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Dystrophic neurites around amyloid plaques of human patients with Gerstmann-Sträussler-Scheinker disease contain ubiquitinated inclusions
Neuroscience Letters, 121 (1991) 55 58 Elsevier Scientific Publishers Ireland Ltd.
55
NSL 07390
Dystrophic neurites around amyloid plaques of human patients with Gerstmann-Strfiussler-Scheinker disease contain ubiquitinated inclusions A n t o n i o Migheli, A n g e l o A t t a n a s i o , M a r i a C l a u d i a Vigliani a n d D a v i d e Schiffer Second Neurologic Clinic, University of Turin, Turin (Italy)
(Received 18 June 1990; Revisedversion received 4 September 1990; Accepted 5 September 1990) Key words." Ubiquitin; Dystrophic neurite; Amyloid; Gerstmann-Strfiussler-Scheinkerdisease; Immunogold; Electron microscopy
Dystrophic neurites have been previously observed around prionic protein-derived amyloid plaques of Gerstmann-Strfiussler-Scheinker (GSS) disease. Ubiquitin (Ubq) immunohistochemistryreveals the presence of dot-like stainings around many of these plaques. In order to determine the nature of ubiquitinated deposits, we performed an immunogold electron microscopestudy on autoptic samples from the cerebellumof a GSS patient. Both pre- and post-embedding staining methods showed Ubq-positive dense bodies and filamentous structures, belonging to dystrophic neurites. They are analogous to ubiquitinated neuritic processesdescribed around cerebellar amyloid plaques of Alzheimer's disease (AD). These results suggest that amyloid deposition is responsible for the degeneration of adjacent axon terminals in both AD and GSS.
Gerstmann-Strfiussler-Scheinker (GSS) disease is a rare familial entity characterized clinically by cerebellar ataxia, pyramidal signs and progressive dementia, and pathologically by spongiform degeneration, gliosis and widespread deposition of amyloid plaques [21]. Together with Kuru and Creutzfeldt-Jakob disease, it belongs to the group of human prion diseases [17]. The amyloid fibrils are related to accumulation of PrP protein, a neuronal m e m b r a n e glycoprotein that also constitutes the scrapie-associated fibrils [9]. Previous ultrastructural studies have described different types of amyloid plaques: (a) primitive, (b) more mature with a stellate aspect and (c) multicentric [19, 22]. The presence of degenerated neuritic processes with accumulation of dense or lamellated osmiophilic bodies, mitochondria and 8-10 nm straight filaments has been noted around primitive plaques, but their significance has not been elucidated [I, 7, 8, 14, 19, 22]. In a case of GSS recently studied by us, the immunohistochemical staining for ubiquitin (Ubq) revealed numerous dot-like deposits around m a n y amyloid plaques (unpublished data). U b q is a 76 amino acid protein involved in a non-lysosomal proteolytic pathway, which has been found to be activated in several neurodegenerative disorders marked by accumulation of abnormal cytoskeletal components [6].
Correspondence: A. Migheli, Clinica Neurologica II, Universit~i di Torino, via Cherasco 15, 10126 Torino, Italy.
0304-3940/91/S 03.50 ,~ 1991 ElsevierScientific Publishers Ireland Ltd.
