Neuroscience Vol. 97, No. 3, pp. 419–424, 2000 419 Copyright 䉷 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Reversal of neuronal deficits in apoE transgenic mice
Pergamon PII: S0306-4522(00)00087-7 www.elsevier.com/locate/neuroscience
REVERSAL OF PRESYNAPTIC DEFICITS OF APOLIPOPROTEIN E-DEFICIENT MICE IN HUMAN APOLIPOPROTEIN E TRANSGENIC MICE S. CHAPMAN,*† T. SABO,*† A. D. ROSES‡ and D. M. MICHAELSON†§ †Department of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978, Israel ‡Glaxo Wellcome Research Division, Five Moore Dr. 5-5616, Research Triangle Park, NC 27709, U.S.A.
Abstract—Apolipoprotein E genotype is an important risk factor of Alzheimer’s disease, which is associated with the degeneration of distinct brain neuronal systems. In the present study we employed apolipoprotein E-deficient mice and human apolipoprotein E3 and apolipoprotein E4 transgenic mice on a null mouse apolipoprotein E background, to examine the extent to which distinct brain neuronal systems are affected by apolipoprotein E and the isoform specificity of this effect. This was pursued by histological and autoradiographic measurements utilizing neuron specific presynaptic markers. The results thus obtained revealed significant reductions in the levels of brain cholinergic and noradrenergic nerve terminals in young apolipoprotein E-deficient mice and no changes in brain dopaminergic nerve terminals. These cholinergic and noradrenergic presynaptic derangements were ameliorated similarly in human apolipoprotein E3 and apolipoprotein E4 transgenic mice. In the case of the cholinergic system, this resulted in complete reversal of the presynaptic deficits, whereas in the case of the noradrenergic neurons the amelioration was partial. These findings suggest that brain cholinergic and noradrenergic neurons are markedly more dependent on brain apolipoprotein E than brain dopaminergic neurons and that the isoform specificity of these effects is not apparent at a young age under nonchallenged conditions. 䉷 2000 IBRO. Published by Elsevier Science Ltd. Key words: Alzheimer’s disease, apolipoprotein E, transgenic mice, brain, cholinergic, dopaminergic.
Apolipoprotein E (apoE) genotype is an important risk factor of Alzheimer’s disease (AD). 23,7 Accordingly, the allele apoE4 which is the AD risk factor was found to be associated with decreased age of onset of the disease, which can start up to 15 years earlier in subjects homozygotic to the apoE4 allele. 22 Previous in vivo and in vitro model studies 20,12,18 and recent experiments in which apoE-deficient mice and human apoE transgenic mice were investigated prior to and following brain injury 5,16,24,21 suggest that apoE plays an important role in neuronal maintenance and repair. The cellular and molecular mechanisms underlying this effect and its apoE isoform specificity are not yet known. Histopathological assessments of AD brains revealed that the disease is associated with a distinct pattern of neuronal degeneration, and that some neuronal systems, such as basal forebrain cholinergic neurons and the locus-coeruleus noradrenergic projections, are particularly affected, whereas other pathways, such as the nigrostriatal dopaminergic projections, are relatively spared. 9 It has recently been suggested that the extent of cholinergic damage in AD is elevated in patients who carry the apoE4 allele. 26,20 Furthermore, diseases such as Parkinson’s disease, which are not associated with cholinergic hypofunction but rather with the neurodegeneration of brain dopaminergic neurons, seem not to be affected by the apoE genotype. 13 These observations suggest that distinct neurons differ in the extent to which they depend on apoE for their maintenance and repair and that the isoform
specific pathophysiological effects of apoE are mediated by a specific subpopulation of brain neurons. Recent animal model findings in support of this hypothesis were obtained with apoE-deficient mice in which it was shown that basal forebrain cholinergic projections and locus-coeruleus noradrenergic projections are markedly affected by apoE deficiency whereas the nigrostriatal dopaminergic neurons are not. 4 In the present study we employed human apoE3 and apoE4 transgenic mice on a null mouse apoE background and examined the extent to which the distinct neuronal derangements of apoE deficient mice are ameliorated by the human apoE3 and apoE4 transgenes. EXPERIMENTAL PROCEDURES
