NeurobiologyofAging,Vol. 13, pp. 537-542, 1992

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In Vivo Neurotoxicity of Beta-Amyloid [/3(1-40)] and the ¢ (25-35) Fragment N. W. KOWALL,*'~ A. C. MCKEE,t I B. A. Y A N K N E R •

A N D M. F. BEAL*

*Neurology Service and ?Department of Pathology, Massachusetts General Hospital and ~Department of Neurology, Childrens Hospital, Boston MA 02115 Received 15 May 1992; Accepted 15 J u n e 1992 KOWALL, N. W., A. C. MCKEE, B. A. YANKNER AND M. F. BEAL. In vivo neurotoxicity ofbeta-amyloid [/3(1-40)]and the /3(25-35)Jragmenl. NEUROBIOL AGING 13(5) 537-542, 1992.--We examined the histologicalchanges produced by injections of beta-amyloid [/3(1-40)], and control peptides in rat and monkey cerebral cortex./3(25-35) injections were also studied in rat cortex. Standard immunoperoxidase procedures were used to detect the distribution of tau, MAP2, /3(1-40) and ALZ 50 immunoreactivity. All injections produced localized necrosis at the injection site surrounded by a zone of neuronal loss and gliosis. In rat cortex, lesions produced by solubilized/3(1-40)and/3(25-35) in water were generally larger than those produced by control peptides. Tau and ALZ 50 antibodies labeled neurites and diffuselypositive perikarya around N 1-40) injections, whereas MAP2 staining was reduced, parallelingthe distribution of neuronal loss and gliosis. In aged primate cortex,/3(1-40) lesion size was dose dependent. Hyalinized, ALZ 50 positive neurons, and abnormal neurites were prominent around the injection site. Although/3amyloid is acutely neurotoxic in both rat and monkey cerebral cortex, neuronal degenerationin the primate more closely resembles that found in AD. /3-Amyloid Cell death

Alzheimer's disease Primate Rat

Neurotoxicity

Cytoskeleton

THE cause of neuronal degeneration in Alzheimer's disease (AD) is unknown, but attention has recently focused on the potential role of/3-amyloid [/3(1-40)], a 38-42 amino acid peptide found in senile plaques that is derived from a transmembrane precursor glycoprotein (8,20,22). Several lines of evidence suggest that abnormal degradation of the amyloid precursor protein (APP) leads to extracellular deposition of/3( 1-40)--a pathological alteration that precedes the onset of dementia in Down's syndrome and AD [reviewed in (8)] (13,15). Recently, point mutations in the APP molecule have been found in several families with autosomal dominant AD (4,7,16), indicating that abnormalities of APP may directly lead to AD in some cases, possibly due to the deleterious effects of/3(1-40). In order to test the hypothesis that/3(1-40) causes neuronal degeneration in AD, Yankner and colleagues examined the effects of/3(1-40) and related peptides in vitro in a dispersed culture system (21 ). They found that solubilized/3(1-40) and the/3(2535) fragment increased the rate of cell death in vitro. Subsequently, others reported that/3(I-40) enhanced glutamate toxicity (11,14) and that aged/3(1-40) was directly neurotoxic in vitro (18). We initially reported the neurotoxicity of soluhilized /3(140) injected locally in rat cerebral cortex and hippocampus (12). More recently, Frautschy et al. reported that senile plaque cores are neurotoxic in vivo in the rat (6) and Neve et al. found that the carboxyterminal of APP produces neurodegeneration in mice

ALZ 50

Tau

Protein

Aging

(17). We undertook the present studies to further examine the cytoskeletal alterations produced by/3(1-40) and to examine the potential neurotoxicity of the/3(25-35) fragment in vivo. In addition, we report preliminary observations on/3(1-40) toxicity in primate cortex. METHOD

Method OfPreparation and Source ~?fPeptide Previous studies indicate that the chemical and physical state offl(1-40) may alter its neurotoxicity (18). The/3(1-40) peptide used in these studies has been characterized by mass spectroscopy and direct amino acid sequencing (Yankner, unpublished observations). Care was taken to ensure that excitatory amino acids, i,e., glutamate and aspartate, did not contaminate the preparation. Our initial studies were performed using solubilized/3( 140) in acetonitrile with 1% trifluroacetic acid./3(1-40), reverse peptide [/3(40-1)], and A37 control peptides were synthesized by Dr. Larry Duffy. The scrambled peptide (CA4) was provided by Dr. Dennis Selkoe. More recently, we used/3(25-35) purchased from Bachem (Torrance, CA) which is soluble in deionized double-distilled water. The peptides were stored in aliquots at - 8 0 ° C until thawed and used on the day of the experiment. The concentration of peptide injected was 3 nmol in a volume of 1 ~1.

