Summary. Corpora amylacea (CA) accumulation in the central nervous system (CNS) is associated with both normal aging and neurodegenerative conditions ...
Acta Neuropathol (1993) 85:233 - 240
Acta Neuropathologm @ Springer-Verlag1993
Regular papers Immunochemical identification of ubiquitin and heat-shock proteins in corpora amylacea from normal aged and Alzheimer's disease brains* S. Ciss61, G. Perry 2, G. Lacoste-Royal 1, T. Cabana 3, and D. Gauvreau 1 1 INRS-Sant6 and D6partment des Sciences Biologique, Universit6 de Montr6al, Montreal, Qu6bee, Canada 2 Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA 3 D6partement des Sciences Biologique, Universit6 de Montreal, Montr6al, Quebec, Canada Received November 10, 1991/Revised,accepted June 12, 1992
Summary. Corpora amylacea (CA) accumulation in the central nervous system (CNS) is associated with both normal aging and neurodegenerative conditions such as Alzheimer's disease (AD). CA is reported to be primarily composed of glucose polymers, but approximately 4 % of the total weight of CA is consistently composed of protein. CA protein resolved on sodium dodecylsulfatepolyacrylamide gel electrophoresis showed a broad range of polypeptides ranging from 24 to 133 kDa, with four abundant bands. Immunoblots of the profile of polypeptides solubilized from purified CA, showed positive ubiquitin (Ub) immunoreactivity for all the bands. Antisera to heat-shock proteins (hsp) 28 and 70 reacted selectively with bands of 30 and 67 kDa. These results show that Ub is associated with the primary protein components of CA and that the polypeptides are likely to be Ub conjugates. Immunostaining experiments were performed to specifically characterize the protein components of CA in brain tissue sections as well as those of CA purified from both AD and normal aged brains. In all cases CA showed positive reactions with antibodies to Ub, with antibodies raised against either paired helical filaments or hsp 28 or 70, the most prominent staining being with antibodies to Ub, hsp 28 or hsp 70. The presence of Ub and hsp 28 and 70, which are actively induced after stress, suggests that accumulation of altered proteins, possibly attributed to an increased frequency of unusual post-translational modifications or to a sustained physiological stress (related to both normal aging and neurodegenerative process), may be involved in the pathogenesis of CA. Key words: Aging - Corpora amylacea - Heat-shock proteins - Ubiquitin - Alzheimer's disease * Supported by doctoral fellowshipsfrom the Graduate School of Universit6 de Montr6al and INRS-Sant6 and NIH grants AG07552 and AG09282. S. C. is a recipient of these fellowships Correspondence to: G. Perry (address see above) Reprint requests to: S. Ciss6 (address see above)
Corpora amylacea (CA) is one of the abnormal inclusions found in human astrocytes and neuronal cell bodies and their processes [3, 4, 32]. They are associated with normal aging of the central nervous system [1, 12] and neurodegenerative diseases such as Lafora disease [18, 23, 34], Atzheimer's disease (AD) [15, 16], and progressive supranuclear palsy (PSP) [6]. They are composed of glucose polymers and a small quantity of proteins [35-37, 40, 42]. The nature and the mode of formation of CA have been widely studied. Studies have mostly focused on the characterization of their sugar components by histological [3, 4, 32], histochemical [1, 25, 29], and biochemical [35-37] methods. Our approach to studying CA formation is to search for CA-associated proteins that could be important in their pathogenesis. A direct biochemical analysis by amino acid sequencing has shown that ubiquitin (Ub), a small protein of 8.5 kDa, is a component of CA polypeptides [11]. This finding is interesting since it is known that in some physiological conditions, Ub binds to free amino groups of acceptor proteins and triggers these proteins for rapid proteolysis [13, 20, 33]. Ub also appears