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Exogenous induction of cerebral β-amyloidosis in βAPP-transgenic mice
Peptides 23 (2002) 1241–1247
Exogenous induction of cerebral -amyloidosis in APP-transgenic mice Lary C. Walker a,∗ , Michael J. Callahan a , Feng Bian a , Robert A. Durham a , Alex E. Roher b , William J. Lipinski a b
a CNS Pharmacology, Pfizer Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA Haldeman Laboratory for Alzheimer’s Disease Research, Sun Health Research Institute, Sun City, AZ 85372, USA
Received 19 April 2001; accepted 3 June 2001
Abstract A key commonality of most age-related neurodegenerative diseases is the accumulation of aggregation-prone proteins in the brain. Except for the prionoses, the initiation and propagation of these proteopathies in vivo remains poorly understood. In a previous study, we found that the deposition of the amyloidogenic peptide A can be induced by injection of dilute extracts of Alzheimeric neocortex into the brains of Tg2576 transgenic mice overexpressing the human -amyloid precursor protein. The present study was undertaken to assess the pathology after long-term (12 months) incubation, and to clarify the distinctive anatomical distribution of seeded A-immunoreactivity. All mice were injected at 3 months of age; 5 months later, as expected, A deposits were concentrated mostly in the injected hemisphere. After 12 months, abundant, transgene-derived A deposits were present bilaterally in the forebrain, but plaque load was still clearly greater in the extract-injected hemisphere. There was also evidence of tau hyperphosphorylation in axons of the corpus callosum that had been injured by the injection, most prominently in transgenic mice, but also, to a lesser degree, in non-transgenic mice. Five months following injection of AD-extract, an isolated cluster of A-immunoreactive microglia was sometimes evident in the ipsilateral entorhinal cortex; the strong innervation of the hippocampus by entorhinal cortical neurons suggests the possible spread of seeded pathology from the injection site via neuronal transport mechanisms. Finally, using India Ink to map the local dispersion of injectate, we found that A induction is especially potent in places where the injectate is sequestered. The AD-seeding model can illuminate the emergence and spread of cerebral -amyloidosis and tau hyperphosphorylation, and thus could enhance our understanding of AD and its pathogenic commonalties with other cerebral proteopathies. © 2002 Published by Elsevier Science Inc. Keywords: Alzheimer; Amyloid; Angiopathy; A; Conformational disease; Neurodegeneration; Prion; Proteopathy; Senile plaque; Spongiform encephalopathy; Tau
1. Introduction The abnormal conformation and assembly of specific proteins is a central feature of virtually all degenerative diseases of the central nervous system [5,11,15,16,31,32,37]. These cerebral proteopathies include Alzheimer’s disease (AD), the prion diseases, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and a number of other neurodegenerative disorders [31,32]. A full understanding of the proteopathic cascade in vivo would accelerate the development of therapies for this large class of debilitating and intractable illnesses. A distinguishing feature of the prion diseases has been their transmissibility (within limits dictated by the animal species and agents involved) [6,20]. In contrast, the full pathology of AD has not yet been conveyed from one animal to another [2,6–8,12]. It may be significant in this regard ∗
Corresponding author. Tel.: +1-734-622-7518; fax: +1-734-622-7178. E-mail address: [email protected] (L.C. Walker).
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that prion diseases can occur in a variety of different animals [20], but AD has not been identified in any species other than humans [30]. Many animal species do, however, generate parenchymal and vascular -amyloid deposits as they age, including aged dogs and non-human primates [29,30], as well as transgenic mice overexpressing human APP (e.g. [3,18,19,25]). -Amyloid is composed mainly of the peptide A; although senile plaques and cerebrovascular amyloid per se are not sufficient to cause AD, pathological, biochemical and genetic data support the abnormal assembly of A as a critical element in the pathogenesis of AD [9,19,23,33,34]. Animal models can help to illuminate the modus operandi of A-polymerization in vivo. Mounting evidence suggests that the deposition of -amyloid can be stimulated by the intracerebral infusion of AD-brain material in non-human primates [2,17] and in APP-transgenic mice [12]. A significant advantage of transgenic mice for these studies is that the timecourse of -amyloid formation is markedly shorter than in primates [12]. Another benefit of transgenic
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Fig. 1. Amyloid deposition in the AD-extract-injected hemisphere (A) is significantly greater than in the contralateral hemisphere (B) of an 8-month-old Tg2576 mouse (5-month incubation period). Note the intense immunoreactivity in the hippocampal fissure, around blood vessels, and along the ventromedial subsurface of the hippocampus. The hippocampus was more heavily affected than was the overlying neocortex, possibly due in part to the larger volume of infusate in the hippocampus. A small region of A-immunoreactivity is marked by an arrow at the border of the hippocampal formation and ventral corpus callosum. The corpus callosum often displayed such small, diffuse A-immunoreactive patches that extended, with decrement, a considerable distance away. The contralateral hemisphere contained some A deposits (mostly near the midline), but there was approximately 10 times more A in the injected hemisphere than in the non-injected hemisphere at 8 months of age [12]. Antibody 4G8; hif = hippocampal fissure; magnification bar = 100 m.
mice is suggested by studies of experimentally transmitted prionosis. Prion protein-deficient (Prnp0/0 ) mice are resistant to prions [20], but mice overexpressing transgenic prion protein are especially vulnerable to the transmission of prion disease [21]. Accordingly, to test the hypothesis that APP-overexpressing mice would be prone to the induction of -amyloidosis, we infused Tg2576 mice intracerebrally with dilute AD-cortical extracts at 3 months of age and found excess accumulation of A in specific anatomical localities by 8 months of age ([12]; Fig. 1). The goals of the present study were to determine the long-term (12 months) effects of AD-extract seeding on A-deposition and tau hyperphosphorylation in transgenic mice, and to elucidate the distinctive anatomical pattern of seeded A-immunoreactivity in the hippocampus and neocortex.
