12
,Vcurosciem'c Lc:hr,. i2 l i !991 ) i? :~,
Elsevier Scientific Publishers ltchmd I.td NSL 0738 t
Tyrosine phosphorylation systems in Alzheimer's disease pathology John G. W o o d 1 and Philip Zinsmeister 2 /Department of Anatomy and Cell Biology, Ernory University School o['Medicine and "Department of Biology, Oglethorpe University, Atlanta, GA (U.S.A.) (Received 16 April 1990; Revised version received 27 July 1990; Accepted 20 August 1990)
Ke), words': Phosphotyrosine; Alzheimer's disease pathology; Growth; Degeneration Immunohistochemical techniques have been used to assess the distribution of phosphotyrosine-containing compartments in Alzheimer's disease (AD) pathology. Elevated levels of phosphotyrosine are apparent in the somatodendritic compartment of tangle-bearing neurons, in the neuritic plaque (NP) and in dystrophic neurites coursing through the neuropil. The only neuronal staining observed in non-AD tissue is in developing neurites. This suggests that some neuronal elements involved in AD pathology may be recapitulating a developmental profile or, alternately, that elevated phosphotyrosine levels may reflect a role for tyrosine kinase/phosphatase systems in the degeneration process directly. Cells in the neuritic plaque which strongly resemble microglia also contain elevated levels of phosphotyrosine compared to non-activated ramified microglia in the same tissue section. Thus, tyrosine phosphorylation systems may be involved in the response of microglia to degeneration in AD pathology. Implications of these results are discussed.
Phosphoproteins such as the microtubule-associated protein, tau, are major components of the neurofibrillary tangle (NFT) and neuritic plaque (NP) cytoskeletal pathologies of Alzheimer's disease (AD; cf. 6, 8, 18). Tau phosphorylation may also be modified in AD [4, 6, 12, 18], and there has been considerable interest in this family of proteins because of their potential role in the pathogenesis of the NFT and NP. The issue of tau phosphorylation is likely to involve serine/threonine phosphorylation sites since there is no evidence for tyrosine phosphorylation of tau. In fact, the general issue of relative contributions of different protein kinase systems to neurodegeneration mechanisms is relatively unexplored. Because of their homology to viral transforming proteins and their association with growth factor receptors, tyrosine kinases are likely to directly or indirectly modify key regulators of cell growth. It follows then that tyrosine kinase systems could also be pivotal in mechanisms related to responses accompanying neurodegeneration or they could be involved in the degeneration process itself if defective. In order to broaden our knowledge of phosphoprotein involvement in AD pathology, we initiated studies of tyrosine kinase systems by probing AD tissue with anti-phosphotyrosine antibodies as an
Correspondence." J.G. Wood, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. 0304-3940/91/$ 03.50 ',~ 1991 Elsevier Scientific Publishers Ireland Ltd.