Animals Human apoE3 and apoE4 transgenic mice were generated on an apoE-deficient C57BL/6J background utilizing human apoE3 and apoE4 transgene constructs as previously reported. 31 Accordingly, cosmid libraries were constructed from lymphoblasts of humans known to be homozygous carriers for apoE3 or apoE4 after which fragments containing human regulatory sequences and the coding sequences for human apoE were used to produce the transgenic mice. The experiments were performed with the apoE3-453 and apoE4-81 lineages which were backbred with genetically homogeneous apoE-deficient mice (Jackson Labs, Cat. No. N10 JAX) for more than 10 generations, and which were heterozygous for the human apoE transgene and homozygous for mouse apoE deficiency. The use of heterozygous mice and their backbreeding protocol with apoE-deficient mice were pursued to minimize genetic drift and to facilitate the apoE genotyping by polymerase chain reaction (PCR), which is more suitable for differentiating between the different genotypes than for measurements of gene dosage. DNA for PCR genotyping was extracted from a small piece of tail tissue that was digested overnight at 55⬚C with proteinase K (Boehringer, Mannheim, DL). Two microliters of the digested tissue were then added to 40 ml of GeneReleaser (BioVentures, TN, U.S.A.) which was then treated for 5 min in a microwave (900 W). The presence of mouse and human apoE genes was determined by PCR analysis of the extracted DNA as described by
*Permanent address: Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona, Israel. §To whom correspondence should be addressed. Tel.: ⫹ 972-3-6409624; fax: ⫹ 972-3-6407643. E-mail address: [email protected]
(D. M. Michaelson). Abbreviations: AChE, acetycholinesterase; AD, Alzheimer’s disease; apoE, apolipoprotein E; PCR, polymerase chain reaction; VAChT, vesicular acetylcholine transporter. 419
S. Chapman et al.
Xu et al. 31 Differentiation between the human apoE3 and apoE4 transgenes was done by PCR utilizing primers which amplify the DNA region which spans the apoE polymorphic site (AA 112). 3 The PCR products thus obtained (227 bp) were digested with the restriction endonuclease-AflIII (NEB), after which the resulting products (177 and 50 bp for apoE3, or 227 bp for apoE4) were analysed on 2% agarose gels as previously described. 3,30 The mice groups studied included: human apoE3 and apoE4 transgenic mice, control mice (C57BL/6J, Jackson Labs) and apoE-deficient mice which were pooled siblings of the apoE3 and apoE4 transgenic mice. Each mouse group contained six to eight 20-week-old male mice. All experiments were approved by the Tel Aviv University Animal Care and Use Committee and every effort was made to minimize animal suffering and to reduce the number of animals used. Brain autoradiography and histochemistry The mice were killed, after which their brains were rapidly excised and frozen in a mixture of isopentane and dry ice. Frozen coronal sections (20 mm) were cut in a cryostat at ⫺20⬚C, after which they were mounted on gelatin-coated glass slides and stored frozen at ⫺80⬚C until used. The densities of cholinergic, noradrenergic, dopaminergic and serotonergic nerve terminals were each assessed by autoradiographic measurements of the levels of the corresponding neurotransmitterspecific presynaptic transporters as previously described. 4 Cholinergic nerve terminals were visualized with [ 3H]AH5183 (35 Ci/mmol), which is a ligand of the presynaptic vesicular acetylcholine transporter (VAChT), and thus a specific marker of cholinergic nerve terminals. This was performed as described by Aubert et al. 1 [ 3H]Nisoxetine (85.9 Ci/mmol), which is a specific ligand of the noradrenergic presynaptic transporter, 28 [ 3H]GBR12935 (30 Ci/mmol), which binds specifically to the dopaminergic presynaptic transporter, 17 and [ 3H]Paroxetine (16 Ci/mmol), which is a specific ligand of the serotonergic presynaptic transporter, 11 were used to monitor the levels of the corresponding nerve terminals. This was performed autoradiographically, utilizing the tritium-sensitive film RPN 535 Hyperfilm (Amersham). 4 As previously described, the autoradiographs were calibrated by simultaneous exposure to the film with Amersham calibration scales for tritium. The films were developed by hand with the Kodak GBX system and all radiolabeled ligands were purchased from New England Nuclear. Acetylcholinesterase (AChE) activity was visualized histochemically by the Karnovsky method using acetylthiocholine as substrate. 14 Four consecutive sections of each brain at the levels of the septum (bregma 0.9) and of the hippocampus (bregma 1.5) 25 were treated by each of the above techniques. The former sections were used for examination of the frontal cortex, septum and striatum, and the latter for examining the parietal cortex and hippocampus. The circumference of each brain area was delineated utilizing a brain atlas, 25 after which its average level of staining was quantified as outlined below. Both hemispheres were examined in each section, yielding a total of eight replicates per brain area per mouse per staining technique.