In order to determine the nature of Ubq-positive structures around amyloid plaques of GSS, we performed both immunohistochemistry and immunoelectron microscopy (IEM) on autoptic samples from the cerebellum of our GSS case. Immunohistochemistry. Formalin-fixed, paraffinembedded, 5/zm sections were immunostained with antibodies (Abs) to: (a) U b q (an affinity-purifed rabbit Ab which recognizes epitopes along the entire U b q molecule [11], diluted 1/800); (b) neurofilaments (NFs) (monoclonal Abs to phosphorylated and non-phosphorylated epitopes SMI 31 and SMI 33, Sternberger, diluted 1/500; monoclonal Abs to 68, 150 and 200 k D a subunits, Boehringer, diluted 1/5); (c) synaptophysin (monoclonal, Dako, diluted 1/100); (d) glial fibrillary acidic protein (GFAP), polyclonal, Dako, diluted 1/600). The prionic nature of amyloid deposits had been previously assessed immunohistochemically with a polyclonal Ab raised in a rabbit immunized with PrP 27-30 purified from hamster brain [2]. The plaques were not stained by a rabbit polyclonal Ab to the 28 residue synthetic peptide (SP 28) homologous to the aminoterminal region of Alzheimer's disease (AD)fl-protein [3]. Immunoelectron microscopy. Deparaffinized 30 /Lm sections were immunogold-stained with the described Abs using both pre- and post-embedding methods. (a) For pre-embedding, sections were autoclaved at 120°C for 30 min (a pre-requisite for U b q staining [11]), incubated with normal goat serum (1/10, 30 min) and primary Abs (48 h at 4°C), and labeled with goat anti-rabbit
56
Fig. 1. Ubq-positivedots in the proximityof an amyloidplaque in the Purkinjecell layer. PAP-DAB, 400 ×,
or goat anti-mouse Igs conjugated with 5 nm colloidal gold (GAR5 or GAM5, BioCell, 1/100 for 72h at 4°C). The sections were refixed in glutaraldehyde and osmium and fiat-embedded in Epon. Ultrathin sections were counterstained with uranyl acetate and lead citrate. (b) For post-embedding, autoclaved sections were dehydrated and fiat-embedded in LR White [12]. Thin sections mounted on Formvar-coated nickel grids were incubated with normal goat serum (30 min), primary Abs (30 min or overnight at 4°C), labeled with GAR20 or GAM20 (60 min) and counterstained. Washings and dilutions were done in Tris-buffered saline (TBS) buffer; controls were performed by substituting the primary Abs with either buffer or preimmune serum. Results were as follows, Immunohistochemical stain-
ing for Ubq revealed a dot-like positivity surrounding many amyloid plaques. These were mostly located in the molecular layer and, occasionally, in the proximity of Purkinje cells (Fig. 1). No corresponding staining was obtained with the Abs to NFs, synaptophysin and GFAP. With both pre- and post-embedding methods, IEM disclosed several Ubq deposits around primitive amyloid plaques. The most outstanding feature was represented by (a) dense osmiophilic bodies, which were decorated by gold at their periphery, and (b) diffusely labeled uncoated amorphous densities (Fig. 2). Less frequently, round labeled structures were found, made up of a meshwork of 10-15 nm short filaments and densities (Fig. 3). All types of deposits were surrounded in places by mem-
Fig. 2. Ubiquitination of (A) a dense body and (B) amorphous density. Pre-embedding,GAR 5. A, 90,000x ; B, 85,000x.
57
A Fig. 3. Ubq-positive filamentous structures. A: pre-embedding, GAR 5, 120,000 ×. B: post-embedding, GAR 20, 45,000 ×.
brane fragments. Abs to NFs, synaptophysin and G F A P did not stain structures corresponding to the ubiquitinated ones. Our results indicate that the Ubq-positive dots around amyloid plaques of GSS correspond to processes with the ultrastructural features of dystrophic neurites. Moreover, they are similar to ubiquitinated dystrophic neurites which have been demonstrated by IEM around cerebellar amyloid plaques of AD [5]. Although amyloid plaques of GSS are enveloped by astrocytic processes [19], a glial nature for the Ubq-positive processes is ruled out by their lack of staining for GFAP. Failure of a corresponding staining with Abs to synaptophysin and NFs is more intriguing: synaptophysin, which is located in the membrane of synaptic vesicles, might have been lost either because of post-mortem autolytic changes, or because of the progression of the disease itself. As for NFs, the same dissociation between staining for Ubq and lack of staining for NFs has been observed by us in filamentous inclusions of motor neuron disease (MND) [13], and is probably due either to Ubq-dependent masking of N F epitopes, or to abnormal formation of NFs. The dense bodies, which constitute the majority of Ubq-positive structures, can be interpreted as autophagic vacuoles of lysosomal origin, and are analogous to the residual bodies of cultured hepatoma cells, which have been shown by IEM to contain unconjugated Ubq [20]. Since our Ab recognizes both free and conjugated Ubq, the dense bodies are likely to represent sites of degradation of previously Ubq-conjugated altered or damaged proteins. It remains to be assessed what causes the intraneuritic accumulation of abnormal proteins in GSS, as well as in other conditions. Ubiquitinated dystrophic neurites have been described in normal aging brain [10, 15], in AD [5, 16], in diffuse Lewy body disease [4] and in M N D [18].