Requests for reprints should be addressed to Ann C. McKee, M.D., Department of Pathology, Massachusetts General Hospital, Boston, MA 02114.

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Injection Technique The successful production of lesions is critically dependant on proper injection methodology. Care was taken to avoid deep injections into the corpus callosum and superficial injections that leak into the subarachnoid space. Ideally, injections are made into deep cerebral cortex so that leakage up the needle track is confined to the cerebral cortex. A blunt-tipped syringe is required to produce localized injections with minimal backflow. We used a 30-gauge Hamilton syringe with a l-inch stainless steel blunt-tipped needle. Plastic tubing was avoided. Rats were anesthetized with pentobarbital (50 mg/kg, IP) followed by placement in a stereotaxic frame. A burr hole was made 1.8 mm posterior to the Bregma and 3.0 mm lateral to the midline. The needle was lowered until it touched the dura, which then becomes less translucent. The needle was lowered 2.3 mm from that point and 1 ;1 of solution (3 nmol) is injected over 1 rain using a microdrive. The needle was then pulled back 1 mm and allowed to remain in place for 2 rain before being slowly removed. Following the injection, the stereotaxic apparatus was moved to the opposite hemisphere and a second injection was made at the same coordinates. In this way both the active peptide and an internal control injection were examined in a single plane of section. Although the placement of injections in the cerebral cortex is not uniformly successful, with experience, more than 90% of injections can be made accurately.

Tissue Acquisition and Handling and Staining Procedures After l-week survival times, animals were deeply anesthetized with pentobarbital and transcardially perfused. The initial perfusate was 150 ml of normal saline, followed by 300 ml of 4% buffered paraformaldehyde. Brains were carefully removed and postfixed for 4 h in 4% paraformaldehyde at 4°C before transfer into cryprotectant containing 2% dimethylsulfoxide and 20% glycerol in 0.1 M phosphate buffered saline (PBS, pH 7.3) for 48 h. Brains were grossly examined and a 6-8 mm block was selected that included the injection sites and surrounding tissue. Brains were cut at 35 #m on a freezing microtome and collected into six sets of serial sections. Each bin contained a sample spaced at 210 um (four to five samples per mm) for a total of 25-30 sections per bin. Each bin was used for an individual staining procedure. In most cases, one set of sections was stained with anti3-amyloid polyclonal antibodies, mounted, and counterstained with cresyl violet. We tested the quality of immunostaining in tissue stored for up to 8 days after cutting at 4°C and concluded that it is important to stain sections as soon as possible after cutting because staining quality noticeably declines 4 days after cutting. Sections were initially treated in absolute methanol with 0.3% hydrogen peroxide for 30 min followed by three 10-min washes in PBS. The free-floating sections were then incubated for 1 h in 10% normal goat serum (GIBCO Labs, Grand Island, NY) followed by incubation in primary antibody for 18-24 h at room temperature on a rocker. Working dilutions were: 1:1000 for beta-amyloid polyclonal antibody ( 1280, Angela courtesy of Dr. Dennis Selkoe; 1:500 for tau 1, tau 2, tau 5 (courtesy of Dr. L.I. Binder); 1:200 for ALZ50 (courtesy of Dr. P. Davies and H. Ghanbari); 1:50 for MAP2 (5F9) and 1:50 for tau (5E2) (courtesy of Dr. K. S. Kosik) in PBS with 0.3% Triton X-100, 0.8% sodium azide, and 2% normal goat serum. Sections were again washed in PBS (three 10-min washes) followed by a 3-5 h incubation in peroxidase-conjugated goat antimouse IgG (or appropriate secondary, 1:300 in PBS with 2% normal goat serum: Boehringer Mannheim, Indianapolis, IN). Sections underwent another series

