Neuropathology and Applied Neurobiology 1992,18,34 1-350
Ballooned neurons in several neurodegenerative diseases and stroke contain aB crystallin J. LOWE*, D. R. E R R I N G T O N ? , G . LENNOXS, I. PIKE?, I. S P E N D L O V E ? , M. LANDON?, A N D R. J. M A Y E R ? Departments of *Pathology, ?Biochemistry and $Neurology, University of Nottingham Medical School, Queens Medical Centre, Nottingham
LOW J., ERRINGTON D. R., L m o x G., PIKEI., SPENDLOVE I., LANDONM. & MAYERR. J. (1992) Neuropathology and Applied Neurobiology 18,34 1-350 Ballooned neurons in several neurodegenerative diseases and stroke contain aB crystallin
a B crystallin is a protein which has homology with the small cell stress proteins. A characterized antibody to residues 1-10 of a B crystallin was used to immunostain tissues containing ballooned (chromatolytic, achromasic) neurons. The tissues included two cases of classical Pick’s disease, one case of dementia with swollen achromasic neurons in the cortex, two cases of Alzheimer’s disease with large numbers of ballooned neurons, two cases of motor neuron disease, four cases of cortico-basal degeneration, and four cases with areas of brain showing swollen neurons adjacent to recent cerebral infarcts. The anti-aB crystallin showed strong diffuse cytoplasmic immunoreactivity of swollen cortical neurons in all the diseases. Astrocytes and oligodendroglial cells were also stained in normal tissues as previously described. Weak diffuse immunoreactivity with an antibody to ubiquitin-conjugates was also seen in the swollen neurons from cases of neurodegenerative disease but not following infarction. Ballooned neurons have been shown to contain phosphorylated neurofilament epitopes not normally present in the perikaryonal region. The presence of aB crystallin in ballooned neurons, together with previous data which also indicate its close association with intermediate filaments, suggest that a B crystallin may be involved in aggregation and remodelling of neurofilaments in disease. The presence of aB crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect cells which are regenerating following damage; its detection may therefore be a marker for such cells. On a practical level, the antibody greatly facilitates the localization of such abnormal neurons in diagnostic histology. Keywords: aB crystallin, heat shock protein, ubiquitin, intermediate filaments, Pick’s disease, Alzheimer’s disease, motor neuron disease, stroke, dementia, neuronal chromatolysis
INTRODUCTION The crystallins are a family of soluble proteins grouped together because of their physical properties whereby they form compact aggregated crystalline structures which are present in the eye and stable for the whole life of the individual (for review see Wistow & Piatigorsky, 1988). While they are found in high concentration in the lens of the eye, each crystallin molecule is also structurally identical or related to proteins with distinct functions outside the lens; a Correspondence: Dr J. Lowe, Department of Pathology, Queens Medical Centre, Nottingham NG7 2UH, UK.
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phenomenon described as ‘gene sharing’ (Wistow & Piatigorsky, 1988; DeJong el al., 1989). The a crystallins are composed of two subunits designated aA and aB which have structural homology with small heat shock proteins of the hsp20-30 class (Ingolia & Craig, 1982). a B crystallin is expressed in non-lenticular tissues (Baht & Nagineni, 1989; Dubin, Wawrousek & Piatigorsky, 1989; Iwaki, Kume-Iwake & Goldman, 1990) and in the nervous system, where it is normally present in astrocytes and oligodendroglial cells but is not seen in normal adult neurons (Iwaki et af.,1990).The protein is modified post-translationally by phosphorylation; however, the functional significance of this is uncertain. In pathological states, reactive glia show increased immunoreactivity with aB crystallin (Iwaki et af., 1992; Renkawek et a[., 1992). We have previously shown that a B crystallin is a common factor in intermediate filamentcontaining ubiquitinated inclusion bodies. In particular aB crystallin is present in neurons containing cortical Lewy bodies where it is seen both in the perikarya as well as in the inclusion (Lowe et af., 1990; Lowe et af.,1992) and this finding has been confirmed by others (Iwaki et af.,1992). The observation in our previous studies that the antiserum to a B crystallin stains only a proportion of ubiquitin-filament inclusions suggested that the protein might be involved in one phase of inclusion biogenesis, perhaps in initial intermediate filament aggregation. aB crystallin is present in the ubiquitinated inclusions in glial cells in multiple system atrophy (Kato et af.,1991) and it has been shown recently that aB crystallin in Rosenthal fibres is ubiquitinated (Goldman & Corbin, 1991). There appears to be an association between intermediate filaments and aB crystallin in addition to its presence in inclusion bodies; for example, it has been localized to the intermediate filaments in cardiac muscle (Longoni et af., 1990), is seen in the large vimentin-containing dysplastic cells in tuberous sclerosis (Iwaki & Tateishi, 1991), and is closely related to the presence of GFAP in glial tumours (Iwaki et af., 1991). The possibility that aB crystallin is involved in intermediate filament aggregation is supported by experimental observations that the molecule has homology with the hsp20-30 class of proteins (Ingolia & Craig 1982), which are known to aggregate with intermediate filaments (Leicht et af.,1986;Arrigo, Suhan &Welch, 1988),and also has self aggregating properties (Wistow & Piatigorsky, 1988) which might mediate adhesion. We now extend observations on the presence of aB crystallin in the nervous system by showing strong immunoreactivity to this protein in swollen neurons in several conditions. This type of abnormality of neurons, in which cells become swollen and voluminous with pale-staining cytoplasm and a large peripherally placed nucleus, has been described as chromatolysis or achromasia by some workers but the descriptive term ballooning (Dickson et af., 1986) is used here. In addition to neuronal axonal reaction following severance of the axon, the most familiar example of such change is the Pick cell of Pick’s disease (Constantinidis, Richard & Tissot, 1974) but ballooned neurons are also a prominent feature of cortico-basal degeneration (Gibb, Luthert & Marsden, 1989; Riley et af., 1990) as well as being seen in some cases of transmissible prion dementia (Clarke et al., 1986; Nakazato et af., 1990). Several workers have described cases of dementia characterized by ballooned cortical neurons in the absence of classical Pick bodies (Constantinidis et af., 1974; Munoz-Garcia & Ludwin, 1986; Clark et af. 1986a; Clarke et af., 1986b; Dickson et af., 1986) and these cases may overlap with those described in association with primary progressive aphasia (Lippa et af., 1991). A small number of cases, which satisfy diagnostic criteria for Alzheimer’s disease, have also been described as showing ballooned cortical neurons (Dickson et af.,1986; Clarke et af., 1986b). A few swollen cortical neurons have been reported in some cases of frontal lobe degeneration of non-Alzheimer type, in which the most characteristic feature is spongiform degeneration in the frontal and temporal lobes limited to the outer cortical layers (Brun, 1987).