In aging brain, Ubq might be involved in the degradation of age-dependent abnormal proteins [15]. In MND, ubiquitinated dystrophic neurites probably form as a distal response of the axon to the neuronal degeneration [18]. In amyloid-related Ubq deposits, on the other hand, other pathogenetic mechanisms can be postulated. A degeneration of axon terminals, possibly related to a neurotoxic effect of amyloid deposition [23], has been proposed for the formation of dystrophic neurites around cerebellar plaques of AD [5]. Our observations suggest that the amyloid of GSS plays a similar role. Supported by CNR, Special Project "Aging'.
1 Azzarelli, B., Muller, J., Ghelti, B., Dyken, M. and ('onneally, P.M., Cerebellar plaques in familial Alzheimer's disease (Gerstmann Str/iussler Scheinker variant?), Acta Neuropathol., 65 (1985) 235 246. 2 Bendheim, P.E., Barry, R.A., DeArmond, S.J., Sites, D.P. and Prusiner, S.B., Antibodies to a scrapie prion protein, Nature, 310 (1984)418 421. 3 Castafio, E.N., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease •]-protein, Biochem. Biophys. Res. Commun., 141 (1986) 782 789. 4 Dickson, D.W., Crystal, H,, Mattiace, L.A., Kress, Y., Schwagerl, A., Ksiezak-Reding, H., Davies, P. and Yen, S.H.C., Diffuse Lewy body disease: light and electron microscopic immunocytochemistry of senile plaques, Acta Neuropalhol., 78 (19891 572 584. 5 Dickson, D.W., Wertkin, A., Mattiace, L.A., Fier, E.. Kress, Y., Davies, P. and Yen, S.-H., Ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of AIzheimer's disease, Acta Neuropathol., 79 (1990) 486 493. 6 Gallo, J.-M. and Anderton, B.H., Ubiquitous variations in nerves, Nature, 337 (1989) 687 688. 7 Gheni, B., Tagliavini, F., Masters, C.L., Beyreuther, K., Giaccone, G., Verga, L., Farlow, M.R., Conneally, P.M., Dlouhy, S.R., Azzarelli, B. and Bugiani, O., Gerstmann-Str~ussler-Scheinker dis-
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9
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ease. II. Neurofibritlary tangles and plaques with PrP-amyloid coexist in an affected family, Neurology, 39 (1989) 1453-1461. Hudson, A.J., Farrel, M.A., Kalnins, R. and Kaufmann, J.C.E., Gerstmann-Str~iussler-Scheinker disease with coincidental familial onset, Ann. Neurol., 14 (1983) 670~578. Kretzschmar, H.A., Prusiner, S.B., Stowring, L.E. and DeArmond, S.J., Scrapie prion proteins are synthesized in neurons, Am. J. Pathol., 122 (1986) 1-5. Leigh, P.N., Probst, A., Dale, G.E., Power, D.P., Brion, J.-P., Dodson, A. and Anderton, B.H., New aspects of the pathology of neurodegenerative disorders as revealed by ubiquitin antibodies, Acta Neuropathol., 79 (1989) 61-72. Manetto, V., Perry, G., Tabaton, M., Mulvihill, P., Fried, V.A., Smith, H.T., Gambetti, P. and Autilio-Gambetti, L., Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 4501-4505. Migheli, A. and Attanasio, A., Two methods for fiat-embedding in LR White sections cut from paraffin blocks, Stain Technol.. in press. Migheli, A., Autilio-Gambetti, L., Gambetti, P., Mocellini, C., Vigliani, M.C. and Schiffer, D., Ubiquitinated filamentous inclusions in spinal cord of patients with motor neuron disease, Neurosci. Lett., 114 (1990) 5-10. Nochlin, D., Sumi, S.M., Bird, T.D., Snow, A.D., Leventhal, C.M., Beureuther, K. and Masters, C.L., Familial dementia with PrPpositive amyloid plaques: a variant of Gerstmann-Str~iussler syndrome, Neurology, 39 (1989) 910-918.