to form stable conjugates with selected proteins, such as histones H2A and H2B [9], lymphocyte homing receptor [38, 43], platelet-derived growth factor receptor [46], actin [5], and microtubule-associated proteins [28]. It has been further suggested that the stable conjugates of Ub may constitute a new class of specific proteases [17]. Both Ub- and ATP-dependent proteolysis has been implicated in the heat-shock response [7, 8, 10, 13, 27] since overloading of the Ub-dependent and other proteolysis system by abnormal proteins seems to activate heatshock genes [2, 27]. Heat-shock response also seems to be induced by many treatments other than heat, most of which provoke the accumulation of denatured or damaged proteins within the cell. For instance, the abnormal forms of actin III, produced in the flight muscles of certain Drosophila mutants, cause expression of heat-shock protein (hsp) in those cells [21, 27]. Similarly in E. coli,
234 high-level expression of h u m a n tissue p l a s m i n o g e n activator, w h i c h fails t o fold correctly, induces hsp [19]. T h e m e c h a n i s m s responsible for C A biogenesis a n d abnormal protein accumulation, mainly Ub-acceptor proteins, in these inclusions r e m a i n to be elucidated. A l t h o u g h C A have b e e n isolated to h o m o g e n e i t y , the direct b i o c h e m i c a l analysis o f all C A p r o t e i n c o m p o nents by direct a m i n o acid s e q u e n c i n g has b e e n h i n d e r e d by t h e low yields o f p r o t e i n m a t e r i a l . W e have, t h e r e f o r e , e m p l o y e d i m m u n o c h e m i c a l t e c h n i q u e s to identify possible C A p r o t e i n c o m p o n e n t s . H e r e i n we d e m o n s t r a t e the p r e s e n c e o f U b and hsp e p i t o p e s in b o t h C A s m e a r s and tissue sections, as well as o n i m m u n o b l o t s o f purified C A .
Materials and methods
Source of tissue Brain tissue from five cases of normal aging (mean age 76.3 years), free of neurological disease as assessed by autopsy, or AD patients (mean age of 74.6 years) was removed within 9-12 h after death and frozen at - 7 0 ~ until needed.
peroxidase-antiperoxidase (PAP) technique [41] with 0.75 g/1 3',3'diaminobenzidine (DAB) and 0.5 ml/1 H202 as co-substrates. In the latter case, the sections were treated for 30 s with 1% OsO4 to enhance contrast. Sections were dehydrated and mounted with Permount. Smears of purified CA were fixed with cold acetone at - 20 ~ and stained with Vectastain ABC-AP system with BCIP and NBT as co-substrates or the PAP technique, using DAB as co-substrate, except that the smears were not dehydrated and were mounted with Aquamount. The secondary Ab dilutions were 1: 250.
Immunoblotting Polypeptides were solubilized from CA preparations in 2.5 % SDS-2-mercaptoethanol [42], and 1.13 rag/gel was run on 12.5 % or 7.5 %-15 % gradient polyacrylamide gels [24] in a mini-gel apparatus (Bio-Rad) and electrotransferred (2.5 A, 60 V overnight, 4~ either to nitrocellulose (NC) membrane (Millipore), heattreated with steam in an autoclave at 120~ for 30 min before blocking [44] or to a polyvinylidene difluoride membrane (Immobilon, Milhpore).The transfer buffer consisted of 125 mM TRIS, 39 mM glycine, and 20 % methanol. The blots were blocked in 0.1 mg/ml nonfat dry milk for 1 h at room temperature, washed in TRIS-buffered saline (TBS) for 3 rain, dried and cut in 2-mm wide strips. Strips were hydrated with TBS and incubated with the Ab at proper dilutions overnight at 4 ~ Reactions were developed by the Veetastain ABC-AP system or the PAP technique.
Preparation of CA and protein extraction Results CA were isolated according to a method previously described [44], and smeared on pre-cleaned slides. Brain tissue samples thawed to room temperature were fixed in 3.7 % (V/V) formaldehyde in 0.1 M sodium phosphate, pH 7.0, or Bouin's fixative, embedded in paraffin and cut at 8-~tm thickness with a microtome.