2. Method 2.1. Infusion of tissue extract Neocortical tissue samples were obtained at autopsy from four AD cases, one age-matched control, and one young control case, as described in detail previously [12]. Clear, 1% tissue extracts (supernatants of homogenized cortex that had been centrifuged to remove particulate matter) were injected unilaterally into the hippocampus and neocortex of male Tg2576 [10] APP-transgenic mice and into non-transgenic,
littermate controls. In our initial study [12], the mice were injected at 3 months of age and allowed to survive until 8 months of age. This time-frame was chosen because we wanted to assess the effects of the extract injections before the emergence of normal (i.e. transgene-driven) plaques and cerebrovascular deposits. Typically, 8-month-old Tg2576 mice have little or no A-deposition, but by 15 months of age, plaques and amyloid angiopathy can be copious [4]. In the present study, to determine the longer-term effects of seeding, 14, 3-month-old male mice were infused with AD-cortical extract; seven mice were examined at 8 months of age, and seven were examined at 15 months of age. Three additional mice were infused with cortical extract from a 25-year-old control case [12] and analyzed at 15 months of age. All mice were killed under deep sodium pentobarbital anesthesia by transcardial perfusion with phosphate-buffered saline (PBS), pH 7.4, followed by phosphate-buffered, 4% paraformaldehyde (pH 7.2). Following post-fixation for 24 h, the brains were cryoprotected in phosphate-buffered, 25% sucrose (pH 7.4), frozen on dry ice, and sectioned in the coronal plane at 25 m thickness. To investigate the neuronal connectivity of the extractinjection sites, the fluorescent retrograde tracer Fast Blue (Sigma) was injected into the same regions of the hippocampus and neocortex (5 g/l final volume, 1 l per site) of two Tg2576 and two wild-type control mice. A small amount of India Ink suspension (0.8 l per site; Becton Dickinson Microbiology Systems, Sparks, MD) was included in the injectate to map the local disposition of injected
L.C. Walker et al. / Peptides 23 (2002) 1241–1247
material. After 7 days, these mice were perfusion-fixed with 4% paraformaldehyde under deep Nembutal anesthesia. The brains were cryoprotected, frozen on dry ice, and sectioned with a cryostat as described above. 2.2. Histology Tissue sections were stained immunohistochemically using primary monoclonal antibodies 6E10 (Senetek, Maryland Heights, MO) to amino acids 5–14 of A, 4G8 (Senetek) to amino acids 17–24 of A, AT-8 (Polymed, Chicago, IL) to tau phosphorylated at positions 202 and 205, with a monoclonal antibody to glial fibrillary acidic protein (GFAP; Boehringer Mannheim, Indianapolis, IN), and with polyclonal antibodies R163 to the C-terminal 8 amino acids of A40, and R165 to the C-terminal 8 amino acids of A42 (Pankaj Mehta, Institute for Basic Research in Developmental Disabilities, Staten Island, NY). Additional sections were stained with Congo Red and viewed with an Olympus BX60 microscope under cross-polarized light, or with hematoxylin and eosin.
3. Results 3.1. Aβ Infusion of AD-cortical extract into Tg2576 mouse brain reliably induced excess A-deposition, primarily in the ipsilateral hemisphere, both at 5 months (Fig. 1) and 12
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months (Fig. 3) post-infusion. In some 8-month-old transgenic mice (5 months incubation), there was a small region of A-immunoreactive gliosis ventral to the rhinal fissure (rf) in the entorhinal cortex, exclusively in the injected hemisphere (Fig. 2A). The cells were negative for GFAP and had the morphological appearance of microglial cells (Fig. 2A, inset). Retrograde tracing with Fast Blue was used to map neuronal input to the injection site. Neurons in the entorhinal cortex were intensely labeled by Fast Blue (Fig. 2B), as expected from the robust projections of this cortical region to the hippocampal formation [27,28]. There were no obvious differences in the retrograde transport of Fast Blue between transgenic and non-transgenic mice. In the absence of exogenous seeding, normal 15-month-old Tg2576 mice commonly have substantial A-deposition in the cortex and hippocampus, and males and females differ significantly in the amount of A deposited [4]. To reduce the degree of within-group variability, we studied only male mice, in which a mean of approximately 2.5–3% of the area of cortex and hippocampus has been found to be occupied by A-plaques at age 15 months [4]. Hence, as expected, transgenic mice that were injected at 3 months and killed at 15 months of age had obvious cerebral -amyloidosis bilaterally in the neocortex and hippocampus. However, the amyloid burden was appreciably greater in the injected hemisphere than in the non-injected hemisphere (Fig. 3). There was a tendency for AD-extract-injected mice to have greater overall A-deposition than mice infused with extract from the young control case, but this finding must
Fig. 2. (A) Small patches of A-immunoreactivity in the entorhinal cortex ventromedial to the rhinal fissure (rf) in an 8-month-old Tg2576 mouse, 5 months following injection with AD-cortical extract. The A-immunoreactivity in this region appeared to be associated with microglial cells; one cell is noted by an arrowhead, also at higher magnification in the inset. Antibody 6E10. (B) Entorhinal cortical neurons are intensely labeled by the retrogradely transported fluorescent marker Fast Blue in another Tg2576 mouse following injection of the marker into the ipsilateral dorsal hippocampus and neocortex. Magnification bar = 100 m (A + B) and 25 m (inset).
