- Email: [email protected]
Developmental expression of Bcl-2 protein in human cortex1
Developmental Brain Research 119 Ž2000. 225–230 www.elsevier.comrlocaterbres
Research report
Developmental expression of Bcl-2 protein in human cortex L. Fredrik Jarskog b
a,b,)
, John H. Gilmore
1
a,b
a Department of Psychiatry, C.B. a7160, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA UNC Mental Health and Neuroscience Clinical Research Center, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
Accepted 10 November 1999
Abstract Apoptosis is essential for normal human neurodevelopment and is increasingly recognized for its role in various neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Bcl-2 is a 26 kDa membrane-associated protein known to protect neurons against apoptosis. Interestingly, Bcl-2 protein levels are altered in certain neurodegenerative disorders that reveal increased apoptosis. However, little is known about the normal expression of Bcl-2 protein in human brain. Bcl-2 protein levels were determined by ELISA and semiquantitative Western Blotting in the frontal cortex of 20 human post-mortem brains, separated into three groups: six infants Žage: 0.83 " 1.0 years, mean " S.D.., five adolescents Žage: 17.4 " 1.7 years., and nine adults Žage: 41.0 " 9.6 years.. All subjects died of non-CNS related illness and had no history of psychiatric illness. Bcl-2 increased significantly across the age groups in the ELISA Ž p s 0.0058. and the Western Blot Ž p s 0.002. experiments. The ELISA demonstrated significant differences in Bcl-2 levels between infant and adolescent cortex Ž p - 0.05., and between infant and adult cortex Ž p - 0.01. using a post-hoc Tukey’s multiple comparison test. The Western blots demonstrated a similar significant increase in Bcl-2 between infant and adult cortex Ž p - 0.01.. A secondary analysis showed significant correlation between individual ages and Bcl-2 levels Ž r 2 s 0.4933, p s 0.0006.. This study demonstrates that Bcl-2 protein expression in human cortex is developmentally regulated and supports the hypothesis that Bcl-2 is involved in normal aging. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Bcl-2; Apoptosis; Neurodevelopment; Neurodegeneration; Aging
1. Introduction Apoptosis, a form of programmed cell death, is vital for normal human neurodevelopment and is also recognized for its role in various neurodegenerative disorders. Members of the Bcl-2 protein family are known to be important regulators of apoptosis Žreviewed in w2x.. In this family is Bcl-2, a 26 kDa membrane-bound protein that inhibits apoptosis. The bcl-2 gene was originally discovered as a proto-oncogene associated with the tŽ14;18. translocation of human B cell lymphoma w24x. Subsequent investigations found that bcl-2 overexpression make cells highly resistant to cell death by pro-apoptotic stimuli w9,28x. Much research has since focused on the anti-apoptotic role of Bcl-2, but functionally its role may have greater scope
)
Corresponding author. Department of Psychiatry, C.B. a7160, University of North Carolina, Chapel Hill, NC 27599-7160, USA. Fax: q1-919-966-5628; e-mail: [email protected] 1 Presented at the 37th annual meeting of the American College of Neuropsychopharmacology in Las Croabas, Puerto Rico on December 14, 1998.
given a recent report that Bcl-2 promotes regeneration of severed central nervous system ŽCNS. neurons w5x. Bcl-2 protein is found in the outer mitochondrial membrane where it has pore-forming capacity w20x, a property shared with other Bcl-2 proteins including pro-apoptotic Bax and anti-apoptotic Bcl-x L . Investigators have speculated that, in part, Bcl-2 protein exerts its anti-apoptotic function through homodimerization and heterodimerization with other Bcl-2 family proteins to regulate the passage of oxidative mediators Že.g., cytochrome c . through the mitochondrial membrane Žreviewed in w8x.. Animal experiments show that Bcl-2 expression is developmentally regulated. Bcl-2 mRNA and protein expression in murine and rat brain is highest during the embryonic period and tapers postnatally w1,3,6,11,16,21x. However, the developmental expression of Bcl-2 in human brain has not been systematically investigated. Immunohistochemically, Bcl-2 protein appears to decrease from early to late gestation w4x, but it remains present in childhood frontal cortex w16x and in aged adult populations in studies of neurodegenerative disorders w10,17,19,22x. Since little is known about Bcl-2 expression in postnatal human brain
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 7 6 - 5
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
226
development, Bcl-2 protein levels were determined in frontal cortex of infant, adolescent, and adult groups. It was hypothesized that Bcl-2 in human cortex would demonstrate developmental regulation across the life span.
2. Materials and methods 2.1. Human tissue Uniform sections of left prefrontal cortex ŽBrodmann’s areas 9r46. of 20 individuals were obtained at autopsy under authority of the State Medical Examiner of North Carolina and frozen at y808C. All subjects died of nonCNS-related illness and had no known history of psychiatric illness or substance abuse. The subjects ranged in age from 9 weeks to 59 years. The brain samples were divided a priori into infant, adolescent, and adult groups. The groups comprised six infants, five adolescents, and nine adults Žsee Table 1..
Fig. 1. Bcl-2 protein standard curve by ELISA. Bcl-2 protein concentration ŽUrml. vs. optical density absorbance ŽAbs. units at 450 nm, in triplicate Žpresented as mean"S.D... Quadratic curve fit correlation coefficient was 0.999.
the plate was washed and TMBrperoxide was added for color development. The reaction was stopped with sulfuric acid and the optical density was measured at 450 nm using a microplate reader ŽVmax, Molecular Devices, Sunnyvale, CA.. A Bcl-2 standard curve was generated to quantitate the amount of Bcl-2 in Unitsrml Žsee Fig. 1.. Intra-assay coefficient of variance was - 8% for all measurements.
2.2. Tissue homogenization and sonication The tissue Ž100–200 mg. was placed in 10 volumes of 50 mM Tris–HCl buffer ŽpH 7.4. with 0.6 M NaCl, 0.2% Triton X-100, 1% BSA, 1 mM benzamidine, 0.1 mM benzethonium chloride, and 0.1 mM PMSF. Samples were homogenized ŽPowerGen 125, Fisher Scientific, Pittsburgh, PA. on ice for 30 s and sonicated ŽSonic Dismembrator 60, Fisher Scientific, Pittsburgh, PA. for 10 s at 10 mV. Samples were then centrifuged for 15 min at 15,000 rpm and 48C. All chemicals were obtained from Sigma ŽSt. Louis, MO..