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Previous studies have shown that such ballooned neurons contain phosphorylated neurofilaments (Dickson et al., 1986; Nakazato et al., 1990) and similar observations have been recorded in swollen anterior horn neurons in motor neuron disease (ALS) (Munoz et al., 1988; Manetto et al., 1988). In this paper we present results which suggest that the intermediate filament accumulation which is seen in such swollen neurons is accompanied by the presence of a B crystallin. MATERIALS A N D METHODS Antibody to aB crystallin
A polyclonal antiserum against aB crystallin was prepared by immunizing rabbits with a synthetic peptide-haemocyanin conjugate and has been previously characterized. It detects a single 22 kDa polypeptide, corresponding to aB crystallin, on nitrocellulose replicas after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of extracts of heart muscle (known to contain aB crystallin) and also immunodetected a similar single approximately 22 kDa polypeptide in extracts of brain from a case of diffuse Lewy body disease (Lowe et al., 1992). Cases
Cases containing ballooned cortical neurons were selected from the files of the Neuropathology section of the Department of Pathology of the Queens Medical Centre Nottingham. All tissues had been fixed in 10% formalin for at least 6 weeks prior to routine embedding in paraffin wax. The tissues were: 1 Temporal lobe from two cases of classical Pick’s disease with both ballooned Pick cells and classical Pick bodies. 2 Temporal lobe from two severely demented patients with Alzheimer’s disease characterized by widespread cortical plaques as well as numerous neocortical neurofibrillary tangles together with large numbers of ballooned neurons in the anterior temporal lobe in the absence of Pick bodies or Lewy bodies. These two cases appear to be of the same type as the two described by Dickson et al. (1986). 3 Temporal lobe from a case of severe dementia characterized by ballooned neurons in the cortex in the absence of Pick bodies, plaques or tangles. This case appears to be similar to the two documented by Clarke et a]. (1986b). 4 Cervical spinal cord from two cases of motor neuron disease (ALS) in which characteristic ubiquitinated inclusions were present in remaining spinal anterior horn cells together with ballooned neurons and axonal spheroids. 5 Cerebral cortex from four cases of cortico-basal degeneration (kindly provided by Dr P. Luthert at the Institute of Psychiatry, London). 6 Cerebral tissue adjacent to areas of recent (8-14 days) cerebral infarction in which swollen neurons had been seen on haematoxylin and eosin staining (four cases).
Immunostaining
Sections were stained using anti-aB crystallin and, in addition, for cases of Alzheimer’s disease, motor neuron disease, cerebral infarction and Pick’s disease with an antibody which detects
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ubiquitin-protein conjugates (Lowe et af., 1988a). Immunostaining was with a standard streptavidin-biotin method. Briefly, after dewaxing in xylene, sections were treated with 3% H20, in methanol, rehydrated to water, and incubated in the following: (i) normal swine serum (NSS; 20%) in 0.05 M Tris saline, pH 7.6 (TS) for 20 min. (ii) either anti-ubiquitin-protein conjugate (1:lOOO) in 120 NSS for 30 min or anti-aB crystallin (1 :4000) in 1:20 NSS for 30 min. (iii) biotinylated swine anti-rabbit immunoglobulin (Dakopatts, Denmark) (1500) in 120 NSS for 30 min. (iv) avidin/biotin/peroxidase complex (Dakopatts, Denmark) in TS for 30 min. (v) peroxidase substrate (0.05% diaminobenzidine, 0.07% imidazole, 0.01YOH,O, in TS) for 10 min. (vi) enhancer (0.5% CuSO, in TS) for 10min. (vii) sections were counterstained with haematoxylin. Negative controls were performed in each case by omission of the primary antiserum. RESULTS The antibody to ubiquitin-protein conjugates immunostained structures such as Pick bodies, neurofibrillary tangles, inclusions in motor neuron disease, dystrophic neurites, and axonal spheroids as previously described (Leigh et al., 1988; Lowe et af.,1988a,b) The antibody to aB crystallin stained astrocytes and oligodendroglial cells in normal tissues, as previously reported (Iwaki et af., 1990; Lowe et af.,1992). Staining in oligodendroglial cells diminished in intensity with prolonged formalin fixation, and was not detected in tissues that had spent more than 4 months in fixative. The antibody to aB crystallin intensely stained the ballooned cortical neurons in all cases examined and this staining was detectable despite prolonged fixation and the absence of oligodendroglial staining in some instances. Examination of the Alzheimer’s disease cases with swollen neurons showed intense positivity of swollen cells with the antibody to aB crystallin (Figure la). These swollen cells also showed weak, diffuse staining with the anti-ubiquitin antibody (Figure 1b). As noted earlier the Pick’s disease cases showed no staining of inclusions (Lowe et af.,1992), but swollen cells showed diffuse and intense immunoreactivity against aB crystallin. These cells also showed diffuse weak staining with the anti-ubiquitin antibody that was identical to that seen in the Alzheimer cases (Figure lb) and is not illustrated here. The cases of cortico-basal degeneration showed intense aB crystallin immunoreactive swollen neurons (Figure 2a) with staining seen extending down the axon in many affected cells (Figure 2b). Examination of areas of grey matter adjacent to recent cerebral infarcts showed positive immunostaining for aB crystallin of a small porportion of neurons (Figure 3). It was possible to trace a range of morphologies from pyramidal with a concave margin, through pyramidal with a swollen convex margin to swollen cells with an eccentric nucleus and dispersed Nissl substance (Figures 3a-c). In contrast to the swollen neurons in cases of neurodegenerative disease, we were not able to detect ubiquitin immunoreactivity in swollen neurons associated with infarction (Figure 3d). Axonal spheroids adjacent to areas of infarction showed no positive staining with anti-aB crystallin despite staining of adjacent neurons and glia (Figure 4).