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Fig. 3. Ipsilateral (A) and contralateral (B) entorhinal cortices of a 15-month-old Tg2576 mouse injected approximately 1 year earlier with AD-cortical extract. Although A deposits are normally found in cortex and hippocampus of Tg2576 mice at this age, there was more A-immunoreactivity in the injected hemisphere. Antibody 4G8, rf = rhinal fissure, magnification bar = 50 m.
be replicated in a larger sample due to the high variability in plaque density that is normally found in 15-month-old Tg2576 mice [4]. 3.2. Tau There was no evidence of neurofibrillary tangles in any mice. However, particularly at 15 months of age, immunostaining with antibody AT-8 revealed axons immunoreac-
tive for phosphorylated tau in the corpus callosum of ADextract-injected mice (Fig. 4A). The axonal staining centered around the injection site, but, like the A deposits, sometimes extended along the corpus callosum, even crossing the midline into the non-injected hemisphere. AT-8-positive callosal axons were most pronounced in Tg2576 mice, and often corresponded to sites of intense A-immunoreactivity in the injected hemisphere (Fig. 4B). Surprisingly, there was a small amount of tau-immunoreactivity near the injection site
Fig. 4. (A) Phosphorylated tau- (antibody AT-8) immunoreactivity in the corpus callosum (ventral callosal border is marked by arrowheads) of a 15-month-old Tg2576 mouse injected approximately 1 year earlier with AD-cortical extract. The arrow denotes a senile plaque in the ventral neocortex containing a halo of small, AT-8-immunoreactive spheres; such plaques (including AT-8-immunoreactive elements) can be found normally in Tg2576 mice at this age, but tau-immunoreactivity in the callosal axons was associated only with extract-injection sites. Even non-transgenic mice showed some callosal tau-immunoreactivity following AD-extract injections (not shown). (B) In a nearby section from the same mouse, the corpus callosum is beset by numerous deposits of A (antibody 4G8). Magnification bar = 50 m.
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Fig. 5. Distribution of injectate in a Tg2576 mouse infused 7 days previously with India Ink into the hippocampus and neocortex. Note the accumulation of India Ink in the hippocampal fissure, around blood vessels (arrow), in the ventromedial hippocampal subsurface, and also along the corpus callosum. Magnification bar = 200 m.
of non-transgenic mice injected with AD tissue extract (not shown). Injection with control extracts from non-AD cortex produced no apparent phosphorylated tau-axonopathy. 3.3. Disposition of injectate The seeded A-immunoreactivity showed a characteristic anatomical distribution in the hippocampal fissure, around blood vessels, and along the outer subsurface of the hippocampus (see Fig. 1). Previously, we found no immunohistochemical evidence of the dilute AD-extract itself at 5 days, 2 weeks or 4 weeks following injection [12]. The infusion of India Ink allowed us to assess the diffusion and sequestration of material in the injected structures 1 week following surgery. The distribution of India Ink (Fig. 5) was similar to the proximate pattern of seeded A-deposition 5 months after AD-extract infusion, suggesting that the A-immunoreactivity that eventually emerges is most effectively induced where the injectate achieves the greatest local concentrations. The transgenic and non-transgenic mice showed similar patterns of India Ink dispersion and sequestration; furthermore, there was no evidence of differential removal of the material in transgenic versus non-transgenic mice.