15 Pappolla, M.A., Omar, R. and Saran, B., The ~Norrnal" Brain. 'Abnormal' ubiquitinitated deposits highlight an age-related protein change, Am. J. Pathol., 135 (1989) 585-591. 16 Perry, G., Friedman, R., Shaw, G. and Chau, V., Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains, Proc. Natl. Acad. SCI. U.S.A., 84 (1987) 3033-3036. 17 Prusiner, S.B., Prions and degenerative diseases, N. Engt. J. Med., 317 (1987) 1571-1581. 18 Schiffer, D., Autilio-Gambetti, L., Chi6, A., Gambetti, P., Giordana, M.T., Gullotta, F., Migheli, A. and Vigliani, M.C., Immunohistochemistry and immunoeleetron microscopy of ubiquitin in ALS, J. Neuropathol. Exp. Neurol., 49 (1990) 277. 19 Schlote, W., Boetlaard, J.W., Schumm, F. and St6hr, M., Gerstmann-Str/iussler-Seheinker's disease, Acta Neuropathol., 52 (1980) 203-211. 20 Schwartz, A.L., Ciechanover, A., Brandt, A.R. and Geuze, H.J., Immunoelectron microscopic localization of ubiquitin in hepatoma cells, EMBO J., 10 (1989) 2961 2966. 21 Seitelberger, F., Str/iussler's disease, Acta Neuropathol., Suppl. VII (1981) 341-343. 22 Vinters, H.V., Hudson, A.J. and Kaufmann, J.C.E., GerstmannStr~iussler-Seheinker disease: autopsy study of a familial case, Ann. Neurol., 20 (1986) 540-543. 23 Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., OsterGranite, M.L. and Neve, R.L., Neurotoxicity of a fragment of the amytoid precursor associated with Alzheimer's disease, Science, 245 (1989) 417-420.
55
NSL 07390
Dystrophic neurites around amyloid plaques of human patients with Gerstmann-Strfiussler-Scheinker disease contain ubiquitinated inclusions A n t o n i o Migheli, A n g e l o A t t a n a s i o , M a r i a C l a u d i a Vigliani a n d D a v i d e Schiffer Second Neurologic Clinic, University of Turin, Turin (Italy)
(Received 18 June 1990; Revisedversion received 4 September 1990; Accepted 5 September 1990) Key words." Ubiquitin; Dystrophic neurite; Amyloid; Gerstmann-Strfiussler-Scheinkerdisease; Immunogold; Electron microscopy
Dystrophic neurites have been previously observed around prionic protein-derived amyloid plaques of Gerstmann-Strfiussler-Scheinker (GSS) disease. Ubiquitin (Ubq) immunohistochemistryreveals the presence of dot-like stainings around many of these plaques. In order to determine the nature of ubiquitinated deposits, we performed an immunogold electron microscopestudy on autoptic samples from the cerebellumof a GSS patient. Both pre- and post-embedding staining methods showed Ubq-positive dense bodies and filamentous structures, belonging to dystrophic neurites. They are analogous to ubiquitinated neuritic processesdescribed around cerebellar amyloid plaques of Alzheimer's disease (AD). These results suggest that amyloid deposition is responsible for the degeneration of adjacent axon terminals in both AD and GSS.