2.4. SemiquantitatiÕe Bcl-2 Western blot Homogenized and sonicated samples were assayed for total protein concentration using the BCA method ŽPierce., then separated on 12% Tris-glycine polyacrylamide gels using a mini-cell electrophoresis unit ŽXcell II, NOVEX, San Diego, CA.. Equal amounts of protein Ž120 mg. were boiled for 5 min in Tris-glycine SDS sample buffer ŽNOVEX. and applied to the gels. Gels were run with a low-range molecular weight ladder ŽRainbow MW Marker, Amersham Pharmacia. and a Jurkat cell lysate ŽTransduction Laboratories, Lexington, KY. for Bcl-2 control. Separated proteins were transferred to polyvinylidene difluoride ŽPVDF. membranes ŽImmobilon-P, Millipore, MA. at 25 V for 2 h, and complete transfer was ascertained by staining duplicate gels with Coomassie Blue and membranes with Ponceau S Ždata not shown.. Nonspecific protein binding was blocked for 1 h with 5% blocking reagent ŽECL, Amersham Pharmacia. in 0.1% Tween TBS ŽTBST.. Membranes were incubated for 1 h at 258C with a mouse monoclonal anti-human Bcl-2 primary antibody
2.3. Bcl-2 enzyme-linked immunoassay (ELISA) Samples were assayed for Bcl-2 using a commercially available ELISA kit ŽEndogen, Woburn, MA.. Briefly, a 96-well microplate was precoated with a mouse monoclonal anti-human Bcl-2 antibody ŽAb. ŽEndogen. and samples Ž50 ml. were diluted 1:1 Žvrv. with fluorescein isothiocyanate ŽFITC.-labeled secondary Ab and applied in triplicate. The plate was incubated for 2 h at room temperature, washed, then all wells received horseradish peroxidase-labeled anti-FITC Ab. Following 30-min incubation, Table 1 Demographic data of human frontal cortex specimens Age and post-mortem interval are presented as mean" standard deviation.
Infants Adolescents Adults a
N
Gender
Ethnicity
Male
Female
Caucasian
African American
6 5 9
2 4 7
4 1 2
4 2 8
2 3 1
Post-mortem intervals across groups were not significantly different by ANOVA Ž p s 0.3087..
Age Žyears.
Post-mortem interval Žh. a
0.83 " 1.0 17.4 " 1.7 41.0 " 9.6
19.7 " 9.6 14.8 " 4.3 16.3 " 7.0
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
227
Fig. 2. Bcl-2 levels in frontal cortex across age groups by ELISA. Bcl-2 protein levels are expressed in Unitsrgram tissue weight in human frontal cortex of infant, adolescent, and adult age groups. Mean"S.E.M. was 989.3"215.9 Urg in infants Ž ns6., 1833"98.5 Urg in adolescents Ž ns 5., and 2023"212.4 Urg in adults Ž ns9.. Bcl-2 levels increased significantly across the age groups by ANOVA Ž ps 0.0058.. Both adolescent ŽU p- 0.05. and adult ŽUU p- 0.01. groups were significantly higher than the infant group.
Fig. 4. Bcl-2 levels in frontal cortex across age groups by semi-quantitative Western blotting. Bcl-2 bands were measured using optical densitometry and data was normalized to a control adult sample Ždefined as 1.00.. Normalized mean optical densities ŽOD. were 0.55"0.04 Žmean"S.E.M.. in infants, 0.82"0.07 in adolescents, and 0.97"0.08 in adults. Bcl-2 expression increased significantly across age groups by ANOVA Ž ps 0.002.. The adult group was significantly higher than the infant group ŽU p- 0.01. in post-hoc testing.
Ž1:500, Transduction Laboratories. followed by 1-h incubation with a secondary sheep anti-mouse HRP-labeled Ab Ž1:1000, ECL, Amersham Pharmacia.. Membranes were developed using chemiluminescence ŽECL, Amersham Pharmacia., and the protein bands were detected on radiographic film ŽHyperfilm ECL, Amersham Pharmacia. after 20–30 s exposure. Optical densitometry of Bcl-2 bands was performed using the NIH Image system. Band densities were normalized to a control adult sample run on all gels.
medial frontal cortex was dissected and frozen. The tissue was homogenized and sonicated as described in Section 2.2, and semiquantitative Bcl-2 Western blotting was performed as described in Section 2.5. The primary Ab in the Western protocol recognized Bcl-2 protein from rat and human origin.
2.5. Bcl-2 post-mortem stability study 150–200 g Sprague–Dawley rats Ž n s 8, equal sex ratio; Zivic-Miller, Allison Park, PA. were anaesthetized with ether and decapitated. Medial frontal cortex from four rats was dissected and immediately frozen at y808C. Then, to approximate the post-mortem condition of human brain tissue, the heads of four rats were left at room temperature for 6 h and then at 48C for 18 h, at which time
2.6. Statistics ELISA and Western blot data was analyzed by one-way analysis of variance ŽANOVA. across age groups and a post-hoc analysis was performed using a Tukey’s Multiple Comparison Test, with two-tailed p-values considered significant at 0.05. A secondary analysis determined the linear correlation coefficient between age and Bcl-2 expression. The post-mortem stability data was analyzed using an unpaired Student’s t-test, with two-tailed p-values considered significant at 0.05.
3. Results ELISA results demonstrated that Bcl-2 levels in human frontal cortex significantly increased across the three age groups by ANOVA Ž F s 7.081, df s 19, p s 0.0058; see
Fig. 3. Linear regression analysis of Bcl-2 in all subjects vs. age. Bcl-2 protein levels measured by ELISA Žvalues in Unitsrgram tissue; gender: male Žv . female Ž\.. correlated significantly with age Ž r 2 s 0.4933, ps 0.0006..
Fig. 5. Western blot of Bcl-2 expression in human frontal cortex. An equal amount of total protein was added to each lane. The transfer membrane was incubated with a monoclonal anti-human Bcl-2 Ab. The bands shown migrated to 26 kDa. Lane 1 contained Jurkat cell lysate for Bcl-2 control. Lanes 2–8 contained a subset of the brain samples as follows: 2, Adult; 3, Infant; 4, Infant; 5, Adult; 6, Infant; 7, Adult; 8, Adolescent.
228
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
Fig. 6. Bcl-2 post-mortem stability study. Bcl-2 expression was examined in rat cortex subjected to a 24 h post-mortem interval Ž6 h at 258C then 18 h at 48C. and compared to tissue processed immediately after death Ž0 h.. Semiquantitative Western blotting using optical densitometry revealed a nonsignificant 6% reduction in Bcl-2 expression following a 24 h postmortem interval.