When a significant effect (P ⬍ 0.05) was determined by ANOVA, a t-test was performed (Microsoft Excel, one tailed, equal variance) for comparison of the differences between two groups. The results are presented as mean ^ S.E.M., and probability above 95% was considered significant. RESULTS
Brain cholinergic nerve terminals of the four mice groups were visualized autoradiographically with the specific presynaptic cholinergic ligand [ 3H]AH5183. Representative brain sections of the apoE transgenic, apoE-deficient and control mice thus obtained at the level of the hippocampus are depicted in Fig. 1. As can be seen, the density of hippocampal cholinergic nerve terminals was lower in apoEdeficient mice than in the control. The densities of the hippocampal nerve terminals of the apoE3 and apoE4 transgenic mice were similar and were both higher than that of the apoEdeficient mice. Quantification of these results and of the levels of cholinergic nerve terminals of the four mice groups in additional brain areas was performed by computerized densitometry using six to eight mice in each group (Fig. 2). This revealed a significant difference between the four mice groups in the hippocampus (one-way ANOVA, F3,24 3.1; P ⬍ 0.05). The intensity of presynaptic hippocampal cholinergic staining in the apoE-deficient mice was significantly lower than that of control mice (87 ^ 6% of control; P ⬍ 0.05). In contrast, the densities of the cholinergic hippocampal neurons of the apoE3 and apoE4 transgenic mice were higher than the control and were similar (respectively, 106 ^ 4 and 110 ^ 6% of control). The amelioration of the cholinergic deficit in the apoE3 and apoE4 transgenic mice was statistically significant (P ⬍ 0.05 relative to the apoE-deficient mice), whereas the trend of increased
Morphometric densitometry The staining intensities of distinct brain areas of the developed autoradiographs and the AChE-stained sections were quantified as previously described, 8 utilizing an Olympus Cue-2 Image Analysis System (Lake Success, NY, U.S.A.) with software (Cue-2 Densitometry, version 4.0) developed by Galai Corp. (Migdal Ha’Emek, Israel). The system consisted of a photo-microscope fitted with a CCD video camera and a power unit that transmitted the microscopic images to an IBM PC on to a Sony color monitor. The Amersham calibration curve was used to transform the staining intensity of the autoradiographs into the radioactive intensity in mCi units. The net optical densities (e.g., after subtraction of background) of eight replicates of the entire indicated brain area of each mouse thus obtained were averaged and the resulting mean was used for comparing the staining intensity of the different mouse groups. Statistical analysis The mean score for each brain area of each mouse of the four mice groups was analysed using Statistix software and the statistical test one-way ANOVA of the optical density scores over the four groups. ANOVA was performed for each brain area separately.
Fig. 1. Comparison of levels of brain cholinergic nerve terminals in control (A), apoE-deficient (B), apoE3 (C) and apoE4 (D) transgenic mice. The micrographs shown depict representative hippocampal fields of coronal sections from the four mice groups. Frozen sections were processed as described in Experimental Procedures and the cholinergic nerve terminals were visualized autoradiographically utilizing the presynaptic cholinergic marker [ 3H]AH5183 as described by Aubert. 1
Reversal of neuronal deficits in apoE transgenic mice
Fig. 2. Quantitative comparison of the levels of brain cholinergic nerve terminals in control (empty bars), apoE-deficient (black bars), apoE3 transgenic (striped bars) and apoE4 transgenic (crossed bars) mice. The levels of cholinergic nerve terminals in the indicated brain areas were measured autoradiographically utilizing [ 3H]AH5183, which binds specifically to the presynaptic cholinergic marker VAChT. 1 Frozen coronal sections at the level of the hippocampus, the parietal cortex and the striatum, were autoradiographed with [ 3H]AH5183. The resulting intensities of staining of the indicated brain areas were then determined by computerized densitometry as described in Experimental Procedures. Results shown are the mean ^ S.E. of six to eight mice in each group. *P ⬍ 0.05 control vs apoE-deficient mice, #P ⬍ 0.05 apoE-deficient vs either the apoE3 or apoE4 transgenic mice.