Antibodies The following antibodies (Ab) were used: (i) a cocktail made of eight monoclonal Ab (mAb) obtained from mice immunized with Ub cross-linked by glutaraldehyde to rabbit serum albumin [26, 28, 31] (4-2D8, 12-Hll, 7-2E6, 3-3G6, 2-3D7, 5-2E6, 5-2F3 and 4-3H8; a gift of Drs. H. Smith and V. Fried); (ii) antisera raised in rabbits against Ub cross-linked by glutaraldehyde to bovine serum albumin (BSA) [31] (3056; a gift of Drs. H. Smith and V. Fried); (iii) affinity-purified (AFP) Ab from rabbits immunized with Ub cross-linked by glutaraldehyde to keyhole limpet hemocyanin (KLH) [26]; (iv) mAb from mice immunized with paired helical filaments (PHF) (5-25) and (3-39) [45] (these mAb recognize Ub and were a gift of Drs. K. Iqbal and I. Grundke-Iqbal); (v) mice mAb specific for both the constitutive and stress induced form of hsp 70 (hsp 73: N27F3-3, hsp 72: C92F3A-5); (vi) rabbit polyclonal Ab to the low molecular weight hsp 28 protein, highly induced after stress. The Ab in (v) and (vi) were a gift of Dr. W. Welch. Secondary Ab for Vectastain avidin-biotin conjugated alkaline phosphatase (ABC-AP) system were anti-mouse IgG and antirabbit IgG biotin conjugate (Sigma). Immunoabsorption consisted of incubating 4-2D8 at 1 : 10 with 10 mg/ml of Ub (Sigma), at 4 ~ for 16 h prior to immunostaining.
Immunostaining Tissue sections were immunostained using the Vectastain ABC-AP standard kit AK-5000 (Vector Laboratories, Inc., 1984, Burlingame, Calif.) with 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) and 0.3 mg/ml of p-nitro blue tetrazolium chloride (NBT) (Bio-Rad) as co-substrates, or for
Immunostaining All A b t o U b a n d t h o s e raised t o hsp proteins, stained C A structures positively in b o t h brain sections a n d smears (Figs. 1-4). W h i l e the b o r d e r was m o s t consist e n t l y stained, the entire C A were o n o c c a s i o n d e e p l y stained for U b A b (4-2D8, p o o l e d U b m A b ) , with p r o m i n e n c e o f t h e cores in s o m e cases (Fig. l a , c). A b raised t o PHF, w h i c h recognize U b ( 5 - 2 5 a n d 3 - 3 9 ) , r e c o g n i z e d the s a m e structures as t h o s e raised against U b (result n o t shown). C A were differentiated f r o m nuclei, which were also stained b y t h e a n t i b o d y to U b , h a v i n g a m o r e u n i f o r m , less g r a n u l a r stain, being o f t e n larger a n d clustered n e a r the e p e n d y m a and n o t being obviously c o n t a i n e d in a cell body. T h e specificity o f t h e U b i m m u n o r e a c t i v i t y in C A was s h o w n b y a b s o r b i n g the 4-2D8 m A b with U b p r i o r to i m m u n o s t a i n i n g , which resulted in a c o m p l e t e b l o c k a g e o f C A staining (Fig. 2). A n o t h e r i m p o r t a n t aspect is t h a t no C A staining was seen in t h e a b s e n c e o f p r i m a r y a n t i b o d y n o r in the p r e s e n c e o f several m A b or antisera to u n r e l a t e d antigens with t h e s a m e s e c o n d a r y r e a g e n t s e m p l o y e d . A b to hsp r e c o g n i z e d m o s t l y the b o r d e r o f C A a n d a few disperse granule-like structures within the C A (Figs. 3, 4). T h e staining profile o f C A s m e a r s b y b o t h U b a n d hsp proteins was similar, except t h a t s o m e t i m e s the A b to U b r e c o g n i z e d t h e central region of C A .