4. Discussion Our findings show that the intracerebral infusion of dilute, Alzheimeric cortical extracts can activate the excess deposition of A as well as axonal tau hyperphosphorylation in APP-transgenic mice. The premature accumulation
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of seeded A seen in mice at 8 months of age [12] corresponds to a relatively greater amyloid burden in the injected hemisphere of mice at 15 months of age, 1 year following infusion of AD-extract. We also detected a focal cluster of A-immunoreactive microglia in the entorhinal cortex, which retrograde axonal tracing showed to project prominently to the injection site. Finally, we found that the pattern of A-deposition in the hippocampus and overlying structures corresponds closely to the anatomical distribution of the tracer India Ink injected into the same site, suggesting that the differential sequestration of infusate influences the intensity of seeding in the injected area. The stimulation of -amyloidogenesis by exogenous material suggests potential parallels between the mechanisms by which the abnormal accumulation of A and prion protein can be induced in vivo. Indeed, the use of Tg2576 mice to test the in vivo induction of -amyloidosis was partly modeled after a transgenic mouse model of prion disease transmission [21]. However, there appear to be important differences between the exogenous induction of prion disease and -amyloidosis. Prion disease is transmitted in an all-or-none fashion; except in the rare instances of genetic or spontaneous (sporadic) prionosis, unless an animal is properly exposed to the pathogenic agent, prion disease will not occur. In contrast, a variety of mammalian species, including susceptible APP-transgenic mice, will in due course develop some degree of -amyloidosis in the absence of apparent exogenous induction. Assuming that it is not widely “transmitted” by some as yet unidentified agent, -amyloidosis may more properly be said to be actuated or induced by exogenous tissue extracts in our seeding model. A similar situation holds for experimental amyloid A amyloidosis, which can be precipitated by sundry stimuli [13,22,24], but which can be dramatically accelerated by “amyloid-enhancing factor”, i.e. material derived from organs of animals that have been stimulated to produce amyloid A [13,14,22,24]. Conclusive identification of the specific element(s) needed to induce any of these diseases in living animals remains an important, but elusive, goal. In vitro, seeded polymerization tends not to occur below a critical concentration of protein [16]. Moreover, the efficiency with which prion disease can be transmitted to PrP-transgenic mice correlates directly with PrP expression levels in the recipient mice [21]. We speculate that the AD-brain extract contains abundant seeds for the ordered aggregation of A; in the context of high endogenous levels of transgenic “human” A, the seed stimulates the premature aggregation of A into diffuse and compact amyloid deposits in Tg2576 mice [12]. Demonstration that amyloid seeding can be accomplished using synthetic A alone would strengthen the case for a protein alone as the causative agent, but an unusual structural conformation and/or one or more modulating factors (see e.g. [1,20,37]) may be needed to bring about the pathogenic protein state. We were intrigued by the presence of hyperphosphorylated tau-immunoreactive callosal axons centered around the
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injection site, especially in AD-extract-injected mice analyzed at 15 months of age. No AT-8 (tau)-immunoreactive somata were apparent in any part of the brain that would suggest incipient, classical neurofibrillary tangles. Furthermore, a small amount of hyperphosphorylated tau also was evident in injured axons of non-transgenic, ADextract-injected mice. Hence, the pathological significance of axonal tau hyperphosphorylation in this model remains to be determined. In the AD-extract-injected hemisphere of some 8month-old Tg2576 mice, we noticed A-immunoreactive microglial cells in the entorhinal cortex, a considerable distance from the injection site. Microglia are closely linked to -amyloid deposits in animals and humans [26,30,35,36], but whether they deposit or remove A (or both) is still controversial. In 15-month-old mice, approximately 1 year after AD tissue injection, the entorhinal cortex in the injected hemisphere displayed considerably more A deposits than did the contralateral entorhinal cortex. Retrograde tracing studies in transgenic mice confirmed that the entorhinal cortex projects heavily to the extract-injection site, suggesting, at least in this paradigm, that axonal transport mechanisms may contribute to the spread of seeded -amyloid pathology in the brain. The results also imply that microglial cells are involved in the early response of the entorhinal cortex to incipient A accumulation, but the nature of the response is uncertain. The A seeding model could be employed to explore these issues experimentally. In summary, our findings indicate that: (1) cerebral -amyloidosis can be exogenously induced in transgenic mice by AD-brain extracts, resulting in long-term augmentation of amyloid load; (2) neuronal transport mechanisms may contribute to the spread of seeded pathology to other brain regions; and (3) tau hyperphosphorylation is associated with callosal axons passing through the extract-injection site. This mouse seeding model may be useful in identifying the essential ingredients for abnormal -amyloid polymerization in vivo, and could help to define the events involved in the early pathogenesis of AD as well as other cerebral proteopathies.
Acknowledgments We gratefully acknowledge helpful discussions with Michael Kane, Harry LeVine and Roy Schwarz. References [1] Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NME, Romano DM, et al. Dramatic aggregation of Alzheimer A by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 1998;273(21):12817–26. [2] Baker HF, Ridley RM, Duchen LW, Crow TJ, Bruton CJ. Induction of (A4)-amyloid in primates by injection of Alzheimer’s disease brain homogenate. Comparison with transmission of spongiform encephalopathy. Mol Neurobiol 1994;8(1):25–39.
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L.C. Walker et al. / Peptides 23 (2002) 1241–1247 [25] Sommer B. Recent advances in transgenic model development for Alzheimer’s disease. Exp Opin Invest Drugs 1998;7:2017–25. [26] Stalder M, Phinney A, Probst A, Sommer B, Staufenbiel M, Jucker M. Association of microglia with amyloid plaques in brains of APP23 transgenic mice. Am J Pathol 1999;154:1673–84. [27] Steward O, Scoville SA. Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat. J Comp Neurol 1976;169:347–70. [28] Super H, Soriano E. The organization of the embryonic, commissural, and septal connections studied with the lipophilic tracer DiI. J Comp Neurol 1994;344:101–20. [29] Walker LC. Cerebral amyloid angiopathy in aged dogs and non-human primates. In: Verbeek MM, Vinters HV, editors. Cerebral amyloid angiopathy in Alzheimer’s disease and related disorders. Boston: Kluwer Academic Publishers, 2000. p. 313–24. [30] Walker LC, Cork LC. The neurobiology of aging in non-human primates. In: Terry RD, Katzman R, Bick KL, Sisodia SS, editors. Alzheimer disease. Philadelphia: Lippincott, Williams and Wilkins, 1999. p. 233–43. [31] Walker LC, LeVine III H. The cerebral proteopathies. Neurobiol Aging 2000;21:559–61.