hippocampal staining in the transgenic mice relative to the controls was not. Comparison of the densities of cortical cholinergic nerve terminals of the four mice groups (Fig. 2) revealed significant differences (one-way ANOVA, F3,25 3.01; P ⬍ 0.05). In this brain area, however, the difference was due to a significant increase in the levels of cortical cholinergic nerve terminals of the apoE3 and apoE4 transgenic mice groups relative to apoE-deficient and control mice (respectively, 110 ^ 4 and 110 ^ 5% of control, P ⬍ 0.05 for both comparisons), whereas the levels of cortical cholinergic nerve terminals of the apoE-deficient mice were not significantly different from those of the controls. Measurements of the levels of cholinergic interneurons in the striatum revealed a significant difference between the four groups (one-way ANOVA, F3,26 3.15; P ⬍ 0.05), which was due to significant increases in the levels of cholinergic nerve terminals of the apoE3 and apoE4 transgenic mice relative to apoE-deficient and control mice (P ⬍ 0.05). Consistently with previous observations, 8,10 the levels of striatal cholinergic nerve terminals were similar in the control and apoE-deficient mice (Fig. 2). The effects of apoE deficiency and of the apoE transgenes on brain cholinergic neurons were also assessed by histochemical measurements of AChE activity. This revealed a significant difference between the four groups in the parietal cortex (one-way ANOVA, F3,24 6.74; P ⬍ 0.01). This was associated with a significant reduction in AChE activity of the apoE-deficient mice (89 ^ 2% of control; P ⬍ 0.05), which was similarly reversed in the apoE3 (105 ^ 2% of control), and in the apoE4 transgenic mice (106 ^ 2% of control). AChE activity was also measured in the hippocampus and was not found to be statistically different between the four mice groups. The septum, which stains very faintly following autoradiography with the presynaptic marker [ 3H]AH5183, is intensely stained for AChE activity. Measurements of the intensities of AChE staining in the septum in the four mice groups revealed a significant difference (one-way ANOVA, F3,23 4.94; P ⬍ 0.05). This was associated with a significant reduction in septal AChE levels of the apoE-deficient mice (90 ^ 1% of control; P ⬍ 0.05), which was reversed in the apoE3 (98 ^ 5% of control) and the apoE4 transgenic mice (102 ^ 2% of control; P ⬍ 0.05). The AChE histochemical and VAChT autoradiographic results described above
show that cholinergic deficits of apoE deficient mice are equally ameliorated in the apoE3 and apoE4 transgenic mice and that in some instances (e.g., [ 3H]AH5183 binding in the parietal cortex and the striatum), this results in an enhancement of the cholinergic marker to levels above those of control. The levels of noradrenergic nerve terminals in the parietal cortex and the hippocampus were assessed autoradiographically utilizing [ 3H]nisoxetine, which is a specific marker of the presynaptic noradrenergic transporter. Representative micrographs thus obtained with brain sections of the four mice groups are depicted in Fig. 3. As can be seen, the intensities of staining in the parietal cortex and the hippocampus of the apoE-deficient mice were markedly lower than those of control, whereas those of the apoE3 and apoE4 transgenic mice had intermediate intensities. Quantification of these results by computerized densitometry using six to eight mice in each group is depicted in Fig. 4. This revealed a significant difference between the four groups in the hippocampus (one-way ANOVA, F3,26 17.08; P ⬍ 0.0001). The density of the noradrenergic nerve terminals in the apoEdeficient mice was markedly lower than the control (78 ^ 3% of control; P ⬍ 0.01). This deficit was partially ameliorated in both the apoE3 and apoE4 transgenic mice groups which had similar levels of noradrenergic nerve terminals (respectively, 87 ^ 4 and 89 ^ 3% of control, Fig. 4). The differences between hippocampal noradrenergic staining of the apoE3 and apoE4 transgenic mice relative to both the apoE-deficient and control mice were significant (P ⬍ 0.05, Fig. 4). Significant differences in the levels of noradrenergic staining of the four mice groups were also observed at the parietal cortex (one-way ANOVA, F3,26 3.59; P ⬍ 0.05). As can be seen in Fig. 4, the density of cortical noradrenergic nerve terminals of the apoE-deficient mice was markedly lower than that of the controls (86 ^ 5% of control; P ⬍ 0.05). This deficit was almost completely reversed in the apoE3 and apoE4 transgenic mice, whose levels of noradrenergic nerve terminals in the parietal cortex were, respectively, 96 ^ 4 and 97 ^ 5% of control. Autoradiographic measurements of the levels of dopaminergic nerve terminals in the striatum and frontal cortex utilizing the ligand [ 3H]GBR12935, which binds specifically to the presynaptic dopaminergic transporter, revealed no significant