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Figure 1. a, Swollen neuron in the cerebral cortex in a case of Alzheimer’s disease. Anti-aB crystallin/haematoxylin. x 430. b, Faint diffuse ubiquitin-immunoreactivity of swollen neuron. Note immunoreactivity of plaque-related neurites-arrowed. Anti-ubiquitin/haematoxylin. x 430.
Figure 2. a, Swollen cortical neurons in corticobasal degeneration. x 110. b, Detail showing axonal extension of aB crystallin immunoreactivity. Anti-aB crystallin/haematoxylin. x 430.
Cases of motor neuron disease showed intense immunostaining of swollen anterior horn cells for aB crystallin together with staining of normal and reactive glia (Figure 5a). Axonal spheroids and normal anterior horn cells were not immunostained (Figure 5b). Comparison with
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Figure 3. aB crystallin in neurons from adjacent to cerebral infarct. a, Normal morphology.b, Early convex border. c, Swollen cell with eccentric nucleus (anti-aB crystallin/haematoxylin). d, Swollen cell in association with infarction do not show ubiquitin immunoreactivity. Anti-ubiquitin/haematoxylin. All x 430.
Figure 4. In an area of recent infarction axonal spheroids show no aB crystallin immunoreactivity in contrast to adjacent neuronal and glial cells. Anti-aB crystallin/ haematoxylin. x 430.
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Figure 5. a, aB crystallin immunoreactivity in anterior horn cell in motor neuron disease. b, No aB crystallin immunoreactivity in axonal spheroid in motor neuron disease. Note positive glial staining. Anti-aB crystallin/ haematoxylin. Both x 430.
antiubiquitin staining showed that the majority of aB crystallin-containing cells did not contain inclusions. DISCUSSION In addition to its presence in glial cells and certain ubiquitinated inclusions, it is clear that aB crystallin is present in swollen neurons in a variety of diseases. This finding is reinforced by other recent published work in which neurons in Creutzfeldt-Jakob disease, multiple system atrophy and progressive supranuclear palsy have shown aB crystallin immunoreactivity (Iwaki et al., 1992). It is possible that the intermediate filament accumulation, which characterizes swollen neurons, is mediated through interactions with aB crystallin. This suggestion is supported by the facts that aB crystallin has self-aggregating properties (Wistow & Piatigorsky, 1988) and has homology with the hsp20-30 class of proteins (Ingolia & Craig, 1982), which are known to aggregate with intermediate filaments (Leicht et al., 1986; Arrigo et al., 1988). It remains to be determined whether aB crystallin is directly bound to intermediate.filaments. The lack of immunostaining of axonal spheroids with the antibody supports the idea that the presence of aB crystallin is not merely an epiphenomenon consequent upon the accumulation of neurofilaments, known to be abundant in spheroids. Recent reports show that aB crystallin is induced by oncogene expression and heat shock in a mouse fibroblast cell line (Klemenz et al., 1989;Klemenz et al., 1991)and the aB crystallin gene is known to have a heat shock promoter (DeJong e f al., 1989; Dubin et al., 1990). aB crystallin
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is not detectable in normal neurons by routine immunochemical techniques and, following neuronal damage, it is probably induced as part of a cell stress response when, like other stress-induced proteins, it has a role in cytoprotection of cells following damaging stimuli. The molecular pathological features of certain forms of neuronal swelling are now beginning to be understood. The swelling of spinal anterior horn cells following crush injury to the sciatic nerve is accompanied by accumulation of phosphorylated neurofilament in the perikaryon together with a reduction in messenger RNA coding for neurofilament protein (Goldstein et af., 1988; Muma et af., 1990).This suggests that such phosphorylated neurofilament accumulation is the result of altered transport or breakdown rather than overproduction. There is also accumulation of peripherin, a ‘new’ neuronal intermediate filament, in such swollen neurons accompanied by increased expression of its mRNA (Troy et af., 1990). aB crystallin now appears to be a key protein involved in this type of neuronal response and may be associating with intermediate filaments following damage, to allow remodelling and regrowth of cell processes in such swollen cells. The presence of aB crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect regeneration following damage, and its detection may therefore be a marker for such cells. This may be a useful tool in evaluating experimental modes of cerebral ischaemia and in assessing the effects of putative cytoprotective agents. The lack of ubiquitin immunoreactivity in the swollen cells associated with ischaemic damage, compared to that seen in cells in neurodegenerative disease, suggests that the cell biology underlying the response of neuronal swelling may vary according to the damaging stimulus and that continued use of the descriptive term ‘ballooned neuron’ to describe the phenomenon is still appropriate. In conclusion, aB crystallin immunoreactivity appears to be a common feature in swollen neurons in diverse neurological disorders. From studies on the biology of aB crystallin it is likely that this protein is interacting with intermediate filamentsand the fact that it is one of the cell stress proteins suggests that the response may be cytoprotective and a reflection of regenerative activity. On a purely practical level use of an antiserum to aB crystallin greatly facilitates detection of such swollen cells. ‘