indicator of compartments containing tyrosine kinase substrates. Hippocampus and frontal cortex from 5 neuropathologically confirmed cases of Alzheimer's patients aged 71-83 years were fixed by immersion for 2h at room temperature in 4% paraformaldehyde containing 0.1 M lysine, 0.01 M periodate and 0.9% NaC1 [10]. The tissues were embedded in 4% agar and sectioned with a Lancer vibratome into 50/tm slices. All subsequent steps were carried out with gentle agitation of the sections in 50 mM Tris-buffered physiological saline, pH 7.3. Sections were blocked for 1 h at room temperature with 1% normal goat serum and 0,3 % Triton X-100 and then incubated with antibody in blocking buffer overnight at 4°C. Affinity-purified polyclonal antibodies to phosphotyrosine, 0.5 mg/ml, diluted 1:500, were generously provided by Jean Y.J. Wang. Control staining was obtained by adding a preneutralized solution of L-O-phosphotyrosine (Sigma) to the antibody at a final concentration of 10 mM and preincubating the antibody with hapten for 30 min at room temperature. Antibody binding was detected with a Vectastain ABC kit (Vector Laboratories), washed 4 times (15 min each) between steps, and developed with a 0.05 % diaminobenzidine/0.02 % hydrogen peroxide solution on ice for 8-10 min. The panels in Fig. 1 are low power views of AD hippocampus revealing anti-phosphotyrosine immunoreactivity in numerous neuritic plaques and several largely py-
13
Fig. 1. Low power light micrographs illustrating anti-phosphotyrosine staining (a-c,e) of vibratome sections of Alzheimer's disease tissue. At this magnification staining is apparent in neuritic plaques and in pyramidal shaped cell bodies. In e, a cluster of more rounded cells is illustrated which may represent a mixture of tangle-bearing neurons and microglia. Specificityof the immunohistochemical procedures is illustrated in panel d which shows a section stained with anti-phosphotyrosine antibody blocked with I0 mM L-O-phosphotyrosine.All panels × 112. r a m i d a l - s h a p e d cell bodies. In some sections (Fig. le) a d d i t i o n a l staining is observed in r o u n d e d or ellipsoid cells. Larger dystrophic neurites (Fig. la) are visible. N o other stained elements are a p p a r e n t in the low magnifi-
cation views. Specificity of a n t i b o d y staining is indicated by lack of reaction p r o d u c t (Fi~. l d) in sections incubated with h a p t e n - b l o c k e d antiseru:,.1. A d d i t i o n a l details of a n t i - p h o s p h o t y r o s i n e i m m u n o -
14
Fig. 2. Light mierographs illustrating details of anti-phosphotyrosine immunoreactivity. The positive pyramidal-shaped cells bear the typical morphology of tangle-bearing neurons (short arrows) and some are lightly stained (d) whereas others are intensely immunoreactive (cf. a). Much of the NP staining is to neurites which are either relatively thin (a) or thickened (c) and segments of positive dystrophic neurites are seen coursing through the neuropil (also see Fig. la, bottom middle). In the NP several profiles (long arrows) strongly resembling ameboid microglia are illustrated. Staining of ramified microglia is barely detectable at the dilution of antibody employed, and these profiles will probably not be visible after reproduction All panels x 288. r e a c t i v i t y are a p p a r e n t in h i g h e r m a g n i f i c a t i o n views (Fig. 2). A t the d i l u t i o n o f a n t i b o d y used here, ramified m i c r o g l i a [17] a r e b a r e l y detectable. T h e c o n c e n t r a t i o n
o f a n t i b o d y r e q u i r e d to d e m o n s t r a t e t h e m is s u c h t h a t the p a t h o l o g y s t a i n i n g is o v e r d e v e l o p e d a n d d e t a i l s are o b s c u r e d . T h e positive p y r a m i d a l cells e x h i b i t the typical
15
appearance of tangle-bearing neurons. In co-localization experiments the cells are also anti-tau positive (not shown) and, thus, do contain N F T (cf. ref. 18). All cells which are anti-tau positive are also anti-phosphotyrosine positive so most of the tangle-bearing neurons in AD tissue contain phosphotyrosine in the pathology. Non tangle-bearing neurons do not stain for anti-phosphotyrosine and the only other neuronal staining is in dystrophic neurites in the neuropil. Much of the label in the NP is clearly in neuritic processes (see Fig. 2a,c), and immunopositive dystrophic neurites are apparent in the neuropil (Fig, 2a~t). The central amyloid core does not stain (Fig. lb). In favorable views of the NP, intensely immunoreactive cells (Fig. l b,d) which strongly resemble descriptions of ameboid microglia [15] are intermingled with the processes. We also examined tissue sections from age-matched non-demented control humans. Except in those tissues