Fig. 2.. Mean levels " S.E.M. were 989.3 " 215.9 Urg in infants, 1833 " 98.5 Urg in adolescents, and 2023 " 212.4 Urg in adults. Both adolescent Ž p - 0.05. and adult Ž p 0.01. groups had significantly higher Bcl-2 levels compared to infant levels. Although adult levels were higher than adolescent levels, this difference was not significant. A secondary analysis revealed a significant linear correlation between individual ages and Bcl-2 levels Ž r 2 s 0.4933, p s 0.0006; see Fig. 3.. No significant differences emerged in post-mortem intervals across the three age groups by ANOVA Ž F s 0.6630, df s 19, p s 0.3087.. Semiquantitative Western blots confirmed the ELISA results by demonstrating a significant increase in Bcl-2 protein levels from infancy to adolescence and adulthood by ANOVA Ž F s 9.390, df s 18, p s 0.002, see Fig. 4.. Bcl-2 was identified in each sample and the difference in band densities between infant and adult samples was readily visualized ŽFig. 5.. Normalized mean optical densities showed Bcl-2 expression was 50% higher in adolescents and 77% higher in adults compared to infant Bcl-2 expression. Only adult Bcl-2 expression was significantly higher than infant levels Ž p - 0.01. in post-hoc analysis. The band at 26 kDa was further confirmed as Bcl-2 by the co-migration of a 26 kDa band from a Jurkat cell lysate run on each gel ŽFig. 5.. Western blots from the post-mortem stability study of Bcl-2 protein demonstrated no significant degradation over 24 h Žsee Fig. 6.. Mean Bcl-2 expression in tissue processed after a 24 h post-mortem interval was nonsignificantly reduced by 6% Ž F s 1.141, df s 6, p s 0.458..
4. Discussion This is the first study to quantitatively measure the developmental expression of Bcl-2 protein in human cortex across the postnatal life span. Bcl-2 protein demonstrates developmental regulation in frontal cortex, increas-
ing from early infancy to adolescence and into adulthood. The quantitative ELISA results are corroborated by similar results using semiquantitative Western blots. Interestingly, our findings demonstrate a different pattern of Bcl-2 expression in human cortex from that seen in rodent cortex. Most animal studies have concluded that the developmental expression of Bcl-2 in the CNS declines with aging w3,6,16,21x. Those studies compared Bcl-2 expression in fetal, postnatal, and adult rodent brain tissue using immunohistochemistry, in situ hybridization, and immunoblotting. Our results demonstrate that postnatal Bcl-2 expression in human cortex increases with age, countering previous findings from animal studies. This also highlights the importance of conducting developmental studies in human brain tissue since human neurodevelopment can differ from animal models. To our knowledge, only two reports have addressed the developmental expression of Bcl-2 protein in human brain. Using fetal tissue, one report demonstrated that human brain Bcl-2 expression is higher in early gestation and downregulated by the end of gestation w4x. Yachnis et al. w27x found a similar downregulation of Bcl-2 protein from early to late gestation in several CNS areas, and Bcl-2 levels were also low in temporal cortex from two adults. Taken together, the current and prior studies suggest that Bcl-2 protein expression in human cortex follows a biphasic pattern that is high in early gestation, followed by perinatal downregulation, and then a gradual upregulation across the life span. In human fetal cortex, Chan and Yew w4x reported that increased neuronal apoptosis coincides with downregulation of Bcl-2 protein expression across gestation. Since Bcl-2 is an anti-apoptotic protein, they speculated that Bcl-2 levels may require downregulation in order to accommodate the massive developmental surge in apoptosis during later gestation. However, it is also known that the rate of apoptosis is substantially reduced perinatally, since studies of postnatal fetal cortex have failed to detect evidence of apoptosis in normal infants as young as 3 weeks of age w7,23x. The specific role of Bcl-2 protein in regulating developmental apoptosis remains unclear, but given the multitude of interacting pro- and anti-apoptotic proteins that are expressed, a complex regulatory mechanism dependent on temporal and regional factors is likely to exist. Although speculative, our finding of lower Bcl-2 levels during infancy may reveal a window during normal development when the CNS is more vulnerable to proapoptotic insults. Some neurodevelopmental disorders, including schizophrenia and autism, have been hypothesized to involve alterations in apoptosis in response to perinatal stressors such as infection or hypoxia w13,26x. Given the established role of Bcl-2 as an anti-apoptotic regulator in early development, our findings are suggestive that Bcl-2 protein may continue to exert such a function throughout life, with important implications for normal aging. A neuroprotective role has already been proposed
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
for Bcl-2 by virtue of its anti-apoptotic properties w6,16,21,25x, and Castren ´ et al. w3x have speculated that basal levels of bcl-2 mRNA are necessary for the survival of postmitotic neurons. In studies of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, Bcl-2 is present and often upregulated in certain brain regions w10,14,17,19x Žreviewed in Ref. w12x.. The upregulation may represent a compensatory response to the neurodegenerative process. Similarly, normal aging has been associated with free radical-induced oxidative damage, as evidenced by higher oxidized protein levels, lipofuscin, DNA damage, and glycosylated proteins w15,18x. Such chronic oxidative stress may contribute to higher Bcl-2 protein levels in adult cortex in order to maintain neuronal viability. Our result documenting the highest postnatal Bcl-2 level in adulthood is consistent with a role for Bcl-2 protein in normal aging. Finally, to assess the potential confounding effect of post-mortem interval on Bcl-2 protein, we used an animal paradigm designed to approximate the human post-mortem condition. Bcl-2 demonstrated high post-mortem stability for 24 h. Since the mean post-mortem intervals in our three human age groups were all less than 20 h and did not differ significantly, it suggests that our results were not confounded by post-mortem degradation of Bcl-2 protein. In conclusion, Bcl-2 protein demonstrates developmental regulation in human cortex. The current study adds to the accumulating evidence that Bcl-2 continues to have a significant role in adult CNS by virtue of its upregulation from infancy to adulthood. Given the complexity of CNS function, a neuroprotective role for Bcl-2 in normal aging would likely occur in concert with a variety other pro- and anti-apoptotic proteins and other processes affecting neuronal survival. Further studies in human brain tissue that characterize age-dependent expression of other Bcl-2 family proteins during normal development may help elucidate this mechanism. Ultimately, defining the role of Bcl-2 family proteins in the development and maintenance of the human CNS will provide a better understanding of neurodevelopmental and neurodegenerative disorders.
Acknowledgements The authors are indebted to Elzbieta S. Selinger, MD, MSc for her excellent technical assistance. This work was supported by the Foundation of Hope ŽRaleigh, NC. and by Center Grant MH-33127.