ACKNOWLEDGEMENTS This work was partly funded from grants from the Parkinson’s Disease Society, and the Motor Neurone Disease Association. We thank Mr W. Brackenbury for photomicrography and Dr P. Luthert, Institute of Psychiatry, London for access to cases of cortico-basal degeneration. This work was presented at the Winter meeting of the British Neuropathological Society, Southampton in January 1992. REFERENCES Arrigo A.P., Suhan J.P. & Welch W.J. (1988) Dynamic changes in the structure and intracellular locale of the mammalian low-molecular weight heat shock protein. Molecular Cell Biology 8,5059-507 1 Bhat S.P. & Nagineni N. (1989) aB subunit of lens specificprotein a crystallin is present in other ocular and non-ocular tissues. Biochemistry Biophysics Research Communications 158,3 19-325 Brun A. (1987) Frontal lobe degeneration of non-Akheimer type. I. Neuropathology. Archives of Gerontology and Geriatrics 6, 193-208 Clarke A.W., White C.L., Manz H.J., Parhad I.M., Curry B., Whitehouse P.J., Lehmann J. & Coyle J.T. (1986a) Primary degenerative dementia without Alzheimer pathology. Canadian Journal of Neurological Sciences 13, 462470
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Clarke A.W., Manz H.J., White C.L., Lehmann J., Miller D. & Coyle J.T. (1986b) Cortical degeneration with swollen chromatolytic neurons: its relationship to Pick’s disease. Journal of Neuropathology and Experimental Neurology 45,268-284 Constantinidis J., Richard J. & Tissot R. (1974) Pick‘s disease: histologicaland clinical correlates. European Neurology 11, 208-2 17 DeJong W.W., Hendriks W., Mulders J.W.M. & Bloemendal H. (1989) Evolution of the lens crystallins: the stress connection. TIBS9,366368 Dickson D., Yen S-H., Suzuke K.I., Davies P., Garcia J.H. & Hirano A. (1986) Ballooned neurons in select neurodegenerative diseases contain phosphorylated neurofilament epitopes. Acta Neuropathologica (Berlin) 71, 2 16223 Dubin R.A., Wawrousek E.F. & Piatigorsky J. (1989) Expression of the murine aB crystallin gene is not restricted to the lens. Molecular Cell Biology 9, 1083-1091 Dubin R.A., Ally A.H., Chung S. & Piatigorsky J. (1990) Human aB crystallin gene and preferential promoter function in the lens. Genomics 7,594-601 Gibb W.R.G., Luthert P.J. & Marsden C.D. (1989) Corticobasal degeneration. Brain 112,1171-1 192 Goldman J.E. & Corbin E. (1991) Rosenthal fibres contain ubiquitinated aB crystallin. American Journal of Pathology 139,933-938 Goldstein M.E., Weiss S.R., Lazzarini R.A., Schneider P.S., Lees J.F. & Schlaepfer,W.W. (1988) mRNA levels ofall three neurofilament proteins decline following nerve transection. Molecular Brain Research 3,287-292 Ingolia T.D. & Craig E.A. (1982) Four small Drosophila heat shock proteins are related to each other and to mammalian a crystallin. Proceedings of the National Academy of Sciences ( U S A ) 19,2360-2364 Iwaki T., Kume-Iwake A., Liem R.K. & Goldman J.E. (1989) aB crystallin is expressed in non-lenticular tissues and accumulates in Alexander’s disease brain. Cell 57,7 1-78 Iwaki T., Kume-Iwake A. & Goldman J.E. (1990) Cellular distribution of aB crystallin in non-lenticular tissues. Journalof Histochemistry and Cytochemistry 38,3 1-39 Iwaki T. & Tateishi J. (1991) Immunohistochemical demonstration of alphaB crystallin in hamartomas of tuberose sclerosis. American Journal of Pathology 139,1303-1 306 Iwaki T., Iwaki A,, Miyazono M. & Goldman J.E. (1991) Preferential expression ofaB crystallin in astrocytic elements of neuroectodermal tumors. Cancer 68,2230-2240 Iwaki T., Wisniewski T., Iwaki A., Corbin E., Tomokane N., Tateishi J. & Goldman J.E. (1992) Accumulation of aB crystallin in central nervous system &a and neurons in pathologic conditions. American Journalof Pathology 140, 345-356 Kato. S., Nakamura H., Hirano A., Ito H., Llena J. & Yen S.-H. (1991) Argyrophilic ubiquitinated cytoplasmic inclusions of Leu-Fpositive glial cells in olivopontocerebellar atrophy (multiple system atrophy). Acta Neuropathologica (Berlin) 82,488493 Klemenz R., Hofmann S., Jaggi R. & Werenskiold A.K. (1989) The v-mos and c-Ha-ras oncoproteins exert similar effects on the pattern of protein synthesis. Oncogene 4,799-803 Klemenz R., Frohli E., Aoyama A., Hoffmann S., Simpson R.J., Maritz R.L. & Schafer R. (1991) aB crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH-3T3 fibroblasts. Molecular Cell Biology 11,803-8 12 Leicht B.G., Beissmann H., Palter K.B. & Bonner J.J. (1986) Small heat shock proteins of Drosophila associate with the cytoskeleton. Proceedings of the National Academy of Sciences (USA) 83,90-94 Leigh P.N., Anderton B.H., Dodson A., Gallo J.M., Swash M. &Power D.M. (1988) Ubiquitin deposits in anterior horn cells in motor neurone disease. Neuroscience Letters 93,197-203 Lippa, C.F., Cohen, R., Swith T.W. & Drachman, D.A. (1991) Primary progressiveaphasia with focal neuronal achromasia. Neurology 41,882-886 Longoni S., Lattonen S., Bullock G. & Chiesi M. (1990) Cardiac alpha crystallin. Intracellular location. Molecular Cell Biochemistry 97,121-128 Lowe J., Blanchard A., Morrell K., Lennox G., Reynolds L., Billett M., Landon M. & Mayer R.J. (1988a) Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson’s disease, Pick’s disease, and Alzheimer’s disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease. Journal of Pathology 155, 9-15 Lowe J., Lennox G., Jefferson D., Morrell K., McQuire D., Gray T., Landon M., Doherty F. & Mayer R.J. (l988b) A filamentous inclusion body within anterior horn neurons in motor neurone disease defined by immunocytochemical localisation of ubiquitin. Neuroscience Letters 94,203-2 10