exhibiting an occasional plaque and/or tangle, the only anti-phosphotyrosine staining observed is in ramified microglia. Tangles and plaques which are present in normal brain are anti-phosphotyrosine positive. This restriction of label in adult tissue is the same as we previously demonstrated in mature rat brain [17]. The cellular elements stained in AD pathology by an anti-phosphotyrosine antibody are different from those recognized by the same antibody in mature rat brain [16, 17] or in control human tissue. In the first place, the somata and apical dendrites of tangle-bearing neurons are intensely immunoreactive, whereas we have not observed labeled somatodendritic compartments of neurons in non-AD tissue. Similarly, the robust labeling of dystrophic neurites in the NP and neuropil represents an atypical compartmentation in adult brain of phosphotyrosine pools recognized by the particular antibody employed in these studies. It is not clear why phosphotyrosine pools are concentrated in tangle-bearing neurons and dystrophic neurites but several interesting possibilities may be discussed. The only other neuronal staining detectable in non-AD brain tissue is intense immunoreactivity in growing axons of developing rat brain which is no longer detectable after synapse formation [16]. The present results, then, may indicate that some neurons in AD are recapitulating a developmental program. While this seems paradoxical in view of the fact that AD pathology represents neurodegeneration, it is not unprecedented. For example, it has been suggested that enhanced acetylcholinesterase activity in the NP represents a sprouting response into the area of degeneration in the dentate gyrus from other regions such as the septal nuclei [2, 5, 7]. In another study, immunocytochemical techniques were used to demonstrate microtubular reorganization and
dendritic growth response in AD [9]. There is also morphological evidence for sprouting in AD, much of it apparently associated with a dendritic response [1, 3, 11, 13, 14]. Thus, it is entirely reasonable that the elevated levels of phosphotyrosine in neuritic processes of the NP and neuropil may reflect a growth response. If elevated levels of phosphotyrosine in AD neurites indicate a growth response, it is not exactly a recapitulation of the process seen in developing rat brain [16]. Based on morphology (also see ref. 9), many of the immunopositive neurites are likely to be dendritic, and elevated phosphotyrosine during development is in axonal compartments. There are several potential explanations for this apparent discrepancy. In the first place it must be noted that, while some of the neuritic processes may represent regenerating axons entering pathological areas, at least some of the dendritic processes are probably from affected neurons. Affected neurons in AD exhibit defective mechanisms for sorting cytoskeletal proteins (cf. refs. 6, 8, 9, 18, 19), and it is entirely possible that tyrosine kinase systems are inappropriately routed in these cells. If this is the case, it would suggest that dendrites, like axons, may be capable of employing tyrosine kinase systems in the growth process. Another possible explanation is that some dendrites in AD pathology may have greater growth capability than axons and they simply co-op the tyrosine kinase systems. It is also possible that we are detecting tyrosine kinase substrates not normally expressed in neurons. The antibody used detects phosphotyrosine which is undoubtably present in a variety of substrates. In this regard, it will be of interest to determine the tyrosine kinase substrates in AD brain regions compared to brain regions from age-matched non-demented controls. The present results also indicate elevated phosphotyrosine in the cell bodies of tangle-bearing neurons. It may be that these cells turn on tyrosine kinase systems in some phase of the degeneration process as part of a presumably abortive regeneration response, or it may be that tyrosine kinase systems are involved in pathogenesis of the NFT. While it is of interest that levels of phosphotyrosine in these cells vary widely based on a spectrum of faint to intense reaction product (compare Fig. 2a and d), this result would be compatible with either of the above possibilities. In the NP intensely labeled cells are observed which strongly resemble descriptions of ameboid microglia responding to neuronal injury [15]. The presence of phosphotyrosine in this compartment is not, in itself, unusual in that we have shown the antibody used specifically recognizes ramified microglia in vivo and ameboid microglia in vitro [17]. The intensity of label is unusual because levels of phosphotyrosine between the two phenotypes
16