Immunoblotting P o l y p e p t i d e s were solubilized f r o m purified C A , resolved b y S D S - P A G E a n d transferred. I m m u n o s t a i n -
235
Fig. 2. Absorption of the mAb to Ub, 4-2D8, with Ub (10 mg/ml) blocked CA staining (a) compared to untreated antibody as shown in the adjacent section (b)
ing of the protein profile with Ab to Ub revealed the same pattern (Fig. 5). Anti-Ub (rabbit 1-4 and 3056) reacted with polypeptide bands ranging from 30 to over 133 kDa, with the material not entering the separating gel. A n t i - P H F (5-25 and 3-39) stained the same bands (Fig. 6). All these Ab showed stronger reactions on heat-treated NC (results not shown). Mice mAb to both constitutive and stress-induced forms of hsp (N27F3-4 and C92F3A-5) revealed a band of 67 kDa, whereas rabbit polyclonal Ab to the low molecular weight hsp 28 recognized polypeptide bands of 30 kDa and a smear at 67 kDa, the f o r m e r being more reactive (Fig. 7). The apparent contrast in the weak recognition of the 67 kDa band and the strong staining of CA b y A b to hsp 70 could indicate that, in addition to containing hsp 70, CA may also be binding the Ab. However, the same result was
Fig. l a d . Ubiquitin immunocytochemical staining of brain tissue sections and purified corpora amylacea (CA). a Section of hippocampus from normal aging brain showing CA (arrowheads) with positive reaction to anti-ubiquitin (Ub) 4-2D8 (1:1000). b
Section of brain tissue from Alzheimer's disease (AD) patient, showing CA (arrowheads) with positive reaction to a cocktail of monoclonal antibodies (mAb) raised to Ub (1 : 1000). e Purified CA from normal aging brain, showing positive reaction with anti-Ub 4-2D8 (1:1000). d Purified CA from AD brain, showing positive reaction with a cocktail of mAb to Ub (1:1000)
236
Fig. 3a-h. Heat-shock protein (hsp) immunocytochemical staining of brain tissue sections and purified CA. a Section of normal aging brain tissue showing area of positive immunoperoxidase reaction products with anti-hsp N27F3-4. h Section of AD brain tissue showing area of positive immunoperoxidase reaction products with anti-hsp N27F3-4 CA (arrowheads).c Purified CA from normal aging brain, showing positive immunoperoxidase reaction products with anti-hsp N27F3-4. d Purified CA from AD brain, showing positive immunoperoxidase reaction products with anti-
hsp N27F3-4. e Section of normal aging brain tissue, showing area of positive immunoperoxidase reaction products with anti-hsp C92F3A-5. f Section of AD brain tissue, showing area of positive immunoperoxidase reaction products with anti-hsp C92F3A-5. g Purified CA from normal aging brain, showing positive immunoperoxidase reaction products with anti-hsp C92F3A-5. h Purified CA from AD brain, showing positive immunoperoxidase reaction products with anti-hsp C92F3A-5
237
Fig. 4a, b. Immunocytochemical staining of brain tissue sections and purified CA with polyclonal Ab raised against hsp 28 (1:100). a Section of normal aging brain tissue, showing positive immunoperoxidase reaction products on CA. b Purified CA from normal aging brain, showing positive immunoperoxidase reaction products
Discussion
Fig. 5. Immunoblots of purified CA polypeptides stained with antibodies to Ub. The electrophoresis were carried out on 7.5 %-15 % linear gradient gel, in buffer containing 125 mM TRIS-HC1 pH 6.8, 3 % SDS and 2 % 2-mercaptoethanol. Bars indicate the positions of molecular weight markers. The antibodies raised against Ub show positive alkaline phosphatase reaction products on all bands. Migration of molecular weight markers is indicated at the left (kDa). Lane A : Sample stained with india ink to show proteins in the replica. Lane B, C: Samples from normal aging brains; B polyclonai anti-Ub 3056 (1 : 10 000), C mAb anti-Ub 4-2D8 (1:10000). Lane D - E : Samples from AD brains; D polyclonal anti-Ub 3056 (1:10 000), E affinity-purified anti-Ub rabbit 1-4 (1:1oooo)
The first step to understanding the nature and the origin of CA is the identification and characterization of its components. Previous studies have mainly focused on the identification of the glucose components [36, 37, 42]. We have looked at the protein components, an approach which may lead to the identification of normal proteins that may be equally, if not more, important precursors of CA. Immunochemical characterization of polypeptide constituents confirms our sequencing studies, indicating that U b is associated with a majority of CA protein components. Additionally, hsp proteins (in particular the 30 kDa) are found in CA, suggesting that the biogenesis of CA may relate to physiological stress. In
eukaryotic cells stress provokes abnormal protein formation, which induces Ub synthesis. Overloading of the proteolysis system by abnormal proteins likely activates heat-shock genes [2, 13, 19, 27]. Immunoblots were carried out to characterize specifically the protein components of CA. The immunoblot characterization was essential since CA intensively binds some Ab irrespective of their affinity. However, with peptide recognition by specific Ab of CA proteins, we could be assured that the recognition of CA was in fact specific. Antibodies to Ub revealed many bands above 20 kDa in the CA polypeptide profile, whereas antibodies to hsp showed staining of only 30- and 67-kDa
found with two Ab, and the sharpness of the protein band in the immunoblot suggests that the recognition is specific. Interestingly the Ab to Ub also recognized 67and 30-kDa bands.
