that other novel marker(s) of entry into the G1 phase should also be present in this vulnerable neuronal population in AD. The members of the MORF4-related protein family are unique markers of emergence out of quiescence and progression into the G1 phase of the cell cycle such that expression is decreased in quiescent and senescent non-dividing normal human cells and protein levels increase when quiescent cells are stimulated to re-enter the cell cycle (Bertram et al., 1999). Importantly, aside from a role in cell cycle, no other functions are known. We therefore initiated this study to determine whether MORF4-related proteins, novel markers of cell cycle progression, are associated with the neuronal pathophysiology of AD.
2. Materials and methods
2.1. Tissue section preparation Hippocampal tissue samples were obtained postmortem from patients (n= 8, ages 68– 95) with histopathologically-confirmed AD, as well as from non-AD young and aged-matched controls (n =8, ages 63– 85). All clinical and pathological diagnoses were according to standardized criteria (Khachaturian, 1985; Mirra et al., 1991). From the clinical reports available to us, we found no obvious differences in agonal status or other potential confounders between the groups. Tissue was fixed in methacarn (methanol:chloroform:acetic acid in a 6:3:1 v/v/v) or buffered forma-
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
5
lin for 16 h at 4 °C. Tissue was subsequently dehydrated through graded ethanol and xylene solutions and embedded in paraffin. Microtome sections 6 mm thick were prepared and placed on silane-coated slides (Sigma, St. Louis, MO).
2.2. Immunocytochemistry Immunocytochemistry with an anti-chromodomain region of the MORF4 family of proteins peptide antibody (Bertram et al., 1999) was performed essentially as previously described (Sternberger, 1986; Smith et al., 1994). Briefly, following immersion in xylene and hydration through graded ethanol solutions, endogenous peroxidase activity was eliminated by incubation of the sections of 3% hydrogen peroxidase for 30 min. To reduce non-specific binding, sections were incubated for 30 min at room temperature in 10% normal goat serum (NGS) in Tris-buffered saline (TBS; 50 mM Tris– HCl, 150 mM NaCl, pH 7.6). After rinsing briefly with 1% NGS, sections were incubated overnight in either MORF4 antisera (1/100) or a mouse monoclonal antibody, AT8, to altered cytoskeletal t protein (Smith et al., 1994). The sections were then incubated in either goat anti-rabbit or goat antimouse antisera (ICN Biomedicals, Costa Mesa, CA), followed by species-specific peroxidase anti-peroxidase complex (Sternberger Monoclonals Inc., Lutherville, MD and ICN Biomedicals, Costa Mesa, CA). Antibodies were localized using 3,3 diaminobenzidine (DAB) as a chromogen (DAKO Corporation, Carpinteria, CA). To ensure the specificity of the reactivity observed, immunostaining was also performed in parallel using antibody that had been pre-incubated with the purified immunizing peptide (50 mg/ml) (Bertram et al., 1999).
2.3. Immunoblot analysis Tissue samples were solubilized in RIPA buffer, protein concentration determined and 20 mg protein loaded on SDS PAGE gels. The proteins were transferred to membranes and probed with 1:1000 of our primary antibody and 1:2000 secondary rabbit polyclonal antibody and visualized with ECL according to the manufacturer’s instructions (Santa Cruz, Santa Cruz, CA).
3. Results The antisera made against the MORF4 family chromodomain region specifically labels large pyramidal neurons in AD cases, but not age-matched controls (Fig. 1). There was significant overlap of the immunostaining profiles of MORF4-related protein and phosphorylated t in neurofibrillary pathology assessed with the antibody AT8, which recognizes t protein only when serine 202 and threonine 205 are phosphorylated. In marked contrast, age-matched normal controls did not show any immunoreactivity. The specificity of this select immunolocalization of MORF4related protein was confirmed by the almost complete abolition of immunostaining using antibody pre-incubated with the immunizing peptide.
6
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
Fig. 1. Antisera to the chromodomain region of MORF4-related proteins specifically recognize intraneuronal neurofibrillary pathology in cases of AD (A), whereas, conversely, there is little specific localization in control cases.
Immunoblot analyses of homogenates of AD and control cortex reveals a single 52 kDa band in 3/3 AD cases and 1/3 control cases recognized by the antibody to MORF4 (Fig. 2). We determined that these bands were specifically eliminated in immunoblot analyses performed with the antibody pre-incubated with the peptide used to obtain the antiserum, indicating that the antisera is specific to the chromodomain region of this family of proteins. Recently, two chromodomain proteins with molecular weights corresponding to about 52 kDa and with homology to the peptide region against which our antibody was raised (Fig. 3) were cloned, namely MOF (Neal et al., 2000; Eisen et al., 2001) and an alternatively spliced form of MSL3L1 (Prakash et al., 1999). MOF is a histone acetyl transferase (HAT) and MSL3L1 was cloned during studies to identify the microopthalmia gene.