from rat tissue are roughly equivalent. In AD pathology, phosphotyrosine levels in the presumptive ameboid microglia are clearly significantly higher than in ramified microglia which are barely detectable at the dilutions of antibody used for these studies. This would indicate that microglia in AD pathology either turn on tyrosine kinase activities or turn off tyrosine phosphatase activities. This issue is somewhat difficult to address biochemically using heterogeneous brain tissue, but it is possible to measure tyrosine kinase and phosphatase activities in homogenates of AD and control tissue for comparative analysis. Another approach to address this question would be to compare substrates recognized by the antibody in cultured microglia and in AD pathology. Regardless of the mechanism for accumulating elevated levels of phosphotyrosine in the presumptive ameboid microglia in AD, the results do suggest that phosphotyrosine may be involved in the response of microglia to AD pathology, possibly through activation of receptors on the microglial cell surface. While much attention has understandably focused on the tau family of phosphoproteins in the NFT and NP, the present results suggest a potential role for tyrosine kinase substrates in these pathological structures. The exact nature of that role and the substrates involved remain to be elucidated. Supported by National Institutes of Health Grant AG 06383 and NS 27731. We thank J. Soria and S. Chaiyachati for excellent technical assistance, and we thank Dr. Jean Y.J. Wang for generous donation of the anti-phosphotyrosine antibody. l Arendt, T., Zvegintseva, H.G. and Leontovich, T.A., Dendritic changes in the basal nucleus of Meynert and the diagonal band nucleus in Alzheimer's disease---a quantitative Golgi investigation, Neuroscience, 19 (1986) 1265-1278. 2 Cotman, C.W., Synaptic plasticity vs pathology in Alzheimer's disease: implications for transplantation. In F. Gage, A. Privat, Y. Christian (Eds.), Neuronal Grafting and Alzheimer's Disease, Springer, Berlin, 1989, pp. 54-62. 3 Ferrer, I., Aymami, A., Rovira, A. and Grau Veciana, J.M., Growth of abnormal neurites in atypical Alzheimer's disease. A study with a Golgi method, Acta Neuropathol., 59 (1983) 167 170.
4 Flament, S. and Delacourte, A., Abnormal tau species are produced during Alzheimer's disease neurodegeneration process, FEBS Lett., 247 (1989) 213-216. 5 Geddes, J.W., Anderson, K.J. and Cotman, C.W., Senile plaques as aberrant sprout stimulating structures, Exp. Neurol., 94 (1986) 767 776. 6 Grundke-Iqbal, I., lqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M. and Binder, L.I. Abnormal phosphoryLation of the microtubule-associated protein tau in Alzheimer cytoskeletat pathology, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 49134917. 7 Hyman, B.T., Kromer, L.J. and van Hoesen, G., Reinnervation of the hippocampal perforant pathway zone in Alzheimer's disease, Ann. Neurol., 21 (1987) 259 267. 8 Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 40444048. 9 McKee, A.C., Kowall, N.W. and Kosik, K.S., Microtubular reorganization and dendritic growth response in Alzheimer's disease, Ann. Neurol., 26 (1989) 652-659. 10 McLean, I.W. and Nakane, P.W., Periodate-lysine paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. 11 Paula-Barbosa, M.M., Cardosa, R.M., Guimaraes, M.L. and Cruz, C., Dendritic degeneration and regrowth in the cerebral cortex of patients with Alzheimer's disease, J. Neurol. Sci., 45 (1980) 129 134. 12 Pollock, N.J. and Wood, J.G., Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation, J. Histochem. Cytochem., 36 (1988) l 117 1121. 13 Probst, A., Basler, V., Bron, B. and Ulrich, J., Neuritic plaques in senile dementia of the Alzheimer type: a Golgi analysis in the hippocampal region, Brain Res., 268 (1983) 249-254. 14 Scheibel, A.B. and Tomiyasu, U., Dendritic sprouting in Alzheimer's presenile dementia, Exp. Neurol., 60 (1978) 1-8. 15 Streit, W.J. and Kreutzberg, G.W., Response of endogenous glial cells to motor neuron loss induced by toxic ricin, J. Comp. Neurot., 268 (1988) 248-263. 16 Tillotson, M.L. and Wood, J.G., Tyrosine phosphorylation in the postnatal rat brain: a developmental, immunohistochemical study, J. Comp. Neurol., 282 (1989) 133-141. 17 Tillotson, M.L. and Wood, J.G., Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo, Glia, 2 (1989) 412-419. 18 Wood, J.G., Mirra, S.S., Pollock, N.J. and Binder, L.I., Neurofibrillary tangles of Alzheimer's disease share antigenic determinants with the axonal microtubule-associated protein, tau, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4040-4044. 19 Wood, J.G., Zinsmeister, P., lmmunohistochemical evidence for reorganization of tau in plaques and tangles in Alzheimer's disease, Histochem. J., 21 (1989) 659-662.