References w1x S. Abe-Dohmae, N. Harada, K. Yamada, R. Tanaka, bcl-2 gene is highly expressed during neurogenesis in the central nervous system, Biochem. Biophys. Res. Commun. 191 Ž1993. 915–921.
229
w2x J.M. Adams, S. Cory, The Bcl-2 protein family: arbiters of cell survival, Science 281 Ž1998. 1322–1326. w3x E. Castren, ´ Y. Ohga, M.P. Berzaghi, G. Tzimagiorgis, H. Thonen, D. Lindholm, bcl-2 messenger RNA is localized in neurons of the developing and adult rat brain, Neuroscience 61 Ž1994. 165–177. w4x W.Y. Chan, D.T. Yew, Apoptosis and Bcl-2 oncoprotein expression in the human fetal central nervous system, Anat. Rec. 252 Ž1998. 165–175. w5x D.F. Chen, G.E. Schneider, J.C. Martinou, S. Tonegawa, Bcl-2 promotes regeneration of severed axons in mammalian CNS, Nature 385 Ž1997. 434–439. w6x I. Ferrer, A. Tortosa, E. Condom, R. Blanco, A. Macaya, A. Planas, Increased expression of bcl-2 immunoreactivity in the developing cerebral cortex of the rat, Neurosci. Lett. 179 Ž1994. 13–16. w7x H.A. Gelbard, H.J. James, L.R. Sharer, S.W. Perry, Y. Saito, A.M. Kazee, B.M. Blumberg, L.G. Epstein, Apoptotic neurons in brains from paediatric patients with HIV-1 encephalitis and progressive encephalopathy, Neuropathol. Appl. Neurobiol. 21 Ž1995. 208–217. w8x D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 Ž1998. 1309–1312. w9x D. Hockenberry, G. Nunez, ˜ C. Milliman, R.D. Schreiber, S.J. Korsmeyer, Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death, Nature 348 Ž1990. 334–336. w10x Y. Kitamura, S. Shimohama, W. Kamoshima, T. Ota, Y. Matsuoka, Y. Nomura, M.A. Smith, G. Perry, P.J. Whitehouse, T. Taniguchi, Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer’s disease, Brain Res. 780 Ž1998. 260–269. w11x D.P. LeBrun, R.A. Warnke, M.L. Cleary, Expression of bcl-2 in fetal tissues suggests a role in morphogenesis, Am. J. Pathol. 142 Ž1993. 743–753. w12x M. Leist, P. Nicotera, Apoptosis, excitotoxicity, and neuropathology, Exp. Cell Res. 239 Ž1998. 183–201. w13x R.L. Margolis, D.M. Chuang, R.M. Post, Programmed cell death: implications for neuropsychiatric disorders, Biol. Psychiatry 35 Ž1994. 946–956. w14x K.-A. Marshall, S.E. Daniel, N. Cairns, P. Jenner, B. Halliwell, Upregulation of the anti-apoptotic protein Bcl-2 may be an early event in neurodegeneration: studies on Parkinson’s and incidental lewy body disease, Biochem. Biophys. Res. Commun. 240 Ž1997. 84–87. w15x P. Mecocci, M.F. Beal, R. Cecchetti, M.C. Polidori, A. Cherubini, F. Chionne, L. Avellini, G. Romano, U. Senin, Mitochondrial membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain, Mol. Chem. Neuropathol. 31 Ž1997. 53–64. w16x D.E. Merry, D.J. Veis, W.F. Hickey, S.J. Korsmeyer, Bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS, Development 120 Ž1994. 301–311. w17x M. Mogi, M. Harada, T. Kondo, Y. Mizuno, H. Narabayashi, P. Riederer, T. Nagatsu, Bcl-2 protein is increased in the brain from Parkinsonian patients, Neurosci. Lett. 215 Ž1996. 137–139. w18x R.E. Mrak, W.S.T. Griffin, D.I. Graham, Aging-associated changes in human brain, J. Neuropathol. Exp. Neurol. 56 Ž1997. 1269–1275. w19x T. Satou, B.J. Cummings, C.W. Cotman, Immunoreactivity for Bcl-2 protein within neurons in the Alzheimer’s disease brain increases with disease severity, Brain Res. 697 Ž1995. 35–43. w20x S.L. Schendel, Z. Xie, M.O. Montal, S. Matsuyama, M. Montal, J.C. Reed, Channel formation by anti-apoptotic protein bcl-2, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 5113–5118. w21x S. Shimohama, S. Fujimoto, Y. Sumida, H. Tanino, Differential expression of rat brain Bcl-2 family proteins in development and aging, Biochem. Biophys. Res. Commun. 252 Ž1998. 92–96. w22x J.H. Su, G. Deng, C.W. Cotman, Bax protein expression is increased in Alzheimer’s brain: correlations with DNA damage, Bcl-2 expression, and brain pathology, J. Neuropathol. Exp. Neurol. 56 Ž1997. 86–93. w23x J.C. Tronosco, R.R. Sukhov, C.H. Kawas, V.E. Koliatsos, In situ
230
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
labeling of dying cortical neurons in normal aging and in Alzheimer’s disease: correlations with senile plaques and disease progression, J. Neuropathol. Exp. Neurol. 55 Ž1996. 1134–1142. w24x Y. Tsujimoto, L.R. Finger, J. Yunis, P.C. Nowell, C.M. Croce, Cloning of the chromosome breakpoint of neoplastic B cells with the tŽ14;18. chromosome translocation, Science 226 Ž1984. 1097–1099. w25x S. Vyas, F. Javoy-Agid, M.-T. Herrero, O. Strada, F. Boissiere, U. Hibner, Y. Agid, Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders, J. Neurochem. 69 Ž1997. 223–231. w26x B.T. Woods, Is schizophrenia a progressive neurodevelopmental
disorder? Toward a unitary pathogenetic mechanism, Am. J. Psychiatry 155 Ž1998. 1661–1670. w27x A.T. Yachnis, S.Z. Powell, J.J. Olmsted, T.A. Eskin, Distinct neurodevelopmental patterns of Bcl-2 and Bcl-x expression are altered in glioneuronal hamartias of the human temporal lobe, J. Neuropathol. Exp. Neurol. 56 Ž1997. 186–198. w28x L.-T. Zhong, T. Sarafian, D.J. Kane, A.C. Charles, S.P. Mah, R.H. Edwards, D.E. Bredesen, bcl-2 inhibits death of central neural cells induced by multiple agents, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 4533–4537.