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Fig. 6. Immunoblots of purified CA polypeptides stained with antibodies to paired helical filaments (PHF) which recognize Ub. Same conditions as Fig. 5. The antibodies raised against PHF recognize the same bands as anti-Ub. Lane A : India ink staining to show proteins present in the replica. Lane B: Sample from normal aging brain, showing bands with alkaline phosphatase reaction products with anti-PHF 3-39 mAb (1:1000). Lane C: Sample from normal aging brain, showing bands with positive alkaline phosphatase reaction products with anti-PHF 5-25 mAb (1:1000)
polypeptides. Since U b is a small protein of 8.5 kDa, these findings are consistent with the formation of U b conjugates. T h e finding that Ab to Ub, in particular those raised to PHF, and to hsp reacted with the bands of similar molecular mass suggests that U b is conjugated to the altered forms of these proteins, or to U b itself. It has been reported that the amount and stability of Tritoninsoluble high molecular mass U b conjugates increases following heat shock [10]. In addition, the rate of protein degradation decreases during the same process [10, 28] and in cellular aging [22, 39]. T h e increase in stable U b conjugates and the decrease of protein degradation have been attributed to the overloading of the U b - d e p e n d e n t proteolysis system due to the presence of increased amounts of abnormal proteins [10, 39]. T h e presence of altered proteins may be attributed
Fig. 7. hsp immunoblots of purified CA polypeptides. The electrophoresis were carried out on 12.5 % polyacrylamide gel under the same conditions as Fig. 5 and 6. The antibodies raised against hsp recognize bands of 67 and 28 kDa. Lane A : India ink staining to show proteins present in replica. Lane B: Sample from normal aging brain,showing bands with positive alkaline phosphate reaction products with monoclonal anti-hsp N27F3-4 (1: 250). Lane C: Sample from normal aging brain, showing bands with positive alkaline phosphatase reaction products with monoclonal anti-hsp C92F3A-5 (1:250). Lane D: Sample from normal aging brain, showing bands with positive alkaline phosphatase reaction products with anti-hsp 28 (1 : 250)
to unusual translational modifications occurring with increased frequency during aging [22], to sustained physiological stress related to the aging process, or to a combination of the two events. This hypothesis is thought to be correlated somehow by the strong reactivity of Ab to hsp 28 protein in both tissue sections and smears, this protein being highly induced after stress. The biological significance of CA inclusions is unknown, as is the role of U b and hsp proteins in these inclusions. Nevertheless, the finding of similar U b conjugates and hsp in CA and neurofibrillary pathology suggests that similar mechanisms may be operative in both. T h e r e is evidence that U b is involved in the disposal of short-lived and altered proteins that are found after a variety of injuries [13, 14] via the Ubdependant proteolytic system. Hsp induced in response to protein damage play an important role in the limiting or repair of such damage by binding to exposed hydrophobic surfaces [13].The major hsp (hsp 70) seems to aid in the repair of heat-damaged or stress-damaged nucleoli [30]. Since CA have been reported to be made
239 o f g l u c o s e p o l y m e r s , it is t e m p t i n g t o s u g g e s t t h a t t h e i r b i o g e n e s i s is r e l a t e d t o p h y s i o l o g i c a l stress d u e t o a l t e r a t i o n o f e n z y m a t i c p r o t e i n s i n v o l v e d in t h e m e t a bolic pathway of glucose. Identification of the Ub c o n j u g a t e s in t h e s e i n c l u s i o n s m i g h t p r o v i d e n e w insight into their mechanism of formation. Acknowledgements. We wish to thank Dr. Raymond Farmer (H6pital L.-H. Lafontaine, Montrdal, Canada) for providing aged human brain tissues; Drs.Victor Fried and Harry Smith (NewYork Medical College, Valhalla, N.Y.) for monoclonal antibodies to ubiquitin; Drs. Khalid Iqbal and Inge Grundke-Iqbal (Institute for Basic Research in Developmental Disorders, Staten Island, N.Y.) for antibodies 5-25 and 3-39; Dr. William Welch (San Francisco General Hospital, San Francisco, Calif.) For antibodies to heatshock proteins; Dr. Yves Robitaille (Institut Neurologique de Montrral, Montrral, Canada) who generously provided Alzheimer brain tissues; Dr. Josdphine Nalbantoglu (INRS-Santr, PointeClaire, Qudbec, Canada) for helpful comments and discussions concerning the manuscript; Ms. Louise Pelletier for the photography; and Ms. Manon Ldger for secretarial work.