,Vcurosciem'c Lc:hr,. i2 l i !991 ) i? :~,
Elsevier Scientific Publishers ltchmd I.td NSL 0738 t
Tyrosine phosphorylation systems in Alzheimer's disease pathology John G. W o o d 1 and Philip Zinsmeister 2 /Department of Anatomy and Cell Biology, Ernory University School o['Medicine and "Department of Biology, Oglethorpe University, Atlanta, GA (U.S.A.) (Received 16 April 1990; Revised version received 27 July 1990; Accepted 20 August 1990)
Ke), words': Phosphotyrosine; Alzheimer's disease pathology; Growth; Degeneration Immunohistochemical techniques have been used to assess the distribution of phosphotyrosine-containing compartments in Alzheimer's disease (AD) pathology. Elevated levels of phosphotyrosine are apparent in the somatodendritic compartment of tangle-bearing neurons, in the neuritic plaque (NP) and in dystrophic neurites coursing through the neuropil. The only neuronal staining observed in non-AD tissue is in developing neurites. This suggests that some neuronal elements involved in AD pathology may be recapitulating a developmental profile or, alternately, that elevated phosphotyrosine levels may reflect a role for tyrosine kinase/phosphatase systems in the degeneration process directly. Cells in the neuritic plaque which strongly resemble microglia also contain elevated levels of phosphotyrosine compared to non-activated ramified microglia in the same tissue section. Thus, tyrosine phosphorylation systems may be involved in the response of microglia to degeneration in AD pathology. Implications of these results are discussed.
Phosphoproteins such as the microtubule-associated protein, tau, are major components of the neurofibrillary tangle (NFT) and neuritic plaque (NP) cytoskeletal pathologies of Alzheimer's disease (AD; cf. 6, 8, 18). Tau phosphorylation may also be modified in AD [4, 6, 12, 18], and there has been considerable interest in this family of proteins because of their potential role in the pathogenesis of the NFT and NP. The issue of tau phosphorylation is likely to involve serine/threonine phosphorylation sites since there is no evidence for tyrosine phosphorylation of tau. In fact, the general issue of relative contributions of different protein kinase systems to neurodegeneration mechanisms is relatively unexplored. Because of their homology to viral transforming proteins and their association with growth factor receptors, tyrosine kinases are likely to directly or indirectly modify key regulators of cell growth. It follows then that tyrosine kinase systems could also be pivotal in mechanisms related to responses accompanying neurodegeneration or they could be involved in the degeneration process itself if defective. In order to broaden our knowledge of phosphoprotein involvement in AD pathology, we initiated studies of tyrosine kinase systems by probing AD tissue with anti-phosphotyrosine antibodies as an
Correspondence." J.G. Wood, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. 0304-3940/91/$ 03.50 ',~ 1991 Elsevier Scientific Publishers Ireland Ltd.