Gerstmann-Strfiussler-Scheinker (GSS) disease is a rare familial entity characterized clinically by cerebellar ataxia, pyramidal signs and progressive dementia, and pathologically by spongiform degeneration, gliosis and widespread deposition of amyloid plaques [21]. Together with Kuru and Creutzfeldt-Jakob disease, it belongs to the group of human prion diseases [17]. The amyloid fibrils are related to accumulation of PrP protein, a neuronal m e m b r a n e glycoprotein that also constitutes the scrapie-associated fibrils [9]. Previous ultrastructural studies have described different types of amyloid plaques: (a) primitive, (b) more mature with a stellate aspect and (c) multicentric [19, 22]. The presence of degenerated neuritic processes with accumulation of dense or lamellated osmiophilic bodies, mitochondria and 8-10 nm straight filaments has been noted around primitive plaques, but their significance has not been elucidated [I, 7, 8, 14, 19, 22]. In a case of GSS recently studied by us, the immunohistochemical staining for ubiquitin (Ubq) revealed numerous dot-like deposits around m a n y amyloid plaques (unpublished data). U b q is a 76 amino acid protein involved in a non-lysosomal proteolytic pathway, which has been found to be activated in several neurodegenerative disorders marked by accumulation of abnormal cytoskeletal components [6].
Correspondence: A. Migheli, Clinica Neurologica II, Universit~i di Torino, via Cherasco 15, 10126 Torino, Italy.
0304-3940/91/S 03.50 ,~ 1991 ElsevierScientific Publishers Ireland Ltd.
In order to determine the nature of Ubq-positive structures around amyloid plaques of GSS, we performed both immunohistochemistry and immunoelectron microscopy (IEM) on autoptic samples from the cerebellum of our GSS case. Immunohistochemistry. Formalin-fixed, paraffinembedded, 5/zm sections were immunostained with antibodies (Abs) to: (a) U b q (an affinity-purifed rabbit Ab which recognizes epitopes along the entire U b q molecule [11], diluted 1/800); (b) neurofilaments (NFs) (monoclonal Abs to phosphorylated and non-phosphorylated epitopes SMI 31 and SMI 33, Sternberger, diluted 1/500; monoclonal Abs to 68, 150 and 200 k D a subunits, Boehringer, diluted 1/5); (c) synaptophysin (monoclonal, Dako, diluted 1/100); (d) glial fibrillary acidic protein (GFAP), polyclonal, Dako, diluted 1/600). The prionic nature of amyloid deposits had been previously assessed immunohistochemically with a polyclonal Ab raised in a rabbit immunized with PrP 27-30 purified from hamster brain [2]. The plaques were not stained by a rabbit polyclonal Ab to the 28 residue synthetic peptide (SP 28) homologous to the aminoterminal region of Alzheimer's disease (AD)fl-protein [3]. Immunoelectron microscopy. Deparaffinized 30 /Lm sections were immunogold-stained with the described Abs using both pre- and post-embedding methods. (a) For pre-embedding, sections were autoclaved at 120°C for 30 min (a pre-requisite for U b q staining [11]), incubated with normal goat serum (1/10, 30 min) and primary Abs (48 h at 4°C), and labeled with goat anti-rabbit
56
Fig. 1. Ubq-positivedots in the proximityof an amyloidplaque in the Purkinjecell layer. PAP-DAB, 400 ×,
or goat anti-mouse Igs conjugated with 5 nm colloidal gold (GAR5 or GAM5, BioCell, 1/100 for 72h at 4°C). The sections were refixed in glutaraldehyde and osmium and fiat-embedded in Epon. Ultrathin sections were counterstained with uranyl acetate and lead citrate. (b) For post-embedding, autoclaved sections were dehydrated and fiat-embedded in LR White [12]. Thin sections mounted on Formvar-coated nickel grids were incubated with normal goat serum (30 min), primary Abs (30 min or overnight at 4°C), labeled with GAR20 or GAM20 (60 min) and counterstained. Washings and dilutions were done in Tris-buffered saline (TBS) buffer; controls were performed by substituting the primary Abs with either buffer or preimmune serum. Results were as follows, Immunohistochemical stain-
ing for Ubq revealed a dot-like positivity surrounding many amyloid plaques. These were mostly located in the molecular layer and, occasionally, in the proximity of Purkinje cells (Fig. 1). No corresponding staining was obtained with the Abs to NFs, synaptophysin and GFAP. With both pre- and post-embedding methods, IEM disclosed several Ubq deposits around primitive amyloid plaques. The most outstanding feature was represented by (a) dense osmiophilic bodies, which were decorated by gold at their periphery, and (b) diffusely labeled uncoated amorphous densities (Fig. 2). Less frequently, round labeled structures were found, made up of a meshwork of 10-15 nm short filaments and densities (Fig. 3). All types of deposits were surrounded in places by mem-
Fig. 2. Ubiquitination of (A) a dense body and (B) amorphous density. Pre-embedding,GAR 5. A, 90,000x ; B, 85,000x.