S. Chapman et al.
Fig. 5. Quantitative comparison of the levels of brain dopaminergic nerve terminals in control (empty bars), apoE-deficient (black bars), apoE3 transgenic (striped bars) and apoE4 transgenic (crossed bars) mice. The levels of dopaminergic nerve terminals in the indicated brain areas were measured autoradiographically utilizing [ 3H]GBR12935, which binds specifically to the presynaptic dopaminergic transporter. Frozen coronal sections at the level of the hippocampus and the parietal cortex were autoradiographed with [ 3H]GBR12935 and the resulting staining intensities of the indicated brain areas were determined by computerized densitometry as described in Experimental Procedures. Results shown are means ^ S.E. of six to eight mice in each group. Fig. 3. Comparison of levels of brain noradrenergic nerve terminals in control (A), apoE-deficient (B), apoE3 (C) and apoE4 (D) transgenic mice. The micrographs shown depict representative hippocampal fields of coronal sections from the four mice groups. Frozen sections were processed as described in Experimental Procedures and the noradrenergic nerve terminals were visualized autoradiographically utilizing the presynaptic noradrenergic marker [ 3H]nisoxetine as described by Tejani-Butt. 28
differences between the four groups (Fig. 5). Similar results were obtained with [ 3H]paroxetine, which binds specifically to the presynaptic serotonergic transporter and which revealed that the levels of cortical serotonergic nerve terminals were not affected either by apoE deficiency or by the human apoE transgenes (not shown). Taken together, these results show that the human apoE transgenes can
Fig. 4. Quantitative comparison of the levels of brain noradrenergic nerve terminals in control (empty bars), apoE-deficient (black bars), apoE3 transgenic (striped bars) and apoE4 transgenic (crossed bars) mice. The levels of noradrenergic nerve terminals in the indicated brain areas were measured autoradiographically utilizing [ 3H]nisoxetine, which binds specifically to the presynaptic noradrenergic transporter. 28 Frozen coronal sections at the level of the hippocampus and the parietal cortex were autoradiographed with [ 3H]nisoxetine and the resulting staining intensities of the indicated brain areas were determined by computerized densitometry as described in Experimental Procedures. Results shown are the mean ^ S.E. of six to eight mice in each group. *P ⬍ 0.05 apoE-deficient vs control mice, #P ⬍ 0.05 apoE3 or apoE4 transgenic mice vs apoE-deficient or control mice.