indicator of compartments containing tyrosine kinase substrates. Hippocampus and frontal cortex from 5 neuropathologically confirmed cases of Alzheimer's patients aged 71-83 years were fixed by immersion for 2h at room temperature in 4% paraformaldehyde containing 0.1 M lysine, 0.01 M periodate and 0.9% NaC1 [10]. The tissues were embedded in 4% agar and sectioned with a Lancer vibratome into 50/tm slices. All subsequent steps were carried out with gentle agitation of the sections in 50 mM Tris-buffered physiological saline, pH 7.3. Sections were blocked for 1 h at room temperature with 1% normal goat serum and 0,3 % Triton X-100 and then incubated with antibody in blocking buffer overnight at 4°C. Affinity-purified polyclonal antibodies to phosphotyrosine, 0.5 mg/ml, diluted 1:500, were generously provided by Jean Y.J. Wang. Control staining was obtained by adding a preneutralized solution of L-O-phosphotyrosine (Sigma) to the antibody at a final concentration of 10 mM and preincubating the antibody with hapten for 30 min at room temperature. Antibody binding was detected with a Vectastain ABC kit (Vector Laboratories), washed 4 times (15 min each) between steps, and developed with a 0.05 % diaminobenzidine/0.02 % hydrogen peroxide solution on ice for 8-10 min. The panels in Fig. 1 are low power views of AD hippocampus revealing anti-phosphotyrosine immunoreactivity in numerous neuritic plaques and several largely py-
13
Fig. 1. Low power light micrographs illustrating anti-phosphotyrosine staining (a-c,e) of vibratome sections of Alzheimer's disease tissue. At this magnification staining is apparent in neuritic plaques and in pyramidal shaped cell bodies. In e, a cluster of more rounded cells is illustrated which may represent a mixture of tangle-bearing neurons and microglia. Specificityof the immunohistochemical procedures is illustrated in panel d which shows a section stained with anti-phosphotyrosine antibody blocked with I0 mM L-O-phosphotyrosine.All panels × 112. r a m i d a l - s h a p e d cell bodies. In some sections (Fig. le) a d d i t i o n a l staining is observed in r o u n d e d or ellipsoid cells. Larger dystrophic neurites (Fig. la) are visible. N o other stained elements are a p p a r e n t in the low magnifi-
cation views. Specificity of a n t i b o d y staining is indicated by lack of reaction p r o d u c t (Fi~. l d) in sections incubated with h a p t e n - b l o c k e d antiseru:,.1. A d d i t i o n a l details of a n t i - p h o s p h o t y r o s i n e i m m u n o -
14
Fig. 2. Light mierographs illustrating details of anti-phosphotyrosine immunoreactivity. The positive pyramidal-shaped cells bear the typical morphology of tangle-bearing neurons (short arrows) and some are lightly stained (d) whereas others are intensely immunoreactive (cf. a). Much of the NP staining is to neurites which are either relatively thin (a) or thickened (c) and segments of positive dystrophic neurites are seen coursing through the neuropil (also see Fig. la, bottom middle). In the NP several profiles (long arrows) strongly resembling ameboid microglia are illustrated. Staining of ramified microglia is barely detectable at the dilution of antibody employed, and these profiles will probably not be visible after reproduction All panels x 288. r e a c t i v i t y are a p p a r e n t in h i g h e r m a g n i f i c a t i o n views (Fig. 2). A t the d i l u t i o n o f a n t i b o d y used here, ramified m i c r o g l i a [17] a r e b a r e l y detectable. T h e c o n c e n t r a t i o n
o f a n t i b o d y r e q u i r e d to d e m o n s t r a t e t h e m is s u c h t h a t the p a t h o l o g y s t a i n i n g is o v e r d e v e l o p e d a n d d e t a i l s are o b s c u r e d . T h e positive p y r a m i d a l cells e x h i b i t the typical
15
appearance of tangle-bearing neurons. In co-localization experiments the cells are also anti-tau positive (not shown) and, thus, do contain N F T (cf. ref. 18). All cells which are anti-tau positive are also anti-phosphotyrosine positive so most of the tangle-bearing neurons in AD tissue contain phosphotyrosine in the pathology. Non tangle-bearing neurons do not stain for anti-phosphotyrosine and the only other neuronal staining is in dystrophic neurites in the neuropil. Much of the label in the NP is clearly in neuritic processes (see Fig. 2a,c), and immunopositive dystrophic neurites are apparent in the neuropil (Fig, 2a~t). The central amyloid core does not stain (Fig. lb). In favorable views of the NP, intensely immunoreactive cells (Fig. l b,d) which strongly resemble descriptions of ameboid microglia [15] are intermingled with the processes. We also examined tissue sections from age-matched non-demented control humans. Except in those tissues exhibiting