Fig. 2. Western analysis of AD (lanes 1 –3) and normal samples (lanes 4 – 6). HeLa cells transfected with MRG 15 driven by the CMV promoter demonstrate the position of the MRG 15 protein at 37 kDa (lanes 7, 8) and the cross reactivity of the antibody with a protein about 52 kDa (lane 8).
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
7
Fig. 3. Amino acid alignment of the chromodomain region of human MRG15, MSL3L1 and MOF. The chromodomain regions of MRG15 (GeneBank Accession Number AF100615), the two splicing variants of MSL3L1 (GeneBank Accession Number AF117065) and MOF proteins were aligned using Clustal analysis. The sequence of the peptide used to obtain the antibody is underlined. Identical residues are shown in bold and similar residues in italics.
4. Discussion Primary neurons in the normal adult brain are generally viewed as being quiescent. However, multiple lines of evidence implicate emergence from this quiescent state, and subsequent attempted re-entry into the cell division cycle, as a proximal pathophysiology in AD (reviewed in Raina et al., 2000). While, thus far, there is no evidence that successful nuclear division occurs in these neurons, proteins associated with exit from the cell cycle are apparently upregulated (Arendt et al., 1998; McShea et al., 1999a,b). These include members of the cell cycle engine, namely CDKs, along with their cognate cyclins in vulnerable neurons (reviewed in Raina et al. 1999a, 2000) and indeed, these neurons in AD are morphologically similar to cells that are cycling, rather than terminally differentiated non-dividing cells (Raina et al., 2000). In such a scenario, one would predict that novel markers, whose presence is obligatory in the G1 phase of the cell cycle, would also be seen in the vulnerable neurons of AD. In this study, we found that a 52 kDa protein MORF4-related cell cycle protein is associated with intraneuronal neurofibrillary pathology in select neuronal population that are vulnerable in AD, while all age matched controls have no immunostaining. Immunoblot analysis confirmed these findings although one of three control individuals did express detectable levels of the protein, which may be an indicator of pathology that might arise in the future. Two such proteins, 52 kD in size, have recently been cloned. One gene, MOF, encodes a HAT and the other being MSL3L1, which was cloned during studies localizing the microopthalmia gene. These, together with MRG15, all encode chromodomain regions. Chromodomains are known to be involved in protein–
8
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
protein interactions and form complexes that remodel chromatin, making transcription factors accessible to gene promoters. Such complexes often contain HATs. The best-studied chromodomain protein is Drosophila msl3 (male specific lethal) that is involved in dosage compensation and up-regulation of transcription on the X chromosome in males. The chromodomain regions of msl3, MRG15, MOF, and MSL3L1 are very similar and the prediction is that these proteins will be present in transcriptional activating complexes that most likely will include a HAT activity. The presence of a positive transcriptional controlling protein in association with intraneuronal neurofibrillary pathology in vulnerable neurons of AD is consistent with the many changes in gene expression that occur and adds credence to the role of proximal mitotic mechanisms in AD. This return to mitotic competence in AD can have profound implications for both screening and therapeutics to modify the nature history of this disease.
Acknowledgements This work was supported by grants from the National Institutes of Health (R01 NS38648 to M.A.S. and R37 AG05333 to O.M.P.S.) and the Alzheimer’s Association (Stephanie B. Overstreet Scholars, IIRG-00-2163, to M.A.S.).
References Arendt, T., Holzer, M., Grossmann, A., Zedlick, D., Bruckner, M.K., 1995. Increased expression and subcellular translocation of the mitogen activated protein kinase and mitogen-activated protein kinase in Alzheimer’s disease. Neuroscience 68, 5 – 18. Arendt, T., Rodel, L., Gartner, U., Holzer, M., 1996. Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer’s disease. Neuroreport 7, 3047 – 3049. Arendt, T., Holzer, M., Gartner, U., 1998. Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer’s disease. J. Neural Transm. 105, 949 – 960. Bertram, M.J., Berube, N.G., Hang-Swanson, X., Ran, Q., Leung, J.K., Bryce, S., Spurgers, K., Bick, R.J., Baldini, A., Ning, Y., Clark, L.J., Parkinson, E.K., Barrett, J.C., Smith, J.R., Pereira-Smith, O.M., 1999. Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol. Cell. Biol. 19, 1479 – 1485. Busser, J., Geldmacher, D.S., Herrup, K., 1998. Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer’s disease brain. J. Neurosci. 18, 2801 – 2807. Eisen, A., Utley, R.T., Nourani, A., Allard, S., Schmidt, P., Lane, W.S., Lucchesi, J.C., Coˆ te´ , J., 2001. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation. J. Biol. Chem. 276, 3484 – 3491. Elledge, S.J., 1996. Cell cycle checkpoints: preventing an identity crisis. Science 274, 1664 – 1672. Gerst, J.L., Raina, A.K., Pirim, I., McShea, A., Harris, P.L.R., Siedlak, S.L., Takeda, A., Petersen, R.B., Smith, M.A., 2000. Altered cell-matrix associated ADAM in Alzheimer disease. J. Neurosci. Res. 59, 680 –684. Khachaturian, Z.S., 1985. Diagnosis of Alzheimer’s disease. Arch. Neurol. 42, 1097 – 1105. McShea, A., Harris, P.L.R., Webster, K.R., Wahl, A.F., Smith, M.A., 1997. Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer’s disease. Am. J. Pathol. 150, 1933 – 1939. McShea, A., Wahl, A.F., Smith, M.A., 1999a. Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med. Hypotheses 52, 525 – 527.