ameliorate the cholinergic and noradrenergic derangements of apoE-deficient mice and that this effect is similar in both the apoE3 and apoE4 transgenic mice. DISCUSSION
The present study revealed that the specific presynaptic derangements of brain projecting cholinergic and noradrenegic neurons in apoE-deficient mice are ameliorated in apoE transgenic mice that contain either human apoE3 or apoE4 genes. By contrast, brain dopaminergic nerve terminals are unaffected either by mouse apoE deficiency or by the human apoE transgene. It has recently been shown, utilizing the general neuronal presynaptic marker synaptophysin and apoE transgenic mice similar to those presently employed, that 12-month-old apoE-deficient mice have reduced cortical synaptophysin levels, 16 and that this deficit is ameliorated in both human apoE3 and human apoE4 transgenic mice. 29 The present findings extend these observations and suggest that the susceptibility to apoE deficiency and its amelioration by the human apoE transgenics are neuron specific. Accordingly, the synaptic derangements monitored by the general presynaptic marker synaptophysin represent the weighted average of brain neurons whose nerve terminals are either apoE independent or apoE dependent to varying degrees. It should be noted that the general presynaptic deficiencies monitored by synaptophysin are significant only in aged apoE-deficient mice, and that the corresponding beneficial effects of the human apoE transgenes were therefore detectable only in aged mice. 16,29 In contrast, the presently observed neuronspecific ameliorating effects of the human apoE transgenes were already apparent in young mice. This suggests that the effects of apoE on distinct neuronal systems are age dependent. Accordingly, the presently observed effects of the human apoE transgenes on brain cholinergic and noradrenergic neurons may reflect a distinct role for apoE in the maintenance of these neurons, whereas the ameliorating effects of the apoE transgene in brain neurons of aging mice may be due to the involvement of apoE in neuronal repair mechanisms
Reversal of neuronal deficits in apoE transgenic mice
whose impact is expected to increase during aging. The observed specificity of the effects of apoE on neuronal maintenance may be due to differences in the levels of apoE receptors (e.g., LRP, VLDL, apoER2) of distinct brain neuronal systems, whose activation can have a marked effect on neuronal lipid metabolism and signaling. 6 Alternatively, this neuronal specificity may be due to differences in the levels of expression of apoE in distinct brain areas or to neuronspecific differences in energy metabolism and lipid turnover, which result in differential vulnerability to apoE deficiency. The profile of neuronal derangements presently observed with the genetically homogeneous C57BL apoE-deficient mice is qualitatively similar to that previously reported with a different line of mice on a heterogeneous C57BL/OLA129 background. 19 However, whereas in the genetically heterogeneous mice the extent of cholinergic derangement was larger than that of the noradrenergic neurons, 4 these neuronal systems were similarly affected in the genetically homogeneous mice. This suggests that the phenotypic expression of apoE deficiency might be affected by the genetic background of the mice. While this study showed that human apoE genes ameliorate the presynaptic derangements that are induced by apoE deficiency, this paradigm revealed no isoform-specific differences between the human apoE3 and apoE4 transgenes. However, it was recently shown, utilizing transgenic mice similar to those presently employed, that focal ischemia induces larger infarct volumes in the human apoE4 transgenic mice than in the apoE3 transgenic mice. 24 Furthermore, preliminary findings suggest that apoE4 transgenic mice are more susceptible to closed head injury than apoE3 transgenic mice are (unpublished observations). It thus seems that, while apoE is required for the maintenance of distinct neuronal systems, the isoform specificity of this effect becomes apparent and is accentuated during repair following injury. In addition to the presently studied presynaptic derangements, apoE-deficient mice have a decreased density of cortical neurites 16 which, unlike the presynaptic deficit, is reversed
specifically in the human apoE3 transgenic mice and not by the human apoE4 transgene. 29 However, utilizing a different line of apoE transgenic mice, whose human apoE genes are under the control of the neuron-specific enolase promoter, 21 it was shown that both the synaptic and dendritic deficits of these mice are reversed in human apoE3 but not in human apoE4 transgenic mice. 2 This suggests that the influence of apoE on neuronal maintenance and repair and its isoform specificity are possibly affected by factors such as the promoter system, which regulates the expression and the cellular compartmentation of the apoE transgene. In addition, the apoE-dependent phenotype might be dependent on the gene dosage of the apoE transgene. The recent development, by gene targeting, of human apoE-transgenic mice which retain the murine apoE-regulatory sequences 15,27 and the availability of human apoE-transgenic mice whose apoE expression is under the control of human neuronal 21 and non-neuronal 29 promoters now provides the means for careful experimental examination of this problem. In summary, the present study shows that cholinergic and noradrenergic derangements induced by apoE deficiency are ameliorated by both the human apoE3 and apoE4 transgenes. Taken together with previous observations, this suggests that apoE is specifically required for the normal function of distinct neuronal systems and that the isoform specific pathological effects of apoE are most pronounced following aging and brain insult. The molecular mechanisms underlying the neuron and isoform specific effects of apoE on neuronal maintenance and repair are yet to be determined. Acknowledgements—We thank Duke University and Glaxo Wellcome for kindly providing the transgenic mice. This work was supported partly by grants from the Joint German and Israeli research projects sponsored by the German and Israeli Ministries of Science (Grant 1626); from the Harry Stern National Center for Alzheimer’s Disease and Related Disorders; from the Jo and Inez Eichenbaum Foundation and from the Revah-Kabelli Fund. D.M.M. is the incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration.