References 1. Alder N (1953) On the nature, origin and distribution of the corpora amylacea of the brain with observations on some new staining reactions. J Ment Sci 99:689-697 2. Ananthan J, Goldberg AL, Voellmy R (1986) Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232:522-524 3. Anzil AP, Herrlinger H, Blinzinger K, Kronski D (1974) Intraneuritic corpora amylacea. Virchows Arch [A] 364:297-304 4. Averback P (1981) Parasynaptic corpora amylacea in the striatum. Arch Pathol Lab Med 105:334-335 5. Ball E, Karlik CC, Beall CJ, Saville DL, Sparrow JC, Bullard B, Fyrberg E A (1987) Arthrin, a myofibrillar protein of insect flight muscle, is actin-ubiquitin conjugate. Cell 51:221-228 6. Behrman S, Caroll JD, Janota I, Matthews WB (1969) Progressive supranuclear palsy. Clinico-pathological study of four cases. Brain 92:663-678 7. Bond U, Schlesinger MJ (1985) Ubiquitin is a heat-shock protein in chicken embryo fibroblasts. Mol Cell Biol 5:949-956 8. Bond U, Schlesinger MJ (1986) The chicken ubiquitin gene contains a heat-shock promoter and expresses an unstable mRNA in heat shock cells. Mol Cell Biol 6:4602-4610 9. Busch H, Goldnopf IL (1981) Ubiquitin-protein conjugates. Mol Cell Biochem 40:173-187 10. Carlson N, Rogers S, Rechsteiner M (1987) Microinjection of ubiquitin: changes in protein degradation in Hela cells subjected to heat shock. J Cell Biol 104:547-555 11. Ciss6 S, Lacoste-Royal G, LaperrierreT, CabanaT, Gauvreau D (1991) Ubiquitin is a component of polypeptides purified from corpora amylacea of aged human brain. Neurochem Res 16:429-433 12. Ellis RS (1920) Norms for some structural changes in the human cerebellum from birth to old age. J Comp Neurol 32:1-3313 13. Finley D,Varshavsky A (1985) The ubiquitin system: functions and mechanisms. Trends Biochem Sci 10:343-346 14. Finley D, Ciechanover A,Varshavsky A (1984) Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell 37:43-55 15. Fleming PD, Rogers J (1986) Neuropathology of olfactory system in Alzheimer's disease and normal aging. Soc Neurosci Abstr 12:1314
16. Fleming PD, Cordoza ME,Woods SG, Griesbach EJ,Worcester MA (1987) Corpora amylacea increased in Aizheimer's disease. Neurology [Suppl 1] 37:157 17. Fried VA, Smith HT, Hildebrandt E, Weiner K (1987) Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation. Proc Natl Acad Sci USA 84:3685-3689 18. Gambetti P, DiMauro S, Hirt L, Blume RP (1971) Myoclonic epilepsy with Lafora bodies: some ultrastructural, histochemical and biochemical aspects. Arch Neurol 25:483-493 19. Goff SA, Goldberg A L (1985) Production of abnormal proteins in E. coli stimulates transcription of Ion and other heat shock genes. Cell 41:587 20. Hershko A, Ciechanover A (1986) The Ubiquitin pathway for the degradation of intraceUular proteins. Prog Nucleic Acid Res Mol Biol 33:19-56 21. Hiromi Y, HottaY (1985) Actin gene mutations in Drosophila; heatshock activation in the indirect flight muscle. EMBO J 4:1681-1687 22. Jahnger JH, Haas AL, Ciechanover A, Blonin J, Eisenhauer D,Taylor A (1986) The eye lens has an active ubiquitin-protein conjugation system. J Biol Chem 261:13760-13767 23. Janeway R, Ravens JR, Pearce LA, Odor DL, Suzuki K (1967) Progressive myoclonus epilepsy with Lafora inclusion bodies. I. Clinical, genetic, histopathologic and biochemical aspects. Arch Neurol 16:565-582 24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 25. Liu HM, Burns AC (1985) Mucopolysaccharides