A.K. Raina et al. / Mechanisms of Ageing and De6elopment 123 (2001) 3–9
9
McShea, A., Zelasko, D.A., Gerst, J.L., Smith, M.A., 1999b. Signal transduction abnormalities in Alzheimer’s disease: evidence of a pathogenic stimuli. Brain Res. 815, 237 – 242. Mirra, S.S., Heyman, A., McKeel, D., Sumi, S.M., Crain, B.J., Brownlee, L.M., Vogel, F.S., Hughes, J.P., van, B.G., Berg, L., 1991. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology 41, 479 – 486. Nagy, Z., Esiri, M.M., Smith, A.D., 1998. The cell division cycle and the pathophysiology of Alzheimer’s disease. Neuroscience 87, 731 – 739. Neal, K.C., Pannuti, A., Smith, E.R., Lucchesi, J.C., 2000. A new human member of the MYST family of histone acetyl transferases with high sequence similarity to Drosophila MOF. Biochim. Biophys. Acta 1490, 170 –174. Padmanabhan, J., Park, D.S., Greene, L.A., Shelanski, M.L., 1999. Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis. J. Neurosci. 19, 8747 – 8756. Park, D.S., Levine, B., Ferrari, G., Greene, L.A., 1997. Cyclin dependent kinase inhibitors and dominant negative cyclin dependent kinase 4 and 6 promote survival of NGF-deprived sympathetic neurons. J. Neurosci. 17, 8975 –8983. Park, D.S., Morris, E.J., Padmanabhan, J., Shelanski, M.L., Geller, H.M., Greene, L.A., 1998. Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents. J. Cell Biol. 143, 457 –467. Park, D.S., Obeidat, A., Giovanni, A., Greene, L.A., 2000. Cell cycle regulators in neuronal death evoked by excitotoxic stress: implications for neurodegeneration and its treatment. Neurobiol. Aging 21, 771 – 781. Prakash, S.K., Van den Veyver, I.B., Franco, B., Volta, M., Ballabio, A., Zoghbi, H.Y., 1999. Characterization of a novel chromo domain gene in Xp22.3 with homology to Drosophila msl-3. Genomics 59, 77 –84. Raina, A.K., Monteiro, M.J., McShea, A., Smith, M.A., 1999a. The role of cell cycle-mediated events in Alzheimer’s disease. Int. J. Exp. Pathol. 80, 71 – 76. Raina, A.K., Takeda, A., Smith, M.A., 1999b. Mitotic neurons: a dogma succumbs. Exp. Neurol. 159, 248–249. Raina, A.K., Zhu, X., Rottkamp, C.A., Monteiro, M., Takeda, A., Smith, M.A., 2000. Cyclin’ toward dementia: cell cycle abnormalities and abortive oncogenesis in Alzheimer disease. J. Neurosci. Res. 61, 128 –133. Smith, M.A., Taneda, S., Richey, P.L., Miyata, S., Yan, S.-D., Stern, D., Sayre, L.M., Monnier, V.M., Perry, G., 1994. Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc. Natl. Acad. Sci. USA 91, 5710 – 5714. Smith, M.A., 1998. Alzheimer disease. In: Bradley, R.J., Harris, R.A. (Eds.), International Review of Neurobiology, Vol. 42, Academic Press, Inc., San Diego, pp. 1 – 54. Stefanis, L., Park, D.S., Yan, C.Y., Farinelli, S.E., Troy, C.M., Shelanski, M.L., Greene, L.A., 1996. Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271, 30663 –30671. Sternberger, L.A., 1986. Immunocytochemistry, 3rd ed. Wiley, New York. Vincent, I., Rosado, M., Davies, P., 1996. Mitotic mechanisms in Alzheimer’s disease? J. Cell Biol. 132, 413–425. Zhu, X., Raina, A.K., Smith, M.A., 1999. Cell cycle events in neurons: proliferation or death? Am. J. Pathol. 155, 327 –329. Zhu, X., Raina, A.K., Boux, H., Simmons, Z.L., Takeda, A., Smith, M.A., 2000. Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int. J. Dev. Neurosci. 18, 433–437.