1. Aubert I., Cecyre D., Gauthier S. and Quirion R. (1996) Comparative ontogenic profile of cholinergic markers, including nicotinic and muscarinic receptors, in the rat brain. J. comp. Neurol. 369, 31–55. 2. Buttini M., Orth M., Bellosta S., Akeefe H., Pitas R. E., Wyss-Coray T., Mucke L. and Mahley R. W. (1999) Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/- mice: isoform-specific effects on neurodegeneration. J. Neurosci. 19, 4867–4880. 3. Chapman J., Estupinan J., Asherov A. and Goldfarb L. G. (1996) A simple and efficient method for apolipoprotein E genotype determination. Neurology 46, 1484–1485. 4. Chapman S. and Michaelson D. M. (1998) Specific neurochemical derangements of brain projecting neurons in apolipoprotein E-deficient mice. J. Neurochem. 70, 708–714. 5. Chen Y., Lomnitski L., Michaelson D. M. and Shohami E. (1997) Motor and cognitive deficits in apolipoprotein E-deficient mice after closed head injury. Neuroscience 80, 1255–1262. 6. Cooper J. A. and Howell B. W. (1999) Lipoprotein receptors: signaling functions in the brain? Cell 97, 671–674. 7. Corder E. H., Saunders A. M., Strittmatter W. J., Schmechel D. E., Gaskell P. C., Small G. W., Roses A. D., Haines I. L. and Pericak-Vance M. A. (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261, 921–923. 8. Fisher A., Brandeis R., Chapman S., Pittel Z. and Michaelson D. M. (1998) M1 muscarinic agonist treatment reverses cognitive and cholinergic impairments of apolipoprotein E-deficient mice. J. Neurochem. 70, 1991–1997. 9. Francis P. T., Cross A. J. and Bowen D. M. (1994) Neurotransmitters and neuropeptides. In Alzheimer’s Disease (eds Terry R. D. and Katzman R. and Bick K. L. Raven Press, New York. 10. Gordon I., Grauer E., Genis I., Sehayek E. and Michaelson D. M. (1995) Memory deficits and cholinergic impairments in apolipoprotein E-deficient mice. Neurosci. Lett. 199, 1–4. 11. Hrdina P. D., Foy B., Hepner A. and Summers R. J. (1990) Antidepressant binding sites in brain: autoradiographic comparison of [ 3H]paroxetine and [ 3H]imipramine localization and relationship to serotonin transporter. J. Pharmac. exp. Ther. 252, 410–418. 12. Ignatius M. J., Gebicke-Harter P. J., Skene J. H., Schilling J. W., Weisgraber K. H., Mahley R. W. and Shooter E. M. (1986) Expression of apolipoprotein E during nerve degeneration and regeneration. Proc. Natn. Acad. Sci. U.S.A. 83, 1125–1129. 13. Inzelberg R., Chapman J., Treves T. A., Asherov A., Kiperwasser S., Hilkewicz O., Virshovski R., Klimowitzki S. and Korczyn A. D. (1998) Apolipoprotein E4 in Parkinson disease and dementia: new data and meta-analysis of published studies. Alzheimers Dis. Assoc. Disord. 12, 45–48.
