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Human APOE protein localized in brains of transgenic mice
ELSEVIER
Neuroscience Letters 219 (1996) 57-59
HEUROSClENC[ IETTEBS
Human APOE protein localized in brains of transgenic mice Barbara H. B o w m a n la, Funmei Yang a, James M. Buchanan a, Gwendolyn S. Adrian a,* , Andrew O. Martinez b, Laila Jansen a, Ming Zhao a, Sally L. Atherton a, James E. Hixson c ~The Department g[ Celhdar and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762, USA bDivision c~[LijO Sciences, University of Texas at San Antonio, San Antonio, Texas, USA ~Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas, USA
Received 1 July 1996; revised version received 17 September 1996; accepted 17 September 1996
Abstract
Transgenic mice carrying the three common human apolipoprotein E (APOE) alleles have been developed. In this study, brains of the transgenic mice have been analyzed by in situ histohybridization, immunohistochemistry, and immunoblots to determine sites of gene expression, to identify specific brain cells associated with human apoE protein, and to determine the relative concentrations of the human apoE. Results indicate that (1) human APOE mRNA and apoE protein occur in the gray and white matter of transgenic mouse brains; (2) in the hippocampus of transgenic brains, human apoE protein reacts immunologically within the same cells as the glial fibrillary acidic protein (GFAP), a specific marker for astrocytes; and (3) concentrations of the apoE isoforms determined in three heterozygous transgenic brains range from 22 to 250 pmol/g wet weight of brain. Keywords." Transgenic mouse; Human apolipoprotein E; Astrocyte; In situ hybridization; Immunohistochemistry; Immunoblot
A correlation between the presence of the human apolipoprotein E (APOE)-4 allele and the risk of developing Alzheimer"s disease (AD) [4] has raised the question of what pathological role is played by apoE4 in the brain. Recent evidence also points toward a relationship between the presence of the A P O E 4 allele and the loss of cognitive performance and occurrence of brain pathology in aging individuals free of A D [7,8,10,1 1]. Therefore, transgenic mice lines expressing each of the three human apoE isoforms in the brain provide a means to search for and follow events that may lead to neuronal destruction and cognitive decline in the m a m m a l i a n brain [1]. The results of the studies presented here demonstrate that the areas of the transgenic brains in which human apoE isoforms occur are similar to sites of endogenous apoE protein in humans and rodents [2,5,9[. Diedrich et al. [5] observed occasional accumulations of
* Corresponding author. Tel.. +l 210 5673800; fax +l 210 5673803. 1Submitted posthumously.
immunoreactive apoE surrounding neuron cell bodies and in association with their processes in the brains of a control mouse and a patient with AD. The source of extracellular apoE protein was probably from neighboring astrocytes, since rodent and human astrocytes specifically display A P O E m R N A when examined by in situ hybridization [9,12]. The purpose of this study was to locate sites of expression of the human A P O E transgenes in the brain, identify cells associated with human apoE protein and to estimate the levels of the human apoE protein in brains of transgenic mice. The transgenic mice utilized in this study carried transgenes containing full-length c D N A s of APOE2, APOE3, or APOE4 under transcriptional control of the human transferrin promoter (-621 to +46 bp of the transferrin 5'-regulatory region). Three-prime to the APOE c D N A sequences is the simian virus 40 polyadenylation signal and small t intron. Details of the constructions are reported in Bowman et al. [1]. The 0.67 kb transferrin promoter was chosen because it has been used with two other structural genes and was shown to direct high levels of expression to brain [1 ]. Yet, the location of brain expression directed by
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved Pll S0304-3940(96) 13 166-9
58
B.H. Bowman
et al. / N e u r o s c i e n c e
,,
~ ~ ~
~,
Letters 219 (1996) 57-59
~, ~ . ~
