Neurofibrillary tangles in the cerebral cortex of sheep

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Neuroscience Letters 170 (1994) 187 190

NEUROSCIENCE LETTERS

Neurofibrillary tangles in the cerebral cortex of sheep Peter T. Nelson ~, Sharon G. Greenberg b, Clifford B. Sapeff* "Committee on Neurobiology and Brain Research btstitute, University q[" Chicago. Chicago, IL 60637. USA ~'Dementia Research, Burke Medical Research Institute, White Plains, N Y 10605, USA "Department qf Neurology and Program in Neuroscience, Beth Israel Hospital and Harvard Medical School, 330 Brooklbw Avenue, Boston, M,t 02215. USA Received 15 December 1993; Accepted 27 January 1994

Abstract Neurofibrillary degeneration, including neurofibrillary tangles (NFTs) and neuritic plaques, is an important pathological hallmark of Alzheimer's disease (AD). Unfortunately, no practical animal model of neurofibrillary degeneration has been described. We report here the presence of structures in the cerebral cortex of sheep, Ovis aries, that resemble Alzheimer NFTs and neuritic plaques. NFT-like structures and clusters of degenerating neurites are stained by silver impregnation and thioflavin-S, and are immunoreactivc with antibodies against tau microtubule-associated proteins. Viewed under the electron microscope, tau-immunoreactive tangles consist of paired helical filaments. Naturally occurring neurofibrillary structures in sheep cortex provide a model for studying the pathobiology of Alzheimer's disease. Key words." Alzheimer's disease; Neurofibrillary tangle; Paired helical filament; Tau; Plaque; Amyloid; Sheep; Animal model

The pathological hallmarks of Alzheimer's disease (AD) are neurofibrillary tangles (NFTs) and amyloid plaques [1]. NFTs, which develop in neuroanatomical foci of cell loss during AD [13], are composed of pathological fibrils called paired helical filaments (PHF) [18]. Identical PHFs are found within dystrophic neurites that surround some amyloid plaques; such plaques are termed 'neuritic' [24]. PHFs are evidently polymers of tau microtubule associated proteins [6], but the causative biochemical antecedents of P H F formation remain unknown. A commercially available animal species in which N F T and neuritic plaques naturally occur could help to reveal information concerning AD pathoetiology and to test potential therapies. We now report that structures which resemble NFTs and neuritic plaques are found in the brains of aging sheep, Ovis aries. We performed immunohistochemical staining on the cortex of sheep as described previously [19]. Alz-50 [25] and PHF-1 [10] are monoclonal antibodies that recognize modified tau proteins in the brains of AD patients,

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as well as certain neuronal systems in normal brain [4,19,20]. In sheep, the characteristic normal staining pattern was observed for Alz-50 and PHF-1 [19], but in addition we found that both antibodies stained cortical structures that were similar to human NFTs (Fig. 1A,B) and distinct from normally stained structures. The Alz50 epitope is located near the amino terminal of the native tau protein [9], whereas PHF-I recognizes a phosphorylated epitope on the carboxyl part of tau [11]. The presence of these two distinct tau epitopes in the sheep NFT-like structures (NFTLSs) strongly implies that tau molecules are a component o f N F T L S s . |n addition to immunoreactive cells, Alz-50 and PHF-1 stained aberrant fibers in sheep brain that resembled AD 'neuropil threads' [2] (Fig. 1E). These structures were more thick and rope-like than normally stained structures in sheep brain. The origin of these immunoreactive neurites is not known. Morphologically distinct from the neuropil thread-like structures, roughly-spherical clusters of dystrophic neurites that could be stained by Alz-50 and PHF-1 were observed in several older sheep. Stained by either antibody, they appeared reminiscent of AD 'neuritic pla-