S. Chapman et al.
14. Karnovsky M. J. and Roots L. A. (1964) A direct cloning thiocholine method for cholinesterases. J. Histochem. Cytochem. 121, 219–222. 15. Knouff C., Hinsdale M. E., Mezdour H., Altenburg M. K., Watanabe M., Quarfordt S. H., Sullivan P. M. and Maeda N. (1999) Apo E structure determines VLDL clearance and atherosclerosis risk in mice. J. clin. Invest. 103, 1579–1586. 16. Masliah E., Samuel W., Veinbergs I., Mallory M., Mante M. and Saitoh T. (1997) Neurodegeneration and cognitive impairment in apoE-deficient mice is ameliorated by infusion of recombinant apoE. Brain Res. 751, 307–314. 17. Mennicken F., Savasta M., Peretti-Renucci R. and Feuerstein C. (1992) Autoradiographic localization of dopamine uptake sites in the rat brain with 3HGBR 12935. J. neural Transm. Gen. Sect. 87, 1–14. 18. Nathan B. P., Bellosta S., Sanan D. A., Weisgraber K. H., Mahley R. W. and Pitas R. E. (1994) Differential effects of apolipoprotein E3 and E4 on neuronal growth in vitro. Science 264, 850–852. 19. Plump A. S., Smith J. D., Hayek T., Aalto-Setala K., Walsh A., Verstyft J. G., Rubin E. M. and Breslow J. L. (1992) Sever hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created. Cell 71, 343–353. 20. Poirier J. (1994) Apolipoprotein E in animal models of CNS injury and in Alzheimer’s disease. Trends Neurosci. 17, 525–530. 21. Raber J., Wong D., Buttini M., Orth M., Bellosta S., Pitas R. E., Mahley R. W. and Mucke L. (1998) Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: increased susceptibility of females. Proc. Natn. Acad. Sci. U.S.A. 95, 10,914–10,919. 22. Roses A. D. (1996) Apolipoprotein E alleles as risk factors in Alzheimer’s disease. A. Rev. Med. 47, 378–400. 23. Saunders A. M., Strittmatter W. J., Schmechel D., St. George-Hyslop P. H., Pericak-Vance M. A., Joo S. H., Rosi B. L., Gusella J. F., Crappermachlachlan D. R., Alberts M. J., Hullete C., Crain B., Goldgaber D. and Roses A. D. (1993) Association of apolipoprotein E allele E4 with late onset familial and sporadic Alzheimer’s disease. Neurology 43, 1467–1472. 24. Sheng H., Laskowitz D. T., Bennett E., Schmechel D. E., Bart R. D., Saunders A. M., Pearlstein R. D., Roses A. D. and Warner D. S. (1998) Apolipoprotein E isoform-specific differences in outcome from focal ischemia in transgenic mice. J. Cereb. Blood Flow Metab. 18, 361–366. 25. Slotnick B. M. and Leonard C. M. (1975) A Stereotaxic Atlas of the Albino Mouse Forebrain. Department of Health, Education and Welfare, Washington, DC. 26. Soininen H., Kosunen O., Helisalmi S., Mannermaa A., Paljarvi L., Talasniemi S., Ryynanen M. and Riekkinen P. S. (1995) A severe loss of choline acetyltransferase in the frontal cortex of Alzheimer patients carrying apolipoprotein epsilon 4 allele. Neurosci. Lett. 187, 79–82. 27. Sullivan P. M., Mezdour H., Quarfordt S. H. and Maeda N. (1998) Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2. J. clin. Invest. 102, 130–135. 28. Tejani-Butt S. M. (1992) [ 3H]Nisoxetine: a radioligand for quantitation of norepinephrine uptake sites by autoradiography or by homogenate binding. J. Pharmac. exp. Ther. 260, 427–436. 29. Veinbergs I., Mallory M., Mante M., Rockenstein E., Gilbert J. R. and Masliah E. (1999) Differential neurotrophic effects of apolipoprotein E in aged transgenic mice. Neurosci. Lett. 265, 218–222. 30. Wenham P. R., Price W. H. and Blandell G. (1991) Apolipoprotein E genotyping by one-stage PCR [Letter]. Lancet 337, 1158–1159. 31. Xu P. T., Schmechel D. E., Rothrock-Cristian T., Burkhart D. S., Qiu H. L., Popko B., Sullivan P., Maeda N. and Saunders A. M. (1996) Human apolipoprotein E2, E3 and E4 isoform-specific transgenic mice: human-like pattern of glial and neuronal immunoreactivity in central nervous system not observed in wild-type mice. Neurobiol. Dis. 1, 229–245. (Accepted 16 February 2000)