KOWALL ET AL.

of PBS washes and were incubated in 3,Y-diaminobenzidine tetrahydrochloride (1 mg/l ml) and 0.005% hydrogen peroxide in 0.05 M Tris HC1 buffer pH 7.6 with 0.1 M imidazole ( 10 ml/ 110 ml Tris buffer) for 1-5 min. Sections were air dried on gelatin-coated slides, cleared in xylene, and coverslipped with Permount (Fisher). In some cases a cresyl violet counterstain was performed. Control procedures included incubation of tissue sections in anti3(l-40) antibody preabsorbed with peptide and the substitution of 1% goat serum for primary antibodies to test the specificity of ALZ 50, tau, and MAP2 antibody staining.

Quantitative Methods We used traditional manual methods to quantitate the extent of neuronal depletion surrounding injection sites, as previously reported (1,5,12). An arbitrary region centered on the injection site at the maximal extent of the lesion was chosen (as determined by the examination of serial sections), and neurons were counted in cresyl violet stained sections in a series of three contiguous fields defined by an eyepiece graticule. At 100×, each 800 um by 800 um field (0.64 mm 2) is subdivided by a grid into 80 um by 80 um squares. The number of neurons was counted in each of these 100 squares in each of three fields and totaled.

Primate Studies The effects of 3 peptide were subsequently investigated in old world primates, species whose brains more closely resemble human brain. Intracerebral microinjections of 3(1-40) and control peptides solubilized in acetonitrile were made into the frontal cortex of five adult cynomolgus monkeys under stereotactic guidance. The 3 peptide was injected at two doses (20 fmol and 3 nmol). Control injections consisted of acetonitrile alone, A37 (3 nmol), CA4 (3 nmol), and reverse peptide (20 fmol). Two animals were 3-year-old males and three were aged ( 17-19-yearold) cynomolgus females. In a sixth monkey, an aged (24-yearold) female rhesus, intracerebral injections of 3(1-40) and 3(401) dissolved in double-distilled deionized water were made. All animals were sacrificed after 10-14 day survivals, and the brains were prepared for routine histology and immunohistochemistry utilizing similar protocols as described for the rat. RESULTS

Cytoskeletal Protein Immunoo,tochemisto, and Lesion Characteristics All injections produced localized tissue destruction with neuronal loss and astrocytosis. Cresyl violet stained sections often showed necrosis at the injection site that was usually more prominent around 3(1-40) injections (Fig. 1). Tau immunoreactivity was increased surrounding beta-amyloid injections and abnormal tau positive thread-like processes, and occasional neuronal perikarya were seen. These changes were much less prominent surrounding control injections. The normal pattern of beaded ALZ 50 immunoreactive fibers and rare neuronal perikarya in the rat cerebral cortex was altered in the vicinity of all injections, but the degree of change was generally much greater surrounding the beta-amyloid lesions. Abnormal ALZ 50 positive neurites and occasional immunoreactive neurons were seen, but neurofibrillary tangles were not present. In general, the extent of altered tau and ALZ 50 immunoreactivity associated with 3(1-40) lesions paralleled the extent of neuronal loss. Nonspecific staining was often present in the necrotic core of the lesion, whereas abnormal immunoreactive cells extended into the tissue surrounding the injection site (Fig. 1). In regions were tau im-