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Lowe J., Landon M., Pike I., Spendlove I., McDermott H. & Mayer R.J. (1990) Dementia with P-amyloid deposition: involvement of aB crystallin supports two main diseases. Lancet 336,515-5 16 Lowe J., McDermott H., Pike I., Spendlove I., Landon M. & Mayer R.J. (1992) aB crystallin expression in non-lenticular tissues and selectivepresence in ubiquitinated inclusion bodies in human disease. Journal of Pathology 166,6148 Manetto V., Abdul-Karim F.W., Perry G., Tabaton M., Autilio-Gambetti L. & Gambetti P. (1989) Selective presence of ubiquitin in intracellular inclusions. American Journal of Pathology 134,505-5 13 Muma N., Hoffman P.N., Slunt H.A., Applegate M.D., Leiberburg I. &Price D.L. (1990) Alteration in levels of mRNAs coding for neurofilament protein subunits during regeneration. Experimental Neurology 107,230-235 Munoz-Garcia D. & Ludwin S. ( 1986) Clinicopathological studies of some non-Alzheimer dementing diseases. Canadian Journal of Neurological Sciences 13,483-489 Munoz D.G., Greene C., Per1 D.P. & Selkoe D.J. (1988) Accumulation of phosphorylated neurofilaments in anterior horn motorneurons of amyotrophic lateral sclerosis. Journal of Neuropathology and Experimental Neurology 47,9-18 Nakazato Y., Hirato J., Yshia I., Hoshi S., Hasegawa M. & Fukuda T. (1990) Swollen cortical neurons in CreutzfeldtJakob disease contain a phosphorylated neurofilament epitope. Journal of Neuropathology and Experimental Neurology 49,197-205 Renkawek K.. deJong W.W., Merck K.B., Frenken C.W.G.M,, vanworkurn F.P.A. & Bosman G.J.C.G.M. (1992) aB crystallin is present in reactive glia in Creutzfeldt-Jakob disease. Aciu Neuropathologica (Berlin) 83,324-327 Riley D.E., Lang A.E., Lewis A,, Resch L., Ashby P., Hornykiewicz 0. & Black S (1990) Cortical-basal ganglionic degeneration. Neurology 40,1203-1212 Troy C.M., Muma N.A., Greene L.A., Price D.L. & Shelanski M.L. (1990) Regulation of peripherin and neurofilament expression in regenerating rat motor neurons. Bruin Research 529,232-238 Wistow G.J. & Piatigorsky J. (1988) Lens crystallins: evolution and expression of proteins for a highly specialised tissue. Annual Review of Biochemistry 57,479-504
Received 9 March 1992 Accepted 2 April 1992
Ballooned neurons in several neurodegenerative diseases and stroke contain aB crystallin J. LOWE*, D. R. E R R I N G T O N ? , G . LENNOXS, I. PIKE?, I. S P E N D L O V E ? , M. LANDON?, A N D R. J. M A Y E R ? Departments of *Pathology, ?Biochemistry and $Neurology, University of Nottingham Medical School, Queens Medical Centre, Nottingham
LOW J., ERRINGTON D. R., L m o x G., PIKEI., SPENDLOVE I., LANDONM. & MAYERR. J. (1992) Neuropathology and Applied Neurobiology 18,34 1-350 Ballooned neurons in several neurodegenerative diseases and stroke contain aB crystallin
a B crystallin is a protein which has homology with the small cell stress proteins. A characterized antibody to residues 1-10 of a B crystallin was used to immunostain tissues containing ballooned (chromatolytic, achromasic) neurons. The tissues included two cases of classical Pick’s disease, one case of dementia with swollen achromasic neurons in the cortex, two cases of Alzheimer’s disease with large numbers of ballooned neurons, two cases of motor neuron disease, four cases of cortico-basal degeneration, and four cases with areas of brain showing swollen neurons adjacent to recent cerebral infarcts. The anti-aB crystallin showed strong diffuse cytoplasmic immunoreactivity of swollen cortical neurons in all the diseases. Astrocytes and oligodendroglial cells were also stained in normal tissues as previously described. Weak diffuse immunoreactivity with an antibody to ubiquitin-conjugates was also seen in the swollen neurons from cases of neurodegenerative disease but not following infarction. Ballooned neurons have been shown to contain phosphorylated neurofilament epitopes not normally present in the perikaryonal region. The presence of aB crystallin in ballooned neurons, together with previous data which also indicate its close association with intermediate filaments, suggest that a B crystallin may be involved in aggregation and remodelling of neurofilaments in disease. The presence of aB crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect cells which are regenerating following damage; its detection may therefore be a marker for such cells. On a practical level, the antibody greatly facilitates the localization of such abnormal neurons in diagnostic histology. Keywords: aB crystallin, heat shock protein, ubiquitin, intermediate filaments, Pick’s disease, Alzheimer’s disease, motor neuron disease, stroke, dementia, neuronal chromatolysis
INTRODUCTION The crystallins are a family of soluble proteins grouped together because of their physical properties whereby they form compact aggregated crystalline structures which are present in the eye and stable for the whole life of the individual (for review see Wistow & Piatigorsky, 1988). While they are found in high concentration in the lens of the eye, each crystallin molecule is also structurally identical or related to proteins with distinct functions outside the lens; a Correspondence: Dr J. Lowe, Department of Pathology, Queens Medical Centre, Nottingham NG7 2UH, UK.