Fig. I. In situ hybridizationof sagittal sectionsof the brain of a transgenicmouse carrying the human APOE3 gene. Labeling of human APOE mRNA appears as silver grains in dark field microscopy of white matter: (a) corpus callosum, (b) fimbriaehippocampus, (c) cerebellumand gray matter: (d) hippocampus, (e) cerebrum• In situ histohybridizationwas carried out as described previously [13]. Bar represents 0.05 mm. the transferrin promoter and the relative amounts of the apoE isoforms expressed has not previously been well examined. In situ histohybridization analysis of the brains of transgenic mice demonstrated that human APOE m R N A was expressed in glial cells of the corpus callosum, fimbriae hippocampi, in the cerebellum, the striatum, the hippocampus and cerebrum (Fig. 1). There was no human APOE m R N A detected in brains of nontransgenic mice. Northern blot analysis [3] suggested previously that levels of human APOE2, 3 and 4 m R N A in brains of transgenic mice were equal or greater than the levels of endogenous mouse APOE m R N A [ 1]. To identify the cell types in the brain associated with human APOE, serial tissue sections of brain were analyzed by immunohistochemistry and were reacted with antibodies to either human apoE or GFAP. As shown in Fig. 2a,b
a
the same astrocytes in consecutive tissue sections of the hippocampus reacted with human apoE and GFAP antibodies, indicating that human apoE protein was present in astrocytes. Homologous sections of brains from nontransgenic mice did not react with the human apoE antibody (not shown)• The levels of human apoE2, 3 and 4 isoforms in brains from three heterozygous transgenic mice from three founder lines are shown in Fig. 3. The highest apoE concentration in Fig. 3 is 249.75 pmol/g of brain homogenate from an APOE3 heterozygous mouse having 40 copies of the human APOE3 transgene. An APOE2 line with 78.43 pmol/g apoE has three copies of the transgene, while 21.48 pmol/g is observed in a APOE4 transgenic line with two copies of the transgene. The human apoE protein concentrations in other transgenic founder lines developed by us also appear to correlate with the transgene copy number.
b
Fig. 2. Immunohistochemistryof cells near the hippocampus of a transgenicmouse carrying the human APOE3 gene. (a) Cell reacting positivelywith human ApoE antibody and (b) on the successive tissue section, with antibody to GFAP, a specific marker for astrocytes. Identificationof brain cells associated with human APOE was performed by immunohistochemistryusing antibody preparations specific for human APOE (Chemicon) and glial fibrillary acidic protein (GFAP) (Novocastra Lab. Ltd.). The analysis was carried out as described by Hsu et al. [6]. Bar represents 0.1 mm.
B.H. B o w m a n et al. / Neuroscience Letters 219 (1996) 5 7 - 5 9
[3]
[4]
J5] Human ApoE
$1
$2
$3
E2
E3
E4
N
[6]
Fig. 3. Immunoblot demonstrating human apoE 2, 3 and 4 proteins in brains of transgenic mice, and absence of human apoE in nontransgenic mouse brains. Brain extracts were immunoprecipitated and apoE was quantitated by immunoblot according to the procedure of Boyles et al. [3]. ~2~I-Protein A labeled blots were scanned with a Beckman Phosphorimager. From left to right, the first three lanes, S l, $2, and $3, contained extracts of nontransgenic mouse (NTG) brain to which authentic human apoE (Calbiochem) 3.0. 1.5, or 0.75 #g, respectively, had been added. Lanes 4 (E2), 5 (E3), and 6 (E4) contained brain extracts of transgenic mice carrying human APOE2, 3 and 4 genes, respectively. Lane 7 (N) contained brain extract from a nontransgenic mouse. Arrow indicates migration of authentic human apoE protein. The research American
was supported
[7]
[8]
[9]
[10]
in p a r t b y a g r a n t f r o m
Health Assistance Foundation
Institutes of Health Grants AG06872,
and by National
EY06012,
AG0650
[l l]
and K 14GM00651. [1] Bowman, B.H., Jansen, L., Yang, F., Adrian, G.S., Zhao, M., Atherton, S.S., Buchanan, J.M., Greene, R., Walter, C., Herbert, D., Weaker, F.J., Chiodo, L.K., Kagan-Hallet, K. and Hixson, J.E., Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles, Proc. Natl. Acad. Sci. USA, 92 (1995) 12115-I2119. [2] Boyles, J.K., Pitas, R.E., Wilson, E., Mabley, R.W. and Taylor,
[12]
[13]
59