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[32] Walker LC, LeVine III H. The cerebral proteopathies: neurodegenerative disorders of protein conformation and assembly. Mol Neurobiol 2000;21:83–95. [33] Walker LC, Pahnke J, Madauss M, Vogelgesang S, Pahnke A, Herbst EW, et al. Apolipoprotein E4 promotes the early deposition of A42 and then A40 in the elderly. Acta Neuropathol 2000;100: 36–42. [34] Warzok RW, Kessler C, Apel G, Schwarz A, Egensperger R, Schreiber D, et al. Apolipoprotein E4 promotes incipient Alzheimer pathology in the elderly. Alzheimer Dis Assoc Disord 1998;12: 33–9. [35] Wegiel J, Wang K-C, Tarnawski M, Lach B. Microglial cells are the driving force in fibrillar plaque formation, whereas astrocytes are a leading factor in plaque degradation. Acta Neuropathol 2000;100: 356–64. [36] Wegiel J, Wang K-C, Imaki H, Rubenstein R, Wronska A, Osuchowski M, et al. The role of microglial cells and astrocytes in fibrillar plaque evolution in transgenic APPsw mice. Neurobiol Aging 2001;22:49–61. [37] Wisniewski T, Aucouturier P, Soto C, Frangione B. The prionoses and other conformational disorders. Amyloid 1998;5:212–24.
Exogenous induction of cerebral -amyloidosis in APP-transgenic mice Lary C. Walker a,∗ , Michael J. Callahan a , Feng Bian a , Robert A. Durham a , Alex E. Roher b , William J. Lipinski a b
a CNS Pharmacology, Pfizer Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA Haldeman Laboratory for Alzheimer’s Disease Research, Sun Health Research Institute, Sun City, AZ 85372, USA
Received 19 April 2001; accepted 3 June 2001
Abstract A key commonality of most age-related neurodegenerative diseases is the accumulation of aggregation-prone proteins in the brain. Except for the prionoses, the initiation and propagation of these proteopathies in vivo remains poorly understood. In a previous study, we found that the deposition of the amyloidogenic peptide A can be induced by injection of dilute extracts of Alzheimeric neocortex into the brains of Tg2576 transgenic mice overexpressing the human -amyloid precursor protein. The present study was undertaken to assess the pathology after long-term (12 months) incubation, and to clarify the distinctive anatomical distribution of seeded A-immunoreactivity. All mice were injected at 3 months of age; 5 months later, as expected, A deposits were concentrated mostly in the injected hemisphere. After 12 months, abundant, transgene-derived A deposits were present bilaterally in the forebrain, but plaque load was still clearly greater in the extract-injected hemisphere. There was also evidence of tau hyperphosphorylation in axons of the corpus callosum that had been injured by the injection, most prominently in transgenic mice, but also, to a lesser degree, in non-transgenic mice. Five months following injection of AD-extract, an isolated cluster of A-immunoreactive microglia was sometimes evident in the ipsilateral entorhinal cortex; the strong innervation of the hippocampus by entorhinal cortical neurons suggests the possible spread of seeded pathology from the injection site via neuronal transport mechanisms. Finally, using India Ink to map the local dispersion of injectate, we found that A induction is especially potent in places where the injectate is sequestered. The AD-seeding model can illuminate the emergence and spread of cerebral -amyloidosis and tau hyperphosphorylation, and thus could enhance our understanding of AD and its pathogenic commonalties with other cerebral proteopathies. © 2002 Published by Elsevier Science Inc. Keywords: Alzheimer; Amyloid; Angiopathy; A; Conformational disease; Neurodegeneration; Prion; Proteopathy; Senile plaque; Spongiform encephalopathy; Tau
1. Introduction The abnormal conformation and assembly of specific proteins is a central feature of virtually all degenerative diseases of the central nervous system [5,11,15,16,31,32,37]. These cerebral proteopathies include Alzheimer’s disease (AD), the prion diseases, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and a number of other neurodegenerative disorders [31,32]. A full understanding of the proteopathic cascade in vivo would accelerate the development of therapies for this large class of debilitating and intractable illnesses. A distinguishing feature of the prion diseases has been their transmissibility (within limits dictated by the animal species and agents involved) [6,20]. In contrast, the full pathology of AD has not yet been conveyed from one animal to another [2,6–8,12]. It may be significant in this regard ∗
Corresponding author. Tel.: +1-734-622-7518; fax: +1-734-622-7178. E-mail address: [email protected] (L.C. Walker).