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phenomenon described as ‘gene sharing’ (Wistow & Piatigorsky, 1988; DeJong el al., 1989). The a crystallins are composed of two subunits designated aA and aB which have structural homology with small heat shock proteins of the hsp20-30 class (Ingolia & Craig, 1982). a B crystallin is expressed in non-lenticular tissues (Baht & Nagineni, 1989; Dubin, Wawrousek & Piatigorsky, 1989; Iwaki, Kume-Iwake & Goldman, 1990) and in the nervous system, where it is normally present in astrocytes and oligodendroglial cells but is not seen in normal adult neurons (Iwaki et af.,1990).The protein is modified post-translationally by phosphorylation; however, the functional significance of this is uncertain. In pathological states, reactive glia show increased immunoreactivity with aB crystallin (Iwaki et af., 1992; Renkawek et a[., 1992). We have previously shown that a B crystallin is a common factor in intermediate filamentcontaining ubiquitinated inclusion bodies. In particular aB crystallin is present in neurons containing cortical Lewy bodies where it is seen both in the perikarya as well as in the inclusion (Lowe et af., 1990; Lowe et af.,1992) and this finding has been confirmed by others (Iwaki et af.,1992). The observation in our previous studies that the antiserum to a B crystallin stains only a proportion of ubiquitin-filament inclusions suggested that the protein might be involved in one phase of inclusion biogenesis, perhaps in initial intermediate filament aggregation. aB crystallin is present in the ubiquitinated inclusions in glial cells in multiple system atrophy (Kato et af.,1991) and it has been shown recently that aB crystallin in Rosenthal fibres is ubiquitinated (Goldman & Corbin, 1991). There appears to be an association between intermediate filaments and aB crystallin in addition to its presence in inclusion bodies; for example, it has been localized to the intermediate filaments in cardiac muscle (Longoni et af., 1990), is seen in the large vimentin-containing dysplastic cells in tuberous sclerosis (Iwaki & Tateishi, 1991), and is closely related to the presence of GFAP in glial tumours (Iwaki et af., 1991). The possibility that aB crystallin is involved in intermediate filament aggregation is supported by experimental observations that the molecule has homology with the hsp20-30 class of proteins (Ingolia & Craig 1982), which are known to aggregate with intermediate filaments (Leicht et af.,1986;Arrigo, Suhan &Welch, 1988),and also has self aggregating properties (Wistow & Piatigorsky, 1988) which might mediate adhesion. We now extend observations on the presence of aB crystallin in the nervous system by showing strong immunoreactivity to this protein in swollen neurons in several conditions. This type of abnormality of neurons, in which cells become swollen and voluminous with pale-staining cytoplasm and a large peripherally placed nucleus, has been described as chromatolysis or achromasia by some workers but the descriptive term ballooning (Dickson et af., 1986) is used here. In addition to neuronal axonal reaction following severance of the axon, the most familiar example of such change is the Pick cell of Pick’s disease (Constantinidis, Richard & Tissot, 1974) but ballooned neurons are also a prominent feature of cortico-basal degeneration (Gibb, Luthert & Marsden, 1989; Riley et af., 1990) as well as being seen in some cases of transmissible prion dementia (Clarke et al., 1986; Nakazato et af., 1990). Several workers have described cases of dementia characterized by ballooned cortical neurons in the absence of classical Pick bodies (Constantinidis et af., 1974; Munoz-Garcia & Ludwin, 1986; Clark et af. 1986a; Clarke et af., 1986b; Dickson et af., 1986) and these cases may overlap with those described in association with primary progressive aphasia (Lippa et af., 1991). A small number of cases, which satisfy diagnostic criteria for Alzheimer’s disease, have also been described as showing ballooned cortical neurons (Dickson et af.,1986; Clarke et af., 1986b). A few swollen cortical neurons have been reported in some cases of frontal lobe degeneration of non-Alzheimer type, in which the most characteristic feature is spongiform degeneration in the frontal and temporal lobes limited to the outer cortical layers (Brun, 1987).