Research report
Developmental expression of Bcl-2 protein in human cortex L. Fredrik Jarskog b
a,b,)
, John H. Gilmore
1
a,b
a Department of Psychiatry, C.B. a7160, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA UNC Mental Health and Neuroscience Clinical Research Center, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
Accepted 10 November 1999
Abstract Apoptosis is essential for normal human neurodevelopment and is increasingly recognized for its role in various neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Bcl-2 is a 26 kDa membrane-associated protein known to protect neurons against apoptosis. Interestingly, Bcl-2 protein levels are altered in certain neurodegenerative disorders that reveal increased apoptosis. However, little is known about the normal expression of Bcl-2 protein in human brain. Bcl-2 protein levels were determined by ELISA and semiquantitative Western Blotting in the frontal cortex of 20 human post-mortem brains, separated into three groups: six infants Žage: 0.83 " 1.0 years, mean " S.D.., five adolescents Žage: 17.4 " 1.7 years., and nine adults Žage: 41.0 " 9.6 years.. All subjects died of non-CNS related illness and had no history of psychiatric illness. Bcl-2 increased significantly across the age groups in the ELISA Ž p s 0.0058. and the Western Blot Ž p s 0.002. experiments. The ELISA demonstrated significant differences in Bcl-2 levels between infant and adolescent cortex Ž p - 0.05., and between infant and adult cortex Ž p - 0.01. using a post-hoc Tukey’s multiple comparison test. The Western blots demonstrated a similar significant increase in Bcl-2 between infant and adult cortex Ž p - 0.01.. A secondary analysis showed significant correlation between individual ages and Bcl-2 levels Ž r 2 s 0.4933, p s 0.0006.. This study demonstrates that Bcl-2 protein expression in human cortex is developmentally regulated and supports the hypothesis that Bcl-2 is involved in normal aging. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Bcl-2; Apoptosis; Neurodevelopment; Neurodegeneration; Aging
1. Introduction Apoptosis, a form of programmed cell death, is vital for normal human neurodevelopment and is also recognized for its role in various neurodegenerative disorders. Members of the Bcl-2 protein family are known to be important regulators of apoptosis Žreviewed in w2x.. In this family is Bcl-2, a 26 kDa membrane-bound protein that inhibits apoptosis. The bcl-2 gene was originally discovered as a proto-oncogene associated with the tŽ14;18. translocation of human B cell lymphoma w24x. Subsequent investigations found that bcl-2 overexpression make cells highly resistant to cell death by pro-apoptotic stimuli w9,28x. Much research has since focused on the anti-apoptotic role of Bcl-2, but functionally its role may have greater scope
)
Corresponding author. Department of Psychiatry, C.B. a7160, University of North Carolina, Chapel Hill, NC 27599-7160, USA. Fax: q1-919-966-5628; e-mail: [email protected] 1 Presented at the 37th annual meeting of the American College of Neuropsychopharmacology in Las Croabas, Puerto Rico on December 14, 1998.
given a recent report that Bcl-2 promotes regeneration of severed central nervous system ŽCNS. neurons w5x. Bcl-2 protein is found in the outer mitochondrial membrane where it has pore-forming capacity w20x, a property shared with other Bcl-2 proteins including pro-apoptotic Bax and anti-apoptotic Bcl-x L . Investigators have speculated that, in part, Bcl-2 protein exerts its anti-apoptotic function through homodimerization and heterodimerization with other Bcl-2 family proteins to regulate the passage of oxidative mediators Že.g., cytochrome c . through the mitochondrial membrane Žreviewed in w8x.. Animal experiments show that Bcl-2 expression is developmentally regulated. Bcl-2 mRNA and protein expression in murine and rat brain is highest during the embryonic period and tapers postnatally w1,3,6,11,16,21x. However, the developmental expression of Bcl-2 in human brain has not been systematically investigated. Immunohistochemically, Bcl-2 protein appears to decrease from early to late gestation w4x, but it remains present in childhood frontal cortex w16x and in aged adult populations in studies of neurodegenerative disorders w10,17,19,22x. Since little is known about Bcl-2 expression in postnatal human brain
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 7 6 - 5
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
226
development, Bcl-2 protein levels were determined in frontal cortex of infant, adolescent, and adult groups. It was hypothesized that Bcl-2 in human cortex would demonstrate developmental regulation across the life span.
2. Materials and methods 2.1. Human tissue Uniform sections of left prefrontal cortex ŽBrodmann’s areas 9r46. of 20 individuals were obtained at autopsy under authority of the State Medical Examiner of North Carolina and frozen at y808C. All subjects died of nonCNS-related illness and had no known history of psychiatric illness or substance abuse. The subjects ranged in age from 9 weeks to 59 years. The brain samples were divided a priori into infant, adolescent, and adult groups. The groups comprised six infants, five adolescents, and nine adults Žsee Table 1..
Fig. 1. Bcl-2 protein standard curve by ELISA. Bcl-2 protein concentration ŽUrml. vs. optical density absorbance ŽAbs. units at 450 nm, in triplicate Žpresented as mean"S.D... Quadratic curve fit correlation coefficient was 0.999.
the plate was washed and TMBrperoxide was added for color development. The reaction was stopped with sulfuric acid and the optical density was measured at 450 nm using a microplate reader ŽVmax, Molecular Devices, Sunnyvale, CA.. A Bcl-2 standard curve was generated to quantitate the amount of Bcl-2 in Unitsrml Žsee Fig. 1.. Intra-assay coefficient of variance was - 8% for all measurements.
2.2. Tissue homogenization and sonication The tissue Ž100–200 mg. was placed in 10 volumes of 50 mM Tris–HCl buffer ŽpH 7.4. with 0.6 M NaCl, 0.2% Triton X-100, 1% BSA, 1 mM benzamidine, 0.1 mM benzethonium chloride, and 0.1 mM PMSF. Samples were homogenized ŽPowerGen 125, Fisher Scientific, Pittsburgh, PA. on ice for 30 s and sonicated ŽSonic Dismembrator 60, Fisher Scientific, Pittsburgh, PA. for 10 s at 10 mV. Samples were then centrifuged for 15 min at 15,000 rpm and 48C. All chemicals were obtained from Sigma ŽSt. Louis, MO..