0196-9781/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 0 5 9 - 1
that prion diseases can occur in a variety of different animals [20], but AD has not been identified in any species other than humans [30]. Many animal species do, however, generate parenchymal and vascular -amyloid deposits as they age, including aged dogs and non-human primates [29,30], as well as transgenic mice overexpressing human APP (e.g. [3,18,19,25]). -Amyloid is composed mainly of the peptide A; although senile plaques and cerebrovascular amyloid per se are not sufficient to cause AD, pathological, biochemical and genetic data support the abnormal assembly of A as a critical element in the pathogenesis of AD [9,19,23,33,34]. Animal models can help to illuminate the modus operandi of A-polymerization in vivo. Mounting evidence suggests that the deposition of -amyloid can be stimulated by the intracerebral infusion of AD-brain material in non-human primates [2,17] and in APP-transgenic mice [12]. A significant advantage of transgenic mice for these studies is that the timecourse of -amyloid formation is markedly shorter than in primates [12]. Another benefit of transgenic
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Fig. 1. Amyloid deposition in the AD-extract-injected hemisphere (A) is significantly greater than in the contralateral hemisphere (B) of an 8-month-old Tg2576 mouse (5-month incubation period). Note the intense immunoreactivity in the hippocampal fissure, around blood vessels, and along the ventromedial subsurface of the hippocampus. The hippocampus was more heavily affected than was the overlying neocortex, possibly due in part to the larger volume of infusate in the hippocampus. A small region of A-immunoreactivity is marked by an arrow at the border of the hippocampal formation and ventral corpus callosum. The corpus callosum often displayed such small, diffuse A-immunoreactive patches that extended, with decrement, a considerable distance away. The contralateral hemisphere contained some A deposits (mostly near the midline), but there was approximately 10 times more A in the injected hemisphere than in the non-injected hemisphere at 8 months of age [12]. Antibody 4G8; hif = hippocampal fissure; magnification bar = 100 m.
mice is suggested by studies of experimentally transmitted prionosis. Prion protein-deficient (Prnp0/0 ) mice are resistant to prions [20], but mice overexpressing transgenic prion protein are especially vulnerable to the transmission of prion disease [21]. Accordingly, to test the hypothesis that APP-overexpressing mice would be prone to the induction of -amyloidosis, we infused Tg2576 mice intracerebrally with dilute AD-cortical extracts at 3 months of age and found excess accumulation of A in specific anatomical localities by 8 months of age ([12]; Fig. 1). The goals of the present study were to determine the long-term (12 months) effects of AD-extract seeding on A-deposition and tau hyperphosphorylation in transgenic mice, and to elucidate the distinctive anatomical pattern of seeded A-immunoreactivity in the hippocampus and neocortex.
2. Method 2.1. Infusion of tissue extract Neocortical tissue samples were obtained at autopsy from four AD cases, one age-matched control, and one young control case, as described in detail previously [12]. Clear, 1% tissue extracts (supernatants of homogenized cortex that had been centrifuged to remove particulate matter) were injected unilaterally into the hippocampus and neocortex of male Tg2576 [10] APP-transgenic mice and into non-transgenic,
littermate controls. In our initial study [12], the mice were injected at 3 months of age and allowed to survive until 8 months of age. This time-frame was chosen because we wanted to assess the effects of the extract injections before the emergence of normal (i.e. transgene-driven) plaques and cerebrovascular deposits. Typically, 8-month-old Tg2576 mice have little or no A-deposition, but by 15 months of age, plaques and amyloid angiopathy can be copious [4]. In the present study, to determine the longer-term effects of seeding, 14, 3-month-old male mice were infused with AD-cortical extract; seven mice were examined at 8 months of age, and seven were examined at 15 months of age. Three additional mice were infused with cortical extract from a 25-year-old control case [12] and analyzed at 15 months of age. All mice were killed under deep sodium pentobarbital anesthesia by transcardial perfusion with phosphate-buffered saline (PBS), pH 7.4, followed by phosphate-buffered, 4% paraformaldehyde (pH 7.2). Following post-fixation for 24 h, the brains were cryoprotected in phosphate-buffered, 25% sucrose (pH 7.4), frozen on dry ice, and sectioned in the coronal plane at 25 m thickness. To investigate the neuronal connectivity of the extractinjection sites, the fluorescent retrograde tracer Fast Blue (Sigma) was injected into the same regions of the hippocampus and neocortex (5 g/l final volume, 1 l per site) of two Tg2576 and two wild-type control mice. A small amount of India Ink suspension (0.8 l per site; Becton Dickinson Microbiology Systems, Sparks, MD) was included in the injectate to map the local disposition of injected
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material. After 7 days, these mice were perfusion-fixed with 4% paraformaldehyde under deep Nembutal anesthesia. The brains were cryoprotected, frozen on dry ice, and sectioned with a cryostat as described above. 2.2. Histology Tissue sections were stained immunohistochemically using primary monoclonal antibodies 6E10 (Senetek, Maryland Heights, MO) to amino acids 5–14 of A, 4G8 (Senetek) to amino acids 17–24 of A, AT-8 (Polymed, Chicago, IL) to tau phosphorylated at positions 202 and 205, with a monoclonal antibody to glial fibrillary acidic protein (GFAP; Boehringer Mannheim, Indianapolis, IN), and with polyclonal antibodies R163 to the C-terminal 8 amino acids of A40, and R165 to the C-terminal 8 amino acids of A42 (Pankaj Mehta, Institute for Basic Research in Developmental Disabilities, Staten Island, NY). Additional sections were stained with Congo Red and viewed with an Olympus BX60 microscope under cross-polarized light, or with hematoxylin and eosin.