IN V I V O T O X I C I T Y

OF [3-AMYLOID

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i ¸ i ii ii ~

G

H

FIG. 1. Patterns of neuronal degeneration produced by high dose/3(1-40) and a 37 amino acid control peptide derived from the amyloid precursor protein (A37) injected into left and fight hemispheres, respectively, after 2-week survival. Injections sites are in deep cerebral cortex and the CAI field of the hippocampus (asterisks). The lateral border of the lesion in CA I induced by/3(1-40) is shown with an arrow in plates B-F. (A) A37 induces minimal gliosis and disruption of the CAI pyramidal cell layer (arrowhead). Cresyl violet stain, bar = 200/~. (B) The contralateral injection produces more extensive disruption of the pyramidal layer of CA I (arrowhead). Gliosis extends into stratum radiatum and lacunosum-moleculare. Bar = 200 u. (C,D) Patterns of tau immunoreactivity associated with /3(1-40) adjacent to the sections shown in panel B and F. The area of enhanced tau immunoreactivity corresponds to the area of neuronal degeneration seen in B. Higher power view (C) shows increased neuropil tau immunoreactivity. Bar in C = 100 u; in D - 200 u. (E,F) Pattern of MAP2 immunoreactivity surrounding/3(1-40) injection adjacent to the sections shown in panel B and D. MAP2 immunoreactivity is depleted in a pattern complementary to the increased local tau immunoreactivity. Bar in E = 100 ~: in D - 200 u. (G) Tau immunoreactivity is barely visible at low magnification around the injection of the control peptide (A37). Bar - 200 ~. (H) Tau immunoreactivity produced by injections of high dose/3(1-40) can be seen grossly while A37 induces minimal changes in the contralateral hemisphere (curved arrows indicate injections). Bar = 1 mm.

qG. 2. (A) Cresyl violet stained section of rat cerebrum, the asterisk indicates a B(25-35) lesion, characterized by focal neuronal loss and gliosis. Original magnification 25X. (B) A/5(25-35) esion in the rat immunostained for ALZ 50, showing enhanced beaded fiber staining and occasional dilated immunoreactive neurites (arrow) in the surrounding neuropil. Original magnification i30X. (C) A control injection site in the rat immunostained for ALZ 50, showing fewer immunoreactive beaded fibers and the absence of tortuous neurites. Original magnification 630×. (D) )ccasional tau positive neuronal perikarya (arrow) were found adjacent to the B(25-35) lesions in the rat, although no structures resembling neurofibrillary tangles were evident. Original nagnification 630X. (E) The control injections in the rat contained virtually no tau positivity. Original magnification 630X. (F) ALZ 50-positive periventricular neurons and their processes ypical of normal rat hypothalamus (asterisk indicates third ventricle). Original magnification 250X. (G) Intensely ALZ 50-positive neuronal perikarya and proximal dendrites found adjacent o the/5(1-40) injections in the aged primates. Immunoreactive neurons were not present around the injections of/~(40-1) in the same vehicle. Original magnification 1000×. (H)) Intensely kLZ 50-positive filamentous, entangled structures were found in some neuronal perikarya in the aged primate, the abnormal filaments often extended into surrounding neurites. Original nagnification 1000X.

IN VIVO TOXICITY OF /3-AMYLOID

541

munoreactivity was increased surrounding /3(1-40) lesions, MAP2 immunoreactivity was reduced (Fig. 1). The adequacy of ALZ 50 staining in each rat was verifying by the demonstration of normal ALZ 50 immunoreactive neurons in the periventricular nucleus of the hypothalamus. If these neurons are not visualized, it is likely that the immunocytochemical procedures have not been successfully performed and the interpretation of lesion characteristics is then not reliable.

Consisleno, qlthe Observation.; Several factors impact on the interpretation of/3(l-40)induced neurotoxicity in vivo. Preparation of the peptide, injection technique, and immunohistochemical procedures may all impact on the assessment of results. In order to verify that/3(1-40) has been deposited in the brain, it is mandatory to demonstrate a focal deposit of fl(l-40) immunoreactivity at the injection site. We studied a total of 113 rats injected with ~(1-40) and related peptides. Of this group, 33 were injected with fl(l-40) into the frontoparietal cortex of one hemisphere and contralateral control peptides in the same vehicle injected into the opposite hemisphere. The normal density of neurons (per mm z) in rat frontoparietal cortex ranges from 1084-1149 (n = 5). The range of cell counts (per mm 2) surrounding peptide injections (3 nmol in acetonitrile-trifluoroacetic acid vehicle in all cases) was as follows: APP residues 261-280 (A37, n = 5): 606-1125; scrambled peptide (CA4, n = 5): 552-860;/3(40-1) (n = 3): 890-1160; /3(1-40) (n = 16): 108-508. Cell counts must be compared to internal contralateral controls to control such variables as section thickness. Statistical analysis of these cell counts by ANOVA showed that A37, CA4, and r(1-40) cell counts were significantly different (p < 0.001 ). Post hoc analysis showed that/3(1-40) cell counts were significantly different from/3(40-1) (p < 0.01, Scheffe test). A group of 22 cases from this series was evaluated blindly and graded qualitatively for the presence of asymmetrical cortical lesions. In 15/22 (68%), there was clear asymmetry evident on qualitative examination. In the remaining seven cases, differences between/3(1-40) and control peptides were not qualitatively apparent. The frontoparietal cortex of 10 animals was injected with /3(25-35) and evaluated as described above (Fig. 2). In one case, the injection was deep into the corpus callosum and striatum. Of the remaining nine, clear asymmetry was present in seven (78%). Of this group, qualitatively large lesions were present in four (visible to the naked eye). Moderate lesions were present in two (obvious with microscopic examination), and a small lesion was seen in one (small, but clearly larger than control). Locally increased tau immunoreactive perikarya and neurites were seen. ALZ 50-positive neurites were swollen, distorted, and increased in frequency around the B(25-35) injection (Fig. 2).