J.M., Apolipoprotein E associated with astrocytic gila of the central nervous system and nonmyelinating gila of the peripheral nervous system, J. Clin. Invest., 76 (1985) 1501-1513. Chomcyznski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskeli, P.C., Small, G.W., Roses, A.D., Haines, J.L. and PericakVance, M.A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer"s disease in late onset families, Science, 261 (1993) 921-923. Diedricb, J.F., Minnigan, H. and Carp, R.1., Whitaker, J.N., Race, R., Frey, W. and Haas, A.T., Neuropathological changes in Scrapie and Alzheimer"s disease are associated with expression of apolipoprotein E and cathepsin D in astrocytcs, J. Virol., 65 (1991) 4759-4768. Hsu, S.M., Raine, L. and Fanger, H., A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies, J. Histochem. Cytochem., 29 (1981) 577-580. Hyman, B.T., Gomez-Isla, T., Briggs, M., Chung, H., Nichols, S., Kohout, F. and Wallace, R., Influence of APOE gcnotype on developing cognitive impairment in the general population, Soc. Neurosci. Abstr., 21 (1995) 1008. Oyama, F., Schimada, H., Oyama, R. and Ibara, Y., Apolipoprotein E genotype, Alzheimer"s pathology and related expression in the aged population, Mol. Brain. Res., 29 (1995) 92-98. Poirier, J., Hess, M., May, P.C. and Finch, C.E., Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus alter endorhinal cortex lesioning, Mol. Brain Res., 11 (1991) 97-106. Polvikoski, T., Sulkave, R., Haltias, M., Kainulainen, K., Vuorco, A., Venkroniemi, A., Nunisto, L., Halonen, P. and Kontulao, K., Apolipoprotein E, dementia, and cortical deposition of/3-amyloid, N. Engl. J. Med., 333 (1995) 1242-1247. Reed, T., Carmelli, D., Swan, G.E., Breitner, J.C., Welsh, K.A., Jarvik, G.P., Deeb, S. and Auwerx, J., Lower cognitive pertormance in normal older adult male twins carrying the apolipoprotein E epsilon 4 allele, Arch. Neurol., 51 (1994) 1189-1192. Yamada, T., Kono, A., Tadamatsu, J., Tateisbi, J. and Gota, 1., Apolipoprotein E mRNA in the brains of patients with Alzheimer's disease, J. Neurol. Sci., 129 (1995) 56-61. Yang, F., Friedrichs, W.E., Navarijo-Ashbaugh, A.L., de Graffenried, L.A., Bowman, B.H. and Coalson, J.J., Inflammatory induction and cell type specific expression of haptoglobin gene in baboon and mouse lung, Lab. Invest., 73 (1995) 433-440.
Neuroscience Letters 219 (1996) 57-59
HEUROSClENC[ IETTEBS
Human APOE protein localized in brains of transgenic mice Barbara H. B o w m a n la, Funmei Yang a, James M. Buchanan a, Gwendolyn S. Adrian a,* , Andrew O. Martinez b, Laila Jansen a, Ming Zhao a, Sally L. Atherton a, James E. Hixson c ~The Department g[ Celhdar and Structural Biology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762, USA bDivision c~[LijO Sciences, University of Texas at San Antonio, San Antonio, Texas, USA ~Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas, USA
Received 1 July 1996; revised version received 17 September 1996; accepted 17 September 1996
Abstract
Transgenic mice carrying the three common human apolipoprotein E (APOE) alleles have been developed. In this study, brains of the transgenic mice have been analyzed by in situ histohybridization, immunohistochemistry, and immunoblots to determine sites of gene expression, to identify specific brain cells associated with human apoE protein, and to determine the relative concentrations of the human apoE. Results indicate that (1) human APOE mRNA and apoE protein occur in the gray and white matter of transgenic mouse brains; (2) in the hippocampus of transgenic brains, human apoE protein reacts immunologically within the same cells as the glial fibrillary acidic protein (GFAP), a specific marker for astrocytes; and (3) concentrations of the apoE isoforms determined in three heterozygous transgenic brains range from 22 to 250 pmol/g wet weight of brain. Keywords." Transgenic mouse; Human apolipoprotein E; Astrocyte; In situ hybridization; Immunohistochemistry; Immunoblot
A correlation between the presence of the human apolipoprotein E (APOE)-4 allele and the risk of developing Alzheimer"s disease (AD) [4] has raised the question of what pathological role is played by apoE4 in the brain. Recent evidence also points toward a relationship between the presence of the A P O E 4 allele and the loss of cognitive performance and occurrence of brain pathology in aging individuals free of A D [7,8,10,1 1]. Therefore, transgenic mice lines expressing each of the three human apoE isoforms in the brain provide a means to search for and follow events that may lead to neuronal destruction and cognitive decline in the m a m m a l i a n brain [1]. The results of the studies presented here demonstrate that the areas of the transgenic brains in which human apoE isoforms occur are similar to sites of endogenous apoE protein in humans and rodents [2,5,9[. Diedrich et al. [5] observed occasional accumulations of
* Corresponding author. Tel.. +l 210 5673800; fax +l 210 5673803. 1Submitted posthumously.