3. Results 3.1. Aβ Infusion of AD-cortical extract into Tg2576 mouse brain reliably induced excess A-deposition, primarily in the ipsilateral hemisphere, both at 5 months (Fig. 1) and 12
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months (Fig. 3) post-infusion. In some 8-month-old transgenic mice (5 months incubation), there was a small region of A-immunoreactive gliosis ventral to the rhinal fissure (rf) in the entorhinal cortex, exclusively in the injected hemisphere (Fig. 2A). The cells were negative for GFAP and had the morphological appearance of microglial cells (Fig. 2A, inset). Retrograde tracing with Fast Blue was used to map neuronal input to the injection site. Neurons in the entorhinal cortex were intensely labeled by Fast Blue (Fig. 2B), as expected from the robust projections of this cortical region to the hippocampal formation [27,28]. There were no obvious differences in the retrograde transport of Fast Blue between transgenic and non-transgenic mice. In the absence of exogenous seeding, normal 15-month-old Tg2576 mice commonly have substantial A-deposition in the cortex and hippocampus, and males and females differ significantly in the amount of A deposited [4]. To reduce the degree of within-group variability, we studied only male mice, in which a mean of approximately 2.5–3% of the area of cortex and hippocampus has been found to be occupied by A-plaques at age 15 months [4]. Hence, as expected, transgenic mice that were injected at 3 months and killed at 15 months of age had obvious cerebral -amyloidosis bilaterally in the neocortex and hippocampus. However, the amyloid burden was appreciably greater in the injected hemisphere than in the non-injected hemisphere (Fig. 3). There was a tendency for AD-extract-injected mice to have greater overall A-deposition than mice infused with extract from the young control case, but this finding must
Fig. 2. (A) Small patches of A-immunoreactivity in the entorhinal cortex ventromedial to the rhinal fissure (rf) in an 8-month-old Tg2576 mouse, 5 months following injection with AD-cortical extract. The A-immunoreactivity in this region appeared to be associated with microglial cells; one cell is noted by an arrowhead, also at higher magnification in the inset. Antibody 6E10. (B) Entorhinal cortical neurons are intensely labeled by the retrogradely transported fluorescent marker Fast Blue in another Tg2576 mouse following injection of the marker into the ipsilateral dorsal hippocampus and neocortex. Magnification bar = 100 m (A + B) and 25 m (inset).
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Fig. 3. Ipsilateral (A) and contralateral (B) entorhinal cortices of a 15-month-old Tg2576 mouse injected approximately 1 year earlier with AD-cortical extract. Although A deposits are normally found in cortex and hippocampus of Tg2576 mice at this age, there was more A-immunoreactivity in the injected hemisphere. Antibody 4G8, rf = rhinal fissure, magnification bar = 50 m.
be replicated in a larger sample due to the high variability in plaque density that is normally found in 15-month-old Tg2576 mice [4]. 3.2. Tau There was no evidence of neurofibrillary tangles in any mice. However, particularly at 15 months of age, immunostaining with antibody AT-8 revealed axons immunoreac-
tive for phosphorylated tau in the corpus callosum of ADextract-injected mice (Fig. 4A). The axonal staining centered around the injection site, but, like the A deposits, sometimes extended along the corpus callosum, even crossing the midline into the non-injected hemisphere. AT-8-positive callosal axons were most pronounced in Tg2576 mice, and often corresponded to sites of intense A-immunoreactivity in the injected hemisphere (Fig. 4B). Surprisingly, there was a small amount of tau-immunoreactivity near the injection site
Fig. 4. (A) Phosphorylated tau- (antibody AT-8) immunoreactivity in the corpus callosum (ventral callosal border is marked by arrowheads) of a 15-month-old Tg2576 mouse injected approximately 1 year earlier with AD-cortical extract. The arrow denotes a senile plaque in the ventral neocortex containing a halo of small, AT-8-immunoreactive spheres; such plaques (including AT-8-immunoreactive elements) can be found normally in Tg2576 mice at this age, but tau-immunoreactivity in the callosal axons was associated only with extract-injection sites. Even non-transgenic mice showed some callosal tau-immunoreactivity following AD-extract injections (not shown). (B) In a nearby section from the same mouse, the corpus callosum is beset by numerous deposits of A (antibody 4G8). Magnification bar = 50 m.
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Fig. 5. Distribution of injectate in a Tg2576 mouse infused 7 days previously with India Ink into the hippocampus and neocortex. Note the accumulation of India Ink in the hippocampal fissure, around blood vessels (arrow), in the ventromedial hippocampal subsurface, and also along the corpus callosum. Magnification bar = 200 m.
of non-transgenic mice injected with AD tissue extract (not shown). Injection with control extracts from non-AD cortex produced no apparent phosphorylated tau-axonopathy. 3.3. Disposition of injectate The seeded A-immunoreactivity showed a characteristic anatomical distribution in the hippocampal fissure, around blood vessels, and along the outer subsurface of the hippocampus (see Fig. 1). Previously, we found no immunohistochemical evidence of the dilute AD-extract itself at 5 days, 2 weeks or 4 weeks following injection [12]. The infusion of India Ink allowed us to assess the diffusion and sequestration of material in the injected structures 1 week following surgery. The distribution of India Ink (Fig. 5) was similar to the proximate pattern of seeded A-deposition 5 months after AD-extract infusion, suggesting that the A-immunoreactivity that eventually emerges is most effectively induced where the injectate achieves the greatest local concentrations. The transgenic and non-transgenic mice showed similar patterns of India Ink dispersion and sequestration; furthermore, there was no evidence of differential removal of the material in transgenic versus non-transgenic mice.