2.4. SemiquantitatiÕe Bcl-2 Western blot Homogenized and sonicated samples were assayed for total protein concentration using the BCA method ŽPierce., then separated on 12% Tris-glycine polyacrylamide gels using a mini-cell electrophoresis unit ŽXcell II, NOVEX, San Diego, CA.. Equal amounts of protein Ž120 mg. were boiled for 5 min in Tris-glycine SDS sample buffer ŽNOVEX. and applied to the gels. Gels were run with a low-range molecular weight ladder ŽRainbow MW Marker, Amersham Pharmacia. and a Jurkat cell lysate ŽTransduction Laboratories, Lexington, KY. for Bcl-2 control. Separated proteins were transferred to polyvinylidene difluoride ŽPVDF. membranes ŽImmobilon-P, Millipore, MA. at 25 V for 2 h, and complete transfer was ascertained by staining duplicate gels with Coomassie Blue and membranes with Ponceau S Ždata not shown.. Nonspecific protein binding was blocked for 1 h with 5% blocking reagent ŽECL, Amersham Pharmacia. in 0.1% Tween TBS ŽTBST.. Membranes were incubated for 1 h at 258C with a mouse monoclonal anti-human Bcl-2 primary antibody
2.3. Bcl-2 enzyme-linked immunoassay (ELISA) Samples were assayed for Bcl-2 using a commercially available ELISA kit ŽEndogen, Woburn, MA.. Briefly, a 96-well microplate was precoated with a mouse monoclonal anti-human Bcl-2 antibody ŽAb. ŽEndogen. and samples Ž50 ml. were diluted 1:1 Žvrv. with fluorescein isothiocyanate ŽFITC.-labeled secondary Ab and applied in triplicate. The plate was incubated for 2 h at room temperature, washed, then all wells received horseradish peroxidase-labeled anti-FITC Ab. Following 30-min incubation, Table 1 Demographic data of human frontal cortex specimens Age and post-mortem interval are presented as mean" standard deviation.
Infants Adolescents Adults a
N
Gender
Ethnicity
Male
Female
Caucasian
African American
6 5 9
2 4 7
4 1 2
4 2 8
2 3 1
Post-mortem intervals across groups were not significantly different by ANOVA Ž p s 0.3087..
Age Žyears.
Post-mortem interval Žh. a
0.83 " 1.0 17.4 " 1.7 41.0 " 9.6
19.7 " 9.6 14.8 " 4.3 16.3 " 7.0
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
227
Fig. 2. Bcl-2 levels in frontal cortex across age groups by ELISA. Bcl-2 protein levels are expressed in Unitsrgram tissue weight in human frontal cortex of infant, adolescent, and adult age groups. Mean"S.E.M. was 989.3"215.9 Urg in infants Ž ns6., 1833"98.5 Urg in adolescents Ž ns 5., and 2023"212.4 Urg in adults Ž ns9.. Bcl-2 levels increased significantly across the age groups by ANOVA Ž ps 0.0058.. Both adolescent ŽU p- 0.05. and adult ŽUU p- 0.01. groups were significantly higher than the infant group.
Fig. 4. Bcl-2 levels in frontal cortex across age groups by semi-quantitative Western blotting. Bcl-2 bands were measured using optical densitometry and data was normalized to a control adult sample Ždefined as 1.00.. Normalized mean optical densities ŽOD. were 0.55"0.04 Žmean"S.E.M.. in infants, 0.82"0.07 in adolescents, and 0.97"0.08 in adults. Bcl-2 expression increased significantly across age groups by ANOVA Ž ps 0.002.. The adult group was significantly higher than the infant group ŽU p- 0.01. in post-hoc testing.
Ž1:500, Transduction Laboratories. followed by 1-h incubation with a secondary sheep anti-mouse HRP-labeled Ab Ž1:1000, ECL, Amersham Pharmacia.. Membranes were developed using chemiluminescence ŽECL, Amersham Pharmacia., and the protein bands were detected on radiographic film ŽHyperfilm ECL, Amersham Pharmacia. after 20–30 s exposure. Optical densitometry of Bcl-2 bands was performed using the NIH Image system. Band densities were normalized to a control adult sample run on all gels.
medial frontal cortex was dissected and frozen. The tissue was homogenized and sonicated as described in Section 2.2, and semiquantitative Bcl-2 Western blotting was performed as described in Section 2.5. The primary Ab in the Western protocol recognized Bcl-2 protein from rat and human origin.
2.5. Bcl-2 post-mortem stability study 150–200 g Sprague–Dawley rats Ž n s 8, equal sex ratio; Zivic-Miller, Allison Park, PA. were anaesthetized with ether and decapitated. Medial frontal cortex from four rats was dissected and immediately frozen at y808C. Then, to approximate the post-mortem condition of human brain tissue, the heads of four rats were left at room temperature for 6 h and then at 48C for 18 h, at which time
2.6. Statistics ELISA and Western blot data was analyzed by one-way analysis of variance ŽANOVA. across age groups and a post-hoc analysis was performed using a Tukey’s Multiple Comparison Test, with two-tailed p-values considered significant at 0.05. A secondary analysis determined the linear correlation coefficient between age and Bcl-2 expression. The post-mortem stability data was analyzed using an unpaired Student’s t-test, with two-tailed p-values considered significant at 0.05.
3. Results ELISA results demonstrated that Bcl-2 levels in human frontal cortex significantly increased across the three age groups by ANOVA Ž F s 7.081, df s 19, p s 0.0058; see
Fig. 3. Linear regression analysis of Bcl-2 in all subjects vs. age. Bcl-2 protein levels measured by ELISA Žvalues in Unitsrgram tissue; gender: male Žv . female Ž\.. correlated significantly with age Ž r 2 s 0.4933, ps 0.0006..
Fig. 5. Western blot of Bcl-2 expression in human frontal cortex. An equal amount of total protein was added to each lane. The transfer membrane was incubated with a monoclonal anti-human Bcl-2 Ab. The bands shown migrated to 26 kDa. Lane 1 contained Jurkat cell lysate for Bcl-2 control. Lanes 2–8 contained a subset of the brain samples as follows: 2, Adult; 3, Infant; 4, Infant; 5, Adult; 6, Infant; 7, Adult; 8, Adolescent.
228
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
Fig. 6. Bcl-2 post-mortem stability study. Bcl-2 expression was examined in rat cortex subjected to a 24 h post-mortem interval Ž6 h at 258C then 18 h at 48C. and compared to tissue processed immediately after death Ž0 h.. Semiquantitative Western blotting using optical densitometry revealed a nonsignificant 6% reduction in Bcl-2 expression following a 24 h postmortem interval.