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Previous studies have shown that such ballooned neurons contain phosphorylated neurofilaments (Dickson et al., 1986; Nakazato et al., 1990) and similar observations have been recorded in swollen anterior horn neurons in motor neuron disease (ALS) (Munoz et al., 1988; Manetto et al., 1988). In this paper we present results which suggest that the intermediate filament accumulation which is seen in such swollen neurons is accompanied by the presence of a B crystallin. MATERIALS A N D METHODS Antibody to aB crystallin
A polyclonal antiserum against aB crystallin was prepared by immunizing rabbits with a synthetic peptide-haemocyanin conjugate and has been previously characterized. It detects a single 22 kDa polypeptide, corresponding to aB crystallin, on nitrocellulose replicas after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of extracts of heart muscle (known to contain aB crystallin) and also immunodetected a similar single approximately 22 kDa polypeptide in extracts of brain from a case of diffuse Lewy body disease (Lowe et al., 1992). Cases
Cases containing ballooned cortical neurons were selected from the files of the Neuropathology section of the Department of Pathology of the Queens Medical Centre Nottingham. All tissues had been fixed in 10% formalin for at least 6 weeks prior to routine embedding in paraffin wax. The tissues were: 1 Temporal lobe from two cases of classical Pick’s disease with both ballooned Pick cells and classical Pick bodies. 2 Temporal lobe from two severely demented patients with Alzheimer’s disease characterized by widespread cortical plaques as well as numerous neocortical neurofibrillary tangles together with large numbers of ballooned neurons in the anterior temporal lobe in the absence of Pick bodies or Lewy bodies. These two cases appear to be of the same type as the two described by Dickson et al. (1986). 3 Temporal lobe from a case of severe dementia characterized by ballooned neurons in the cortex in the absence of Pick bodies, plaques or tangles. This case appears to be similar to the two documented by Clarke et a]. (1986b). 4 Cervical spinal cord from two cases of motor neuron disease (ALS) in which characteristic ubiquitinated inclusions were present in remaining spinal anterior horn cells together with ballooned neurons and axonal spheroids. 5 Cerebral cortex from four cases of cortico-basal degeneration (kindly provided by Dr P. Luthert at the Institute of Psychiatry, London). 6 Cerebral tissue adjacent to areas of recent (8-14 days) cerebral infarction in which swollen neurons had been seen on haematoxylin and eosin staining (four cases).
Immunostaining
Sections were stained using anti-aB crystallin and, in addition, for cases of Alzheimer’s disease, motor neuron disease, cerebral infarction and Pick’s disease with an antibody which detects
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ubiquitin-protein conjugates (Lowe et af., 1988a). Immunostaining was with a standard streptavidin-biotin method. Briefly, after dewaxing in xylene, sections were treated with 3% H20, in methanol, rehydrated to water, and incubated in the following: (i) normal swine serum (NSS; 20%) in 0.05 M Tris saline, pH 7.6 (TS) for 20 min. (ii) either anti-ubiquitin-protein conjugate (1:lOOO) in 120 NSS for 30 min or anti-aB crystallin (1 :4000) in 1:20 NSS for 30 min. (iii) biotinylated swine anti-rabbit immunoglobulin (Dakopatts, Denmark) (1500) in 120 NSS for 30 min. (iv) avidin/biotin/peroxidase complex (Dakopatts, Denmark) in TS for 30 min. (v) peroxidase substrate (0.05% diaminobenzidine, 0.07% imidazole, 0.01YOH,O, in TS) for 10 min. (vi) enhancer (0.5% CuSO, in TS) for 10min. (vii) sections were counterstained with haematoxylin. Negative controls were performed in each case by omission of the primary antiserum. RESULTS The antibody to ubiquitin-protein conjugates immunostained structures such as Pick bodies, neurofibrillary tangles, inclusions in motor neuron disease, dystrophic neurites, and axonal spheroids as previously described (Leigh et al., 1988; Lowe et af.,1988a,b) The antibody to aB crystallin stained astrocytes and oligodendroglial cells in normal tissues, as previously reported (Iwaki et af., 1990; Lowe et af.,1992). Staining in oligodendroglial cells diminished in intensity with prolonged formalin fixation, and was not detected in tissues that had spent more than 4 months in fixative. The antibody to aB crystallin intensely stained the ballooned cortical neurons in all cases examined and this staining was detectable despite prolonged fixation and the absence of oligodendroglial staining in some instances. Examination of the Alzheimer’s disease cases with swollen neurons showed intense positivity of swollen cells with the antibody to aB crystallin (Figure la). These swollen cells also showed weak, diffuse staining with the anti-ubiquitin antibody (Figure 1b). As noted earlier the Pick’s disease cases showed no staining of inclusions (Lowe et af.,1992), but swollen cells showed diffuse and intense immunoreactivity against aB crystallin. These cells also showed diffuse weak staining with the anti-ubiquitin antibody that was identical to that seen in the Alzheimer cases (Figure lb) and is not illustrated here. The cases of cortico-basal degeneration showed intense aB crystallin immunoreactive swollen neurons (Figure 2a) with staining seen extending down the axon in many affected cells (Figure 2b). Examination of areas of grey matter adjacent to recent cerebral infarcts showed positive immunostaining for aB crystallin of a small porportion of neurons (Figure 3). It was possible to trace a range of morphologies from pyramidal with a concave margin, through pyramidal with a swollen convex margin to swollen cells with an eccentric nucleus and dispersed Nissl substance (Figures 3a-c). In contrast to the swollen neurons in cases of neurodegenerative disease, we were not able to detect ubiquitin immunoreactivity in swollen neurons associated with infarction (Figure 3d). Axonal spheroids adjacent to areas of infarction showed no positive staining with anti-aB crystallin despite staining of adjacent neurons and glia (Figure 4).
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Figure 1. a, Swollen neuron in the cerebral cortex in a case of Alzheimer’s disease. Anti-aB crystallin/haematoxylin. x 430. b, Faint diffuse ubiquitin-immunoreactivity of swollen neuron. Note immunoreactivity of plaque-related neurites-arrowed. Anti-ubiquitin/haematoxylin. x 430.
Figure 2. a, Swollen cortical neurons in corticobasal degeneration. x 110. b, Detail showing axonal extension of aB crystallin immunoreactivity. Anti-aB crystallin/haematoxylin. x 430.