57
A Fig. 3. Ubq-positive filamentous structures. A: pre-embedding, GAR 5, 120,000 ×. B: post-embedding, GAR 20, 45,000 ×.
brane fragments. Abs to NFs, synaptophysin and G F A P did not stain structures corresponding to the ubiquitinated ones. Our results indicate that the Ubq-positive dots around amyloid plaques of GSS correspond to processes with the ultrastructural features of dystrophic neurites. Moreover, they are similar to ubiquitinated dystrophic neurites which have been demonstrated by IEM around cerebellar amyloid plaques of AD [5]. Although amyloid plaques of GSS are enveloped by astrocytic processes [19], a glial nature for the Ubq-positive processes is ruled out by their lack of staining for GFAP. Failure of a corresponding staining with Abs to synaptophysin and NFs is more intriguing: synaptophysin, which is located in the membrane of synaptic vesicles, might have been lost either because of post-mortem autolytic changes, or because of the progression of the disease itself. As for NFs, the same dissociation between staining for Ubq and lack of staining for NFs has been observed by us in filamentous inclusions of motor neuron disease (MND) [13], and is probably due either to Ubq-dependent masking of N F epitopes, or to abnormal formation of NFs. The dense bodies, which constitute the majority of Ubq-positive structures, can be interpreted as autophagic vacuoles of lysosomal origin, and are analogous to the residual bodies of cultured hepatoma cells, which have been shown by IEM to contain unconjugated Ubq [20]. Since our Ab recognizes both free and conjugated Ubq, the dense bodies are likely to represent sites of degradation of previously Ubq-conjugated altered or damaged proteins. It remains to be assessed what causes the intraneuritic accumulation of abnormal proteins in GSS, as well as in other conditions. Ubiquitinated dystrophic neurites have been described in normal aging brain [10, 15], in AD [5, 16], in diffuse Lewy body disease [4] and in M N D [18].
In aging brain, Ubq might be involved in the degradation of age-dependent abnormal proteins [15]. In MND, ubiquitinated dystrophic neurites probably form as a distal response of the axon to the neuronal degeneration [18]. In amyloid-related Ubq deposits, on the other hand, other pathogenetic mechanisms can be postulated. A degeneration of axon terminals, possibly related to a neurotoxic effect of amyloid deposition [23], has been proposed for the formation of dystrophic neurites around cerebellar plaques of AD [5]. Our observations suggest that the amyloid of GSS plays a similar role. Supported by CNR, Special Project "Aging'.
1 Azzarelli, B., Muller, J., Ghelti, B., Dyken, M. and ('onneally, P.M., Cerebellar plaques in familial Alzheimer's disease (Gerstmann Str/iussler Scheinker variant?), Acta Neuropathol., 65 (1985) 235 246. 2 Bendheim, P.E., Barry, R.A., DeArmond, S.J., Sites, D.P. and Prusiner, S.B., Antibodies to a scrapie prion protein, Nature, 310 (1984)418 421. 3 Castafio, E.N., Ghiso, J., Prelli, F., Gorevic, P.D., Migheli, A. and Frangione, B., In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease •]-protein, Biochem. Biophys. Res. Commun., 141 (1986) 782 789. 4 Dickson, D.W., Crystal, H,, Mattiace, L.A., Kress, Y., Schwagerl, A., Ksiezak-Reding, H., Davies, P. and Yen, S.H.C., Diffuse Lewy body disease: light and electron microscopic immunocytochemistry of senile plaques, Acta Neuropalhol., 78 (19891 572 584. 5 Dickson, D.W., Wertkin, A., Mattiace, L.A., Fier, E.. Kress, Y., Davies, P. and Yen, S.-H., Ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of AIzheimer's disease, Acta Neuropathol., 79 (1990) 486 493. 6 Gallo, J.-M. and Anderton, B.H., Ubiquitous variations in nerves, Nature, 337 (1989) 687 688. 7 Gheni, B., Tagliavini, F., Masters, C.L., Beyreuther, K., Giaccone, G., Verga, L., Farlow, M.R., Conneally, P.M., Dlouhy, S.R., Azzarelli, B. and Bugiani, O., Gerstmann-Str~ussler-Scheinker dis-