immunoreactive apoE surrounding neuron cell bodies and in association with their processes in the brains of a control mouse and a patient with AD. The source of extracellular apoE protein was probably from neighboring astrocytes, since rodent and human astrocytes specifically display A P O E m R N A when examined by in situ hybridization [9,12]. The purpose of this study was to locate sites of expression of the human A P O E transgenes in the brain, identify cells associated with human apoE protein and to estimate the levels of the human apoE protein in brains of transgenic mice. The transgenic mice utilized in this study carried transgenes containing full-length c D N A s of APOE2, APOE3, or APOE4 under transcriptional control of the human transferrin promoter (-621 to +46 bp of the transferrin 5'-regulatory region). Three-prime to the APOE c D N A sequences is the simian virus 40 polyadenylation signal and small t intron. Details of the constructions are reported in Bowman et al. [1]. The 0.67 kb transferrin promoter was chosen because it has been used with two other structural genes and was shown to direct high levels of expression to brain [1 ]. Yet, the location of brain expression directed by
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved Pll S0304-3940(96) 13 166-9
58
B.H. Bowman
et al. / N e u r o s c i e n c e
,,
~ ~ ~
~,
Letters 219 (1996) 57-59
~, ~ . ~
Fig. I. In situ hybridizationof sagittal sectionsof the brain of a transgenicmouse carrying the human APOE3 gene. Labeling of human APOE mRNA appears as silver grains in dark field microscopy of white matter: (a) corpus callosum, (b) fimbriaehippocampus, (c) cerebellumand gray matter: (d) hippocampus, (e) cerebrum• In situ histohybridizationwas carried out as described previously [13]. Bar represents 0.05 mm. the transferrin promoter and the relative amounts of the apoE isoforms expressed has not previously been well examined. In situ histohybridization analysis of the brains of transgenic mice demonstrated that human APOE m R N A was expressed in glial cells of the corpus callosum, fimbriae hippocampi, in the cerebellum, the striatum, the hippocampus and cerebrum (Fig. 1). There was no human APOE m R N A detected in brains of nontransgenic mice. Northern blot analysis [3] suggested previously that levels of human APOE2, 3 and 4 m R N A in brains of transgenic mice were equal or greater than the levels of endogenous mouse APOE m R N A [ 1]. To identify the cell types in the brain associated with human APOE, serial tissue sections of brain were analyzed by immunohistochemistry and were reacted with antibodies to either human apoE or GFAP. As shown in Fig. 2a,b
a
the same astrocytes in consecutive tissue sections of the hippocampus reacted with human apoE and GFAP antibodies, indicating that human apoE protein was present in astrocytes. Homologous sections of brains from nontransgenic mice did not react with the human apoE antibody (not shown)• The levels of human apoE2, 3 and 4 isoforms in brains from three heterozygous transgenic mice from three founder lines are shown in Fig. 3. The highest apoE concentration in Fig. 3 is 249.75 pmol/g of brain homogenate from an APOE3 heterozygous mouse having 40 copies of the human APOE3 transgene. An APOE2 line with 78.43 pmol/g apoE has three copies of the transgene, while 21.48 pmol/g is observed in a APOE4 transgenic line with two copies of the transgene. The human apoE protein concentrations in other transgenic founder lines developed by us also appear to correlate with the transgene copy number.
b
Fig. 2. Immunohistochemistryof cells near the hippocampus of a transgenicmouse carrying the human APOE3 gene. (a) Cell reacting positivelywith human ApoE antibody and (b) on the successive tissue section, with antibody to GFAP, a specific marker for astrocytes. Identificationof brain cells associated with human APOE was performed by immunohistochemistryusing antibody preparations specific for human APOE (Chemicon) and glial fibrillary acidic protein (GFAP) (Novocastra Lab. Ltd.). The analysis was carried out as described by Hsu et al. [6]. Bar represents 0.1 mm.