Fig. 2.. Mean levels " S.E.M. were 989.3 " 215.9 Urg in infants, 1833 " 98.5 Urg in adolescents, and 2023 " 212.4 Urg in adults. Both adolescent Ž p - 0.05. and adult Ž p 0.01. groups had significantly higher Bcl-2 levels compared to infant levels. Although adult levels were higher than adolescent levels, this difference was not significant. A secondary analysis revealed a significant linear correlation between individual ages and Bcl-2 levels Ž r 2 s 0.4933, p s 0.0006; see Fig. 3.. No significant differences emerged in post-mortem intervals across the three age groups by ANOVA Ž F s 0.6630, df s 19, p s 0.3087.. Semiquantitative Western blots confirmed the ELISA results by demonstrating a significant increase in Bcl-2 protein levels from infancy to adolescence and adulthood by ANOVA Ž F s 9.390, df s 18, p s 0.002, see Fig. 4.. Bcl-2 was identified in each sample and the difference in band densities between infant and adult samples was readily visualized ŽFig. 5.. Normalized mean optical densities showed Bcl-2 expression was 50% higher in adolescents and 77% higher in adults compared to infant Bcl-2 expression. Only adult Bcl-2 expression was significantly higher than infant levels Ž p - 0.01. in post-hoc analysis. The band at 26 kDa was further confirmed as Bcl-2 by the co-migration of a 26 kDa band from a Jurkat cell lysate run on each gel ŽFig. 5.. Western blots from the post-mortem stability study of Bcl-2 protein demonstrated no significant degradation over 24 h Žsee Fig. 6.. Mean Bcl-2 expression in tissue processed after a 24 h post-mortem interval was nonsignificantly reduced by 6% Ž F s 1.141, df s 6, p s 0.458..
4. Discussion This is the first study to quantitatively measure the developmental expression of Bcl-2 protein in human cortex across the postnatal life span. Bcl-2 protein demonstrates developmental regulation in frontal cortex, increas-
ing from early infancy to adolescence and into adulthood. The quantitative ELISA results are corroborated by similar results using semiquantitative Western blots. Interestingly, our findings demonstrate a different pattern of Bcl-2 expression in human cortex from that seen in rodent cortex. Most animal studies have concluded that the developmental expression of Bcl-2 in the CNS declines with aging w3,6,16,21x. Those studies compared Bcl-2 expression in fetal, postnatal, and adult rodent brain tissue using immunohistochemistry, in situ hybridization, and immunoblotting. Our results demonstrate that postnatal Bcl-2 expression in human cortex increases with age, countering previous findings from animal studies. This also highlights the importance of conducting developmental studies in human brain tissue since human neurodevelopment can differ from animal models. To our knowledge, only two reports have addressed the developmental expression of Bcl-2 protein in human brain. Using fetal tissue, one report demonstrated that human brain Bcl-2 expression is higher in early gestation and downregulated by the end of gestation w4x. Yachnis et al. w27x found a similar downregulation of Bcl-2 protein from early to late gestation in several CNS areas, and Bcl-2 levels were also low in temporal cortex from two adults. Taken together, the current and prior studies suggest that Bcl-2 protein expression in human cortex follows a biphasic pattern that is high in early gestation, followed by perinatal downregulation, and then a gradual upregulation across the life span. In human fetal cortex, Chan and Yew w4x reported that increased neuronal apoptosis coincides with downregulation of Bcl-2 protein expression across gestation. Since Bcl-2 is an anti-apoptotic protein, they speculated that Bcl-2 levels may require downregulation in order to accommodate the massive developmental surge in apoptosis during later gestation. However, it is also known that the rate of apoptosis is substantially reduced perinatally, since studies of postnatal fetal cortex have failed to detect evidence of apoptosis in normal infants as young as 3 weeks of age w7,23x. The specific role of Bcl-2 protein in regulating developmental apoptosis remains unclear, but given the multitude of interacting pro- and anti-apoptotic proteins that are expressed, a complex regulatory mechanism dependent on temporal and regional factors is likely to exist. Although speculative, our finding of lower Bcl-2 levels during infancy may reveal a window during normal development when the CNS is more vulnerable to proapoptotic insults. Some neurodevelopmental disorders, including schizophrenia and autism, have been hypothesized to involve alterations in apoptosis in response to perinatal stressors such as infection or hypoxia w13,26x. Given the established role of Bcl-2 as an anti-apoptotic regulator in early development, our findings are suggestive that Bcl-2 protein may continue to exert such a function throughout life, with important implications for normal aging. A neuroprotective role has already been proposed
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
for Bcl-2 by virtue of its anti-apoptotic properties w6,16,21,25x, and Castren ´ et al. w3x have speculated that basal levels of bcl-2 mRNA are necessary for the survival of postmitotic neurons. In studies of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, Bcl-2 is present and often upregulated in certain brain regions w10,14,17,19x Žreviewed in Ref. w12x.. The upregulation may represent a compensatory response to the neurodegenerative process. Similarly, normal aging has been associated with free radical-induced oxidative damage, as evidenced by higher oxidized protein levels, lipofuscin, DNA damage, and glycosylated proteins w15,18x. Such chronic oxidative stress may contribute to higher Bcl-2 protein levels in adult cortex in order to maintain neuronal viability. Our result documenting the highest postnatal Bcl-2 level in adulthood is consistent with a role for Bcl-2 protein in normal aging. Finally, to assess the potential confounding effect of post-mortem interval on Bcl-2 protein, we used an animal paradigm designed to approximate the human post-mortem condition. Bcl-2 demonstrated high post-mortem stability for 24 h. Since the mean post-mortem intervals in our three human age groups were all less than 20 h and did not differ significantly, it suggests that our results were not confounded by post-mortem degradation of Bcl-2 protein. In conclusion, Bcl-2 protein demonstrates developmental regulation in human cortex. The current study adds to the accumulating evidence that Bcl-2 continues to have a significant role in adult CNS by virtue of its upregulation from infancy to adulthood. Given the complexity of CNS function, a neuroprotective role for Bcl-2 in normal aging would likely occur in concert with a variety other pro- and anti-apoptotic proteins and other processes affecting neuronal survival. Further studies in human brain tissue that characterize age-dependent expression of other Bcl-2 family proteins during normal development may help elucidate this mechanism. Ultimately, defining the role of Bcl-2 family proteins in the development and maintenance of the human CNS will provide a better understanding of neurodevelopmental and neurodegenerative disorders.
Acknowledgements The authors are indebted to Elzbieta S. Selinger, MD, MSc for her excellent technical assistance. This work was supported by the Foundation of Hope ŽRaleigh, NC. and by Center Grant MH-33127.
References w1x S. Abe-Dohmae, N. Harada, K. Yamada, R. Tanaka, bcl-2 gene is highly expressed during neurogenesis in the central nervous system, Biochem. Biophys. Res. Commun. 191 Ž1993. 915–921.