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10
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ease. II. Neurofibritlary tangles and plaques with PrP-amyloid coexist in an affected family, Neurology, 39 (1989) 1453-1461. Hudson, A.J., Farrel, M.A., Kalnins, R. and Kaufmann, J.C.E., Gerstmann-Str~iussler-Scheinker disease with coincidental familial onset, Ann. Neurol., 14 (1983) 670~578. Kretzschmar, H.A., Prusiner, S.B., Stowring, L.E. and DeArmond, S.J., Scrapie prion proteins are synthesized in neurons, Am. J. Pathol., 122 (1986) 1-5. Leigh, P.N., Probst, A., Dale, G.E., Power, D.P., Brion, J.-P., Dodson, A. and Anderton, B.H., New aspects of the pathology of neurodegenerative disorders as revealed by ubiquitin antibodies, Acta Neuropathol., 79 (1989) 61-72. Manetto, V., Perry, G., Tabaton, M., Mulvihill, P., Fried, V.A., Smith, H.T., Gambetti, P. and Autilio-Gambetti, L., Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 4501-4505. Migheli, A. and Attanasio, A., Two methods for fiat-embedding in LR White sections cut from paraffin blocks, Stain Technol.. in press. Migheli, A., Autilio-Gambetti, L., Gambetti, P., Mocellini, C., Vigliani, M.C. and Schiffer, D., Ubiquitinated filamentous inclusions in spinal cord of patients with motor neuron disease, Neurosci. Lett., 114 (1990) 5-10. Nochlin, D., Sumi, S.M., Bird, T.D., Snow, A.D., Leventhal, C.M., Beureuther, K. and Masters, C.L., Familial dementia with PrPpositive amyloid plaques: a variant of Gerstmann-Str~iussler syndrome, Neurology, 39 (1989) 910-918.
15 Pappolla, M.A., Omar, R. and Saran, B., The ~Norrnal" Brain. 'Abnormal' ubiquitinitated deposits highlight an age-related protein change, Am. J. Pathol., 135 (1989) 585-591. 16 Perry, G., Friedman, R., Shaw, G. and Chau, V., Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains, Proc. Natl. Acad. SCI. U.S.A., 84 (1987) 3033-3036. 17 Prusiner, S.B., Prions and degenerative diseases, N. Engt. J. Med., 317 (1987) 1571-1581. 18 Schiffer, D., Autilio-Gambetti, L., Chi6, A., Gambetti, P., Giordana, M.T., Gullotta, F., Migheli, A. and Vigliani, M.C., Immunohistochemistry and immunoeleetron microscopy of ubiquitin in ALS, J. Neuropathol. Exp. Neurol., 49 (1990) 277. 19 Schlote, W., Boetlaard, J.W., Schumm, F. and St6hr, M., Gerstmann-Str/iussler-Seheinker's disease, Acta Neuropathol., 52 (1980) 203-211. 20 Schwartz, A.L., Ciechanover, A., Brandt, A.R. and Geuze, H.J., Immunoelectron microscopic localization of ubiquitin in hepatoma cells, EMBO J., 10 (1989) 2961 2966. 21 Seitelberger, F., Str/iussler's disease, Acta Neuropathol., Suppl. VII (1981) 341-343. 22 Vinters, H.V., Hudson, A.J. and Kaufmann, J.C.E., GerstmannStr~iussler-Seheinker disease: autopsy study of a familial case, Ann. Neurol., 20 (1986) 540-543. 23 Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., OsterGranite, M.L. and Neve, R.L., Neurotoxicity of a fragment of the amytoid precursor associated with Alzheimer's disease, Science, 245 (1989) 417-420.