in corpora amylacea of normal and diseased brains. J Neuropathol Exp Neurol 44:335 26. ManettoV, Perry G,Tabaton M, Mulvihill P, Fried V, Smith H, Gambetti E Aubillio-Gambetti L (1988) Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases. Proc Natl Acad Sci USA 85:4501-4505 27. Munro S, Pelham H (1985) What turns on heatshock genes? Nature 317:477-478 28. Murti KG, Smith HT, Fried VA (1988) Ubiquitin is a component of the microtubule network. Proc Natl Acad Sci USA 85:3019-3023 29. Palmucci L, Anzil AP, Luh S (1982) Intra-astrocytic glycogen granules and corpora amylacea stain positively for polyglucosans: a cytochemical contribution on the fine structural polymorphism of particulate polysaccharides. Acta Neuropathol (Bed) 57:99-102 30. Pelham HRB (1984) Hsp 70 accelerates the recovery of nuclear morphology after heat shock. EMBO J 3:3095-3100 31. Perry G, Mulvihill E Fried VA, Smith HT, Grundke-Iqbal I, Iqbal K (1989) Immunochemical properties of ubiquitin conjugates in the paired helical filaments of Alzheimer disease. J Neurochem 52:1523-1528 32. Ramsey HJ (1965) Ultrastructure of copora amylacea. J Neuropathol Exp Neurol 24:25-39 33. Rechsteiner M (1987) Ubiquitin mediated pathways for intracellular proteolysis. Ann Rev Cell Biol 3:1-30 34. Robitaille Y, Carpenter S, Karpati G, DiMauro S (1980) A distinct form of adult polyglucosan body disease with massive involvement of central and peripheral neuronal processes and astrocytes. Brain 103:315-336 35. Roukema PA, Oderkerk CH (1970) Isolation and preliminary characterization of corpora amylacea from human brains. Psychiatr Neurol Neurochir 73:87-89 36. Sakai M, Austin J,Witmer F,Trueb L (1969). Studies of corpora amylacea. I: Isolation and preliminary characterization by chemical and histochemical techniques. Arch Neurol 21:526-544 37. Sakai M, Austin J, Witmer F, Trueb L (1969) Corpora amylacea: isolation, characterization and significance. Trans Am Neurol Assoc 94:336-338 38. Siegelman M, Bond MW, Gallatin WM, St-JohnT, Smith HT,
240
39. 40. 41. 42.
43.
Fried VA,Weissman IL (1986) Cell surface molecule associated with stimulated homing is a ubiquinated branched-chain glycoprotein. Science 231:823-829 Speiser S, Etlinger JD (1983) ATP proteolysis in reticulocytes extracts by repressing an endogenous inhibitor. Proc Natl Acad Sci USA 80:3577-3580 Stam FC, Roukema PA (1973) Histochemical and biochemical aspects of corpora amylacea. Acta Neuropathol (Berl) 25:95-102 Sternberger LA (1986) Immunocytochemistry, 3rd edn. John Wiley, New York Steyaert A, Ciss6 S, Merhi Y, Kalbakji A, Reid N, Gauvreau D, Lacoste-Royal G (1990) Purification and polypeptide composition of corpora amylacea from aged human brain. J Neurosci Methods 31:59-64 St-John T, Gallatin WM, Siegelman M, Smith HT, Fried VA,
Weissman IL (1986) Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species. Science 231:845-850 44. Swerdlow PS, Finley D,Varshavsky A.(1986) Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal Biochem 156:147-153 45. Wang GE Grundke-Iqbal I, Kascsak RJ, Iqbal K, Wisniewski HM (1984) Alzheimer neurofibrillary tangles: monoclonal antibodies to inherent antigen(s). Acta Neuropathol (Berl) 62:268-275 46. YardenY, Escobedo JA, Kuang WJ,Yang-FengTL, Daniel TO, Tremble PM, Chen EY, Ando ME, Harkins RN, Francke U, Fried VA, Ullrich A, Williams LT (1986) Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature 323:226-232