4. Discussion Our findings show that the intracerebral infusion of dilute, Alzheimeric cortical extracts can activate the excess deposition of A as well as axonal tau hyperphosphorylation in APP-transgenic mice. The premature accumulation
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of seeded A seen in mice at 8 months of age [12] corresponds to a relatively greater amyloid burden in the injected hemisphere of mice at 15 months of age, 1 year following infusion of AD-extract. We also detected a focal cluster of A-immunoreactive microglia in the entorhinal cortex, which retrograde axonal tracing showed to project prominently to the injection site. Finally, we found that the pattern of A-deposition in the hippocampus and overlying structures corresponds closely to the anatomical distribution of the tracer India Ink injected into the same site, suggesting that the differential sequestration of infusate influences the intensity of seeding in the injected area. The stimulation of -amyloidogenesis by exogenous material suggests potential parallels between the mechanisms by which the abnormal accumulation of A and prion protein can be induced in vivo. Indeed, the use of Tg2576 mice to test the in vivo induction of -amyloidosis was partly modeled after a transgenic mouse model of prion disease transmission [21]. However, there appear to be important differences between the exogenous induction of prion disease and -amyloidosis. Prion disease is transmitted in an all-or-none fashion; except in the rare instances of genetic or spontaneous (sporadic) prionosis, unless an animal is properly exposed to the pathogenic agent, prion disease will not occur. In contrast, a variety of mammalian species, including susceptible APP-transgenic mice, will in due course develop some degree of -amyloidosis in the absence of apparent exogenous induction. Assuming that it is not widely “transmitted” by some as yet unidentified agent, -amyloidosis may more properly be said to be actuated or induced by exogenous tissue extracts in our seeding model. A similar situation holds for experimental amyloid A amyloidosis, which can be precipitated by sundry stimuli [13,22,24], but which can be dramatically accelerated by “amyloid-enhancing factor”, i.e. material derived from organs of animals that have been stimulated to produce amyloid A [13,14,22,24]. Conclusive identification of the specific element(s) needed to induce any of these diseases in living animals remains an important, but elusive, goal. In vitro, seeded polymerization tends not to occur below a critical concentration of protein [16]. Moreover, the efficiency with which prion disease can be transmitted to PrP-transgenic mice correlates directly with PrP expression levels in the recipient mice [21]. We speculate that the AD-brain extract contains abundant seeds for the ordered aggregation of A; in the context of high endogenous levels of transgenic “human” A, the seed stimulates the premature aggregation of A into diffuse and compact amyloid deposits in Tg2576 mice [12]. Demonstration that amyloid seeding can be accomplished using synthetic A alone would strengthen the case for a protein alone as the causative agent, but an unusual structural conformation and/or one or more modulating factors (see e.g. [1,20,37]) may be needed to bring about the pathogenic protein state. We were intrigued by the presence of hyperphosphorylated tau-immunoreactive callosal axons centered around the
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injection site, especially in AD-extract-injected mice analyzed at 15 months of age. No AT-8 (tau)-immunoreactive somata were apparent in any part of the brain that would suggest incipient, classical neurofibrillary tangles. Furthermore, a small amount of hyperphosphorylated tau also was evident in injured axons of non-transgenic, ADextract-injected mice. Hence, the pathological significance of axonal tau hyperphosphorylation in this model remains to be determined. In the AD-extract-injected hemisphere of some 8month-old Tg2576 mice, we noticed A-immunoreactive microglial cells in the entorhinal cortex, a considerable distance from the injection site. Microglia are closely linked to -amyloid deposits in animals and humans [26,30,35,36], but whether they deposit or remove A (or both) is still controversial. In 15-month-old mice, approximately 1 year after AD tissue injection, the entorhinal cortex in the injected hemisphere displayed considerably more A deposits than did the contralateral entorhinal cortex. Retrograde tracing studies in transgenic mice confirmed that the entorhinal cortex projects heavily to the extract-injection site, suggesting, at least in this paradigm, that axonal transport mechanisms may contribute to the spread of seeded -amyloid pathology in the brain. The results also imply that microglial cells are involved in the early response of the entorhinal cortex to incipient A accumulation, but the nature of the response is uncertain. The A seeding model could be employed to explore these issues experimentally. In summary, our findings indicate that: (1) cerebral -amyloidosis can be exogenously induced in transgenic mice by AD-brain extracts, resulting in long-term augmentation of amyloid load; (2) neuronal transport mechanisms may contribute to the spread of seeded pathology to other brain regions; and (3) tau hyperphosphorylation is associated with callosal axons passing through the extract-injection site. This mouse seeding model may be useful in identifying the essential ingredients for abnormal -amyloid polymerization in vivo, and could help to define the events involved in the early pathogenesis of AD as well as other cerebral proteopathies.
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