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ques' (Fig. IF). These lesions, which we term 'neuritic clusters', were concentrated in certain areas of the isocortex, often in superficial cell layers. One sheep, a 13year-old ewe, had many more of these lesions than other animals (Fig. 2). Adjacent sections stained for AIz-50 and PHF-I contained neuritic dusters in practically identical neuroanatomical locations. These lesions were usually between 40 and 120/~m in diameter. The number of NFTLSs in sheep cortex varied among animals, becoming more numerous with age. Although accurate birth records were not usually available for the animals we used, cues such as dental deterioration were used to approximate the sheep's ages. Young sheep (less than 3 years old) were never observed to have NFTLSs. Among older animals, 5 of 7 sheep between approximately 5 and 10 years of age had NFTLSs, typically with a density of one or two per 50/~m thick hemisection. The neuroanatomical locations of these lesions varied, but most were found either deep in the anterior sulcus of the insular cortex or in the piriform cortex. No tangles were found in the hippocampi of sheep aged 5~-10. We also stained the brains of five older sheep, all ewes over 10 years of age, and all had more N F T L S s per section (20 70 per 50/.tm section). N F T L S s were not observed in any of the sheep to be concentrated in the hippocampus.

Fig. 1. Neurofibrillary degeneration in the sheep cortex as viewed under the light microscope. Neurofibrillary tangle-like structuress (A D) in sheep brains, stained using: (A) Alz-50 antibody; (B) PHF-I antibody: tC) Gallyas' silver stain; and (D) thioflavine S. Lesions resembling, 'neuropil threads' (E) and neuritic plaques (F) could be visualized using PHF-I (shown hereL as well as Alz-50. Bars in A = 20/tm, in F = 50,urn.

Fig. 2. Camera ludica representation of a coronal section from a 13year-old ewe. Neurofibrillary tangle-like structures are represented by triangles, and neuritic plaques by stars. Tangles tended to be found scattered throughout the cerebral cortex, whereas neuritic plaques were found in discrete loci. Am, amygdala: CI, claustrum; 1:, fornix; Hy, hypothalanms: IC. internal capsule; OT, optic tract; PC, piriform cortex: Th. thalamus,

To further assess the lesions in sheep brain, we used histochemical stains for AD neurofibrillary degeneration including the modified Gallyas silver stain [3,7] and thioflavine S. Both techniques seemed less sensitive than tau antibodies in visualizing N F T L S s in the sheep. However, both silver-impregnated and thioflavine-positive structures were found that resembled NFTLSs stained with Alz-50 and PHF-1 (Fig. 1C, D). Hence, in addition to tau immunoreactivity, the sheep lesions were analogous to pathological structures in AD brain using histochemical criteria. We also performed electron microscopic immunocytochemistry using Alz-50 and P H F - l to determine whether the sheep lesions ultrastructurally resembled NFTs in humans with AD. Cells that were stained by either Alz50 or PHF-1 contained many fibrils onto which reaction product was deposited (Fig, 3A). These fibrils streamed through the cytoplasm either in bundles or in more scattered arrays. We compared the dimensions of some unstained fibrils in the immunoreactive cells against PHFs from a human AD brain. The helical periodicity of relalively unstained sheep fibrils (Fig. 3B-D) was 65-80nm. strikingly similar to PHFs in AD (Fig. 3E). Immunohistochemistry using a panel of other antisera that are sensitive to AD neuropathology revealed distinctions between the lesions in sheep and those in human AD brains. For example, we stained for ubiquitin using two monoclonal antibodies ('3 35' [23] and '5-25' [26]) and a polyclonal antiserum IChemicon). Background staming was high for all ubiquitin antisera, but NFTLSs could not be distinguished. We also stained sheep brain sections using antisera ('1280' [14], "1282' [12], and "C8" [21]) against b-amyloid protein (flAP) and the b-amyloid precursor protein tflAPPI in five cases. We fbund no plaque-like flAP deposits using those antisera