contained argyrophilic, thioflavine S fluorescent, Alz 50, and ubiquitin immunoreactive perikaryal, and neuritic alterations (Fig. 2). The morphology of the neuronal and neuritic alterations closely resembled neurofibrillary tangles and dystrophic neurites characteristic of human brain affected by AD. They were not found adjacent to the/3(1-40) lesions in the young monkeys or near control injections, although one reverse peptide injection in aqueous solution produced faint Alz 50 neuronal positivity. A detailed quantitative report of this work is currently being prepared for publication. DISCUSSION There are several potential pitfalls to the interpretation of in vivo lesioning experiments despite our observation that/3(1-40) and/3(25-35) generally produce well-defined cortical lesions that are clearly distinguishable from control peptides. Variability may be due to the physical or chemical state of the peptide in solution, injection technique, or histological procedures. It is important to confirm that/3(1-40) has been successfully injected into the site of interest prior to concluding that it is not neurotoxic. Specific immunocytochemical demonstration of/3(1-40) is the best way to confirm its presence in the lesion. The best internal control is the concurrent contralateral injection of a control peptide dissolved in the same vehicle as the/3(1-40). The problem of nonspecific immunoreactivity can be addressed by using absorption controls and by staining with antisera against other proteins. For example, we found a clear divergence between tau and MAP2 immunoreactivity around the injection sites even though both antibodies are mouse monoclonals used at similar concentrations with identical seconda~ antibodies and subsequent staining procedures. Abnormal ALZ 50 immunoreactive neurons and fibers were not seen in sections incubated without primary antibody. The ALZ 50 antibody does not solely recognize an Alzheimerassociated antigen because normal neuronal populations, such as the paraventricular neurons in the rat (Fig. 2), are labeled. Caution must, therefore, be used in equating ALZ 50 immunoreactivity to Alzheimer-type pathology. Nevertheless, our demonstration of abnormal ALZ 50 immunoreactive neurons in the primate neocortex surrounding/3(1-40) suggests that neurons that do not normally contain ALZ 50 immunoreactivity become positive when exposed to/3(1-40) and not to control peptides. In aged primates, the neuropathological alterations accompanying/3(1-40) lesions resemble the neuronal and neuritic alterations found in AD. The clear differences in the effects of /3(1-40) in the rodent and primate brain underscores the importance of species differences, There is substantial evidence that both connectional organization and individual neuronal characteristics differ greatly among rat, monkey, and human (2,3,19). This is further exemplified by the lack of/3(1-40) accumulation in rats and the paucity of Alzheimer pathology in transgenic mice that overexpress APP (9,10).

Primate Studies

ACKNOWLEDGEMENTS

In the primate brain,/3(1-40) both acetonitrile and aqueous vehicles produced dose-dependent cortical lesions that were significantly larger than those produced by control peptides. In the four aged primates, the cortex surrounding the/3(1-40) lesions

We would like to thank Karen Harrington. Alida Evans, Lawrence Cherkas, and Andrew Singer for expert technical assistance. Supported in part by NIA grants AG 05134, AG09229, NS01240, and a grant from the Alzheimer Association.

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