229
w2x J.M. Adams, S. Cory, The Bcl-2 protein family: arbiters of cell survival, Science 281 Ž1998. 1322–1326. w3x E. Castren, ´ Y. Ohga, M.P. Berzaghi, G. Tzimagiorgis, H. Thonen, D. Lindholm, bcl-2 messenger RNA is localized in neurons of the developing and adult rat brain, Neuroscience 61 Ž1994. 165–177. w4x W.Y. Chan, D.T. Yew, Apoptosis and Bcl-2 oncoprotein expression in the human fetal central nervous system, Anat. Rec. 252 Ž1998. 165–175. w5x D.F. Chen, G.E. Schneider, J.C. Martinou, S. Tonegawa, Bcl-2 promotes regeneration of severed axons in mammalian CNS, Nature 385 Ž1997. 434–439. w6x I. Ferrer, A. Tortosa, E. Condom, R. Blanco, A. Macaya, A. Planas, Increased expression of bcl-2 immunoreactivity in the developing cerebral cortex of the rat, Neurosci. Lett. 179 Ž1994. 13–16. w7x H.A. Gelbard, H.J. James, L.R. Sharer, S.W. Perry, Y. Saito, A.M. Kazee, B.M. Blumberg, L.G. Epstein, Apoptotic neurons in brains from paediatric patients with HIV-1 encephalitis and progressive encephalopathy, Neuropathol. Appl. Neurobiol. 21 Ž1995. 208–217. w8x D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 Ž1998. 1309–1312. w9x D. Hockenberry, G. Nunez, ˜ C. Milliman, R.D. Schreiber, S.J. Korsmeyer, Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death, Nature 348 Ž1990. 334–336. w10x Y. Kitamura, S. Shimohama, W. Kamoshima, T. Ota, Y. Matsuoka, Y. Nomura, M.A. Smith, G. Perry, P.J. Whitehouse, T. Taniguchi, Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer’s disease, Brain Res. 780 Ž1998. 260–269. w11x D.P. LeBrun, R.A. Warnke, M.L. Cleary, Expression of bcl-2 in fetal tissues suggests a role in morphogenesis, Am. J. Pathol. 142 Ž1993. 743–753. w12x M. Leist, P. Nicotera, Apoptosis, excitotoxicity, and neuropathology, Exp. Cell Res. 239 Ž1998. 183–201. w13x R.L. Margolis, D.M. Chuang, R.M. Post, Programmed cell death: implications for neuropsychiatric disorders, Biol. Psychiatry 35 Ž1994. 946–956. w14x K.-A. Marshall, S.E. Daniel, N. Cairns, P. Jenner, B. Halliwell, Upregulation of the anti-apoptotic protein Bcl-2 may be an early event in neurodegeneration: studies on Parkinson’s and incidental lewy body disease, Biochem. Biophys. Res. Commun. 240 Ž1997. 84–87. w15x P. Mecocci, M.F. Beal, R. Cecchetti, M.C. Polidori, A. Cherubini, F. Chionne, L. Avellini, G. Romano, U. Senin, Mitochondrial membrane fluidity and oxidative damage to mitochondrial DNA in aged and AD human brain, Mol. Chem. Neuropathol. 31 Ž1997. 53–64. w16x D.E. Merry, D.J. Veis, W.F. Hickey, S.J. Korsmeyer, Bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS, Development 120 Ž1994. 301–311. w17x M. Mogi, M. Harada, T. Kondo, Y. Mizuno, H. Narabayashi, P. Riederer, T. Nagatsu, Bcl-2 protein is increased in the brain from Parkinsonian patients, Neurosci. Lett. 215 Ž1996. 137–139. w18x R.E. Mrak, W.S.T. Griffin, D.I. Graham, Aging-associated changes in human brain, J. Neuropathol. Exp. Neurol. 56 Ž1997. 1269–1275. w19x T. Satou, B.J. Cummings, C.W. Cotman, Immunoreactivity for Bcl-2 protein within neurons in the Alzheimer’s disease brain increases with disease severity, Brain Res. 697 Ž1995. 35–43. w20x S.L. Schendel, Z. Xie, M.O. Montal, S. Matsuyama, M. Montal, J.C. Reed, Channel formation by anti-apoptotic protein bcl-2, Proc. Natl. Acad. Sci. U. S. A. 94 Ž1997. 5113–5118. w21x S. Shimohama, S. Fujimoto, Y. Sumida, H. Tanino, Differential expression of rat brain Bcl-2 family proteins in development and aging, Biochem. Biophys. Res. Commun. 252 Ž1998. 92–96. w22x J.H. Su, G. Deng, C.W. Cotman, Bax protein expression is increased in Alzheimer’s brain: correlations with DNA damage, Bcl-2 expression, and brain pathology, J. Neuropathol. Exp. Neurol. 56 Ž1997. 86–93. w23x J.C. Tronosco, R.R. Sukhov, C.H. Kawas, V.E. Koliatsos, In situ
230
L.F. Jarskog, J.H. Gilmorer DeÕelopmental Brain Research 119 (2000) 225–230
labeling of dying cortical neurons in normal aging and in Alzheimer’s disease: correlations with senile plaques and disease progression, J. Neuropathol. Exp. Neurol. 55 Ž1996. 1134–1142. w24x Y. Tsujimoto, L.R. Finger, J. Yunis, P.C. Nowell, C.M. Croce, Cloning of the chromosome breakpoint of neoplastic B cells with the tŽ14;18. chromosome translocation, Science 226 Ž1984. 1097–1099. w25x S. Vyas, F. Javoy-Agid, M.-T. Herrero, O. Strada, F. Boissiere, U. Hibner, Y. Agid, Expression of Bcl-2 in adult human brain regions with special reference to neurodegenerative disorders, J. Neurochem. 69 Ž1997. 223–231. w26x B.T. Woods, Is schizophrenia a progressive neurodevelopmental
disorder? Toward a unitary pathogenetic mechanism, Am. J. Psychiatry 155 Ž1998. 1661–1670. w27x A.T. Yachnis, S.Z. Powell, J.J. Olmsted, T.A. Eskin, Distinct neurodevelopmental patterns of Bcl-2 and Bcl-x expression are altered in glioneuronal hamartias of the human temporal lobe, J. Neuropathol. Exp. Neurol. 56 Ž1997. 186–198. w28x L.-T. Zhong, T. Sarafian, D.J. Kane, A.C. Charles, S.P. Mah, R.H. Edwards, D.E. Bredesen, bcl-2 inhibits death of central neural cells induced by multiple agents, Proc. Natl. Acad. Sci. U. S. A. 90 Ž1993. 4533–4537.