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B

D

E

Fig. 3. Uhrastructurally, neurolibrillary tangle-like structures are composed of tau-immunoreactive helical fibrils. An epoxy resin-elnbedded PHF-I stained cell was localized under light microscope (a), then cut and viewed under the electron lnicroscope (A). Note the immunoreactire bundles of fibrils that course through the cytoplasm, lJpofuscin (L), was also fotmd in many non-tangle bearing neurons, Bar = 2#m. Viewed ;.11 higher magnilications, fibrils in sheep (B D) displaycd the 'twisted ribbon' morphology reminiscent of Alzheimer PH Fs. B and C are from the above PHF-1-stained cell: D is florn an AIz-50-stained cell: and E is from an unstained human AD NFT. Bar = 50 nm for 13 E.

in sheep, even after t\~rmic acid pretreatment. In the sheep with neuritic clusters, we performed double-label immnnohistochemistry to discern whether the tau-immunoreactive plaques contained flAR Unlike human sections included as positive controls, flAP was not found to be associated with neuritic clusters in sheep. Hence, although these lesions apparently consist of clusters of tau-immunoreactive dystrophic neurites, they are biochemically distinct fl'om Alzheimer neuritic plaques. Finally, we wished to ascertain whether the AD-like pathology observed in sheep wets related to the spongiform encephalopathy of scrapie. We stained sheep brain sections with two different antisera [16,17] that recognize PrP'L the putative scrapie prion protein. We found no plaque-likc structures that were spccitically

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stained for PrP '~, and staining was not augmented by formic acid pretreatment. Obvious spongiform changes were not observed light microscopically in any brain. Taken as a whole, the present study demonstrates structures in sheep brain that closely resemble cells and neurites undergoing neurofibrillary degeneration in AD brain. The presence of tau-immunoreactive NFTLSs, PHFs, and neuritic plaques has not been previously described in a commercially available animal model, although other aspects of AD-like neuropathology have been demonstrated in animals, flAP-immunoreactive plaques are found in non-human primates [22] and dogs [8], but none of these animals has been shown to develop neurofibrillary degeneration. The only animal that has been shown to develop neurofibrillary pathology similar to AD are aged bears [5]. In these animals, neither delinitive PHFs nor tau-immunoreaclive neuritic plaques were observed: moreover, this species is not readily awlilable t\~r research purposes. We were not able to assess whether the neurofibrillary changes we observed in sheep brains correlated with cognitive deterioration. Although we l\mnd an increasing incidence of neurofibrillary changes with age, our sheep were raised by commercial breeders as farm animals, and so were not particularly old specimens considering that sheep may live 20 years or longer. Even our oldest animals (10 14 years of age) may not have been physiologically elderly, and none had the density of neurofibrillary changes that are typically seen in AD patients. Thus the pathology may not have been sufticiently advanced to cause behavioral changes in these animals. Moreover, although our sheep did not demonstrate blatant behavioral impairment, they were not tested in sophisticated behavioral paradigms. Another difficulty in comparing our sheep model with human AD is the lack of specitic immunoreactive staining for flAP. flAPP, o1 ubiquitin. As the amino acid sequences of both the flAP segment o f f l A P P and ubiquitin in sheep are identical to human [23], the antisera we used would be expected to demonstrate these molecules if they were present. The lack of flAP innnunoreactivity associated with neurofibrillary changes is especially intriguing, suggesting that a different chelnical trigger may initiate neurofibrillary degeneration in this species. It is possible that the same pathogenic substance(s) play a role in neurofibrillary degeneration in human AD as well. The lack of an animal model for human neurofibrillary degeneration has hampe.-ed the investigation of such hypotheses. The presence of neurofibrillary changes in sheep, a relatively inexpensive and widely axailable domesticated animal, may provide a practical model for the investigation of the molecular and genetic basis of N FT lormation. We thank Drs. R. Kascsak. K. Kosik. D. Selkoc and R Daxies for useful discussion and criticism, as well as

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