Cases of motor neuron disease showed intense immunostaining of swollen anterior horn cells for aB crystallin together with staining of normal and reactive glia (Figure 5a). Axonal spheroids and normal anterior horn cells were not immunostained (Figure 5b). Comparison with
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Figure 3. aB crystallin in neurons from adjacent to cerebral infarct. a, Normal morphology.b, Early convex border. c, Swollen cell with eccentric nucleus (anti-aB crystallin/haematoxylin). d, Swollen cell in association with infarction do not show ubiquitin immunoreactivity. Anti-ubiquitin/haematoxylin. All x 430.
Figure 4. In an area of recent infarction axonal spheroids show no aB crystallin immunoreactivity in contrast to adjacent neuronal and glial cells. Anti-aB crystallin/ haematoxylin. x 430.
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Figure 5. a, aB crystallin immunoreactivity in anterior horn cell in motor neuron disease. b, No aB crystallin immunoreactivity in axonal spheroid in motor neuron disease. Note positive glial staining. Anti-aB crystallin/ haematoxylin. Both x 430.
antiubiquitin staining showed that the majority of aB crystallin-containing cells did not contain inclusions. DISCUSSION In addition to its presence in glial cells and certain ubiquitinated inclusions, it is clear that aB crystallin is present in swollen neurons in a variety of diseases. This finding is reinforced by other recent published work in which neurons in Creutzfeldt-Jakob disease, multiple system atrophy and progressive supranuclear palsy have shown aB crystallin immunoreactivity (Iwaki et al., 1992). It is possible that the intermediate filament accumulation, which characterizes swollen neurons, is mediated through interactions with aB crystallin. This suggestion is supported by the facts that aB crystallin has self-aggregating properties (Wistow & Piatigorsky, 1988) and has homology with the hsp20-30 class of proteins (Ingolia & Craig, 1982), which are known to aggregate with intermediate filaments (Leicht et al., 1986; Arrigo et al., 1988). It remains to be determined whether aB crystallin is directly bound to intermediate.filaments. The lack of immunostaining of axonal spheroids with the antibody supports the idea that the presence of aB crystallin is not merely an epiphenomenon consequent upon the accumulation of neurofilaments, known to be abundant in spheroids. Recent reports show that aB crystallin is induced by oncogene expression and heat shock in a mouse fibroblast cell line (Klemenz et al., 1989;Klemenz et al., 1991)and the aB crystallin gene is known to have a heat shock promoter (DeJong e f al., 1989; Dubin et al., 1990). aB crystallin
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is not detectable in normal neurons by routine immunochemical techniques and, following neuronal damage, it is probably induced as part of a cell stress response when, like other stress-induced proteins, it has a role in cytoprotection of cells following damaging stimuli. The molecular pathological features of certain forms of neuronal swelling are now beginning to be understood. The swelling of spinal anterior horn cells following crush injury to the sciatic nerve is accompanied by accumulation of phosphorylated neurofilament in the perikaryon together with a reduction in messenger RNA coding for neurofilament protein (Goldstein et af., 1988; Muma et af., 1990).This suggests that such phosphorylated neurofilament accumulation is the result of altered transport or breakdown rather than overproduction. There is also accumulation of peripherin, a ‘new’ neuronal intermediate filament, in such swollen neurons accompanied by increased expression of its mRNA (Troy et af., 1990). aB crystallin now appears to be a key protein involved in this type of neuronal response and may be associating with intermediate filaments following damage, to allow remodelling and regrowth of cell processes in such swollen cells. The presence of aB crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect regeneration following damage, and its detection may therefore be a marker for such cells. This may be a useful tool in evaluating experimental modes of cerebral ischaemia and in assessing the effects of putative cytoprotective agents. The lack of ubiquitin immunoreactivity in the swollen cells associated with ischaemic damage, compared to that seen in cells in neurodegenerative disease, suggests that the cell biology underlying the response of neuronal swelling may vary according to the damaging stimulus and that continued use of the descriptive term ‘ballooned neuron’ to describe the phenomenon is still appropriate. In conclusion, aB crystallin immunoreactivity appears to be a common feature in swollen neurons in diverse neurological disorders. From studies on the biology of aB crystallin it is likely that this protein is interacting with intermediate filamentsand the fact that it is one of the cell stress proteins suggests that the response may be cytoprotective and a reflection of regenerative activity. On a purely practical level use of an antiserum to aB crystallin greatly facilitates detection of such swollen cells. ‘
ACKNOWLEDGEMENTS This work was partly funded from grants from the Parkinson’s Disease Society, and the Motor Neurone Disease Association. We thank Mr W. Brackenbury for photomicrography and Dr P. Luthert, Institute of Psychiatry, London for access to cases of cortico-basal degeneration. This work was presented at the Winter meeting of the British Neuropathological Society, Southampton in January 1992. REFERENCES Arrigo A.P., Suhan J.P. & Welch W.J. (1988) Dynamic changes in the structure and intracellular locale of the mammalian low-molecular weight heat shock protein. Molecular Cell Biology 8,5059-507 1 Bhat S.P. & Nagineni N. (1989) aB subunit of lens specificprotein a crystallin is present in other ocular and non-ocular tissues. Biochemistry Biophysics Research Communications 158,3 19-325 Brun A. (1987) Frontal lobe degeneration of non-Akheimer type. I. Neuropathology. Archives of Gerontology and Geriatrics 6, 193-208 Clarke A.W., White C.L., Manz H.J., Parhad I.M., Curry B., Whitehouse P.J., Lehmann J. & Coyle J.T. (1986a) Primary degenerative dementia without Alzheimer pathology. Canadian Journal of Neurological Sciences 13, 462470
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Received 9 March 1992 Accepted 2 April 1992