B.H. B o w m a n et al. / Neuroscience Letters 219 (1996) 5 7 - 5 9
[3]
[4]
J5] Human ApoE
$1
$2
$3
E2
E3
E4
N
[6]
Fig. 3. Immunoblot demonstrating human apoE 2, 3 and 4 proteins in brains of transgenic mice, and absence of human apoE in nontransgenic mouse brains. Brain extracts were immunoprecipitated and apoE was quantitated by immunoblot according to the procedure of Boyles et al. [3]. ~2~I-Protein A labeled blots were scanned with a Beckman Phosphorimager. From left to right, the first three lanes, S l, $2, and $3, contained extracts of nontransgenic mouse (NTG) brain to which authentic human apoE (Calbiochem) 3.0. 1.5, or 0.75 #g, respectively, had been added. Lanes 4 (E2), 5 (E3), and 6 (E4) contained brain extracts of transgenic mice carrying human APOE2, 3 and 4 genes, respectively. Lane 7 (N) contained brain extract from a nontransgenic mouse. Arrow indicates migration of authentic human apoE protein. The research American
was supported
[7]
[8]
[9]
[10]
in p a r t b y a g r a n t f r o m
Health Assistance Foundation
Institutes of Health Grants AG06872,
and by National
EY06012,
AG0650
[l l]
and K 14GM00651. [1] Bowman, B.H., Jansen, L., Yang, F., Adrian, G.S., Zhao, M., Atherton, S.S., Buchanan, J.M., Greene, R., Walter, C., Herbert, D., Weaker, F.J., Chiodo, L.K., Kagan-Hallet, K. and Hixson, J.E., Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles, Proc. Natl. Acad. Sci. USA, 92 (1995) 12115-I2119. [2] Boyles, J.K., Pitas, R.E., Wilson, E., Mabley, R.W. and Taylor,
[12]
[13]
59
J.M., Apolipoprotein E associated with astrocytic gila of the central nervous system and nonmyelinating gila of the peripheral nervous system, J. Clin. Invest., 76 (1985) 1501-1513. Chomcyznski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskeli, P.C., Small, G.W., Roses, A.D., Haines, J.L. and PericakVance, M.A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer"s disease in late onset families, Science, 261 (1993) 921-923. Diedricb, J.F., Minnigan, H. and Carp, R.1., Whitaker, J.N., Race, R., Frey, W. and Haas, A.T., Neuropathological changes in Scrapie and Alzheimer"s disease are associated with expression of apolipoprotein E and cathepsin D in astrocytcs, J. Virol., 65 (1991) 4759-4768. Hsu, S.M., Raine, L. and Fanger, H., A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies, J. Histochem. Cytochem., 29 (1981) 577-580. Hyman, B.T., Gomez-Isla, T., Briggs, M., Chung, H., Nichols, S., Kohout, F. and Wallace, R., Influence of APOE gcnotype on developing cognitive impairment in the general population, Soc. Neurosci. Abstr., 21 (1995) 1008. Oyama, F., Schimada, H., Oyama, R. and Ibara, Y., Apolipoprotein E genotype, Alzheimer"s pathology and related expression in the aged population, Mol. Brain. Res., 29 (1995) 92-98. Poirier, J., Hess, M., May, P.C. and Finch, C.E., Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus alter endorhinal cortex lesioning, Mol. Brain Res., 11 (1991) 97-106. Polvikoski, T., Sulkave, R., Haltias, M., Kainulainen, K., Vuorco, A., Venkroniemi, A., Nunisto, L., Halonen, P. and Kontulao, K., Apolipoprotein E, dementia, and cortical deposition of/3-amyloid, N. Engl. J. Med., 333 (1995) 1242-1247. Reed, T., Carmelli, D., Swan, G.E., Breitner, J.C., Welsh, K.A., Jarvik, G.P., Deeb, S. and Auwerx, J., Lower cognitive pertormance in normal older adult male twins carrying the apolipoprotein E epsilon 4 allele, Arch. Neurol., 51 (1994) 1189-1192. Yamada, T., Kono, A., Tadamatsu, J., Tateisbi, J. and Gota, 1., Apolipoprotein E mRNA in the brains of patients with Alzheimer's disease, J. Neurol. Sci., 129 (1995) 56-61. Yang, F., Friedrichs, W.E., Navarijo-Ashbaugh, A.L., de Graffenried, L.A., Bowman, B.H. and Coalson, J.J., Inflammatory induction and cell type specific expression of haptoglobin gene in baboon and mouse lung, Lab. Invest., 73 (1995) 433-440.