an occasional plaque and/or tangle, the only anti-phosphotyrosine staining observed is in ramified microglia. Tangles and plaques which are present in normal brain are anti-phosphotyrosine positive. This restriction of label in adult tissue is the same as we previously demonstrated in mature rat brain [17]. The cellular elements stained in AD pathology by an anti-phosphotyrosine antibody are different from those recognized by the same antibody in mature rat brain [16, 17] or in control human tissue. In the first place, the somata and apical dendrites of tangle-bearing neurons are intensely immunoreactive, whereas we have not observed labeled somatodendritic compartments of neurons in non-AD tissue. Similarly, the robust labeling of dystrophic neurites in the NP and neuropil represents an atypical compartmentation in adult brain of phosphotyrosine pools recognized by the particular antibody employed in these studies. It is not clear why phosphotyrosine pools are concentrated in tangle-bearing neurons and dystrophic neurites but several interesting possibilities may be discussed. The only other neuronal staining detectable in non-AD brain tissue is intense immunoreactivity in growing axons of developing rat brain which is no longer detectable after synapse formation [16]. The present results, then, may indicate that some neurons in AD are recapitulating a developmental program. While this seems paradoxical in view of the fact that AD pathology represents neurodegeneration, it is not unprecedented. For example, it has been suggested that enhanced acetylcholinesterase activity in the NP represents a sprouting response into the area of degeneration in the dentate gyrus from other regions such as the septal nuclei [2, 5, 7]. In another study, immunocytochemical techniques were used to demonstrate microtubular reorganization and
dendritic growth response in AD [9]. There is also morphological evidence for sprouting in AD, much of it apparently associated with a dendritic response [1, 3, 11, 13, 14]. Thus, it is entirely reasonable that the elevated levels of phosphotyrosine in neuritic processes of the NP and neuropil may reflect a growth response. If elevated levels of phosphotyrosine in AD neurites indicate a growth response, it is not exactly a recapitulation of the process seen in developing rat brain [16]. Based on morphology (also see ref. 9), many of the immunopositive neurites are likely to be dendritic, and elevated phosphotyrosine during development is in axonal compartments. There are several potential explanations for this apparent discrepancy. In the first place it must be noted that, while some of the neuritic processes may represent regenerating axons entering pathological areas, at least some of the dendritic processes are probably from affected neurons. Affected neurons in AD exhibit defective mechanisms for sorting cytoskeletal proteins (cf. refs. 6, 8, 9, 18, 19), and it is entirely possible that tyrosine kinase systems are inappropriately routed in these cells. If this is the case, it would suggest that dendrites, like axons, may be capable of employing tyrosine kinase systems in the growth process. Another possible explanation is that some dendrites in AD pathology may have greater growth capability than axons and they simply co-op the tyrosine kinase systems. It is also possible that we are detecting tyrosine kinase substrates not normally expressed in neurons. The antibody used detects phosphotyrosine which is undoubtably present in a variety of substrates. In this regard, it will be of interest to determine the tyrosine kinase substrates in AD brain regions compared to brain regions from age-matched non-demented controls. The present results also indicate elevated phosphotyrosine in the cell bodies of tangle-bearing neurons. It may be that these cells turn on tyrosine kinase systems in some phase of the degeneration process as part of a presumably abortive regeneration response, or it may be that tyrosine kinase systems are involved in pathogenesis of the NFT. While it is of interest that levels of phosphotyrosine in these cells vary widely based on a spectrum of faint to intense reaction product (compare Fig. 2a and d), this result would be compatible with either of the above possibilities. In the NP intensely labeled cells are observed which strongly resemble descriptions of ameboid microglia responding to neuronal injury [15]. The presence of phosphotyrosine in this compartment is not, in itself, unusual in that we have shown the antibody used specifically recognizes ramified microglia in vivo and ameboid microglia in vitro [17]. The intensity of label is unusual because levels of phosphotyrosine between the two phenotypes
16
from rat tissue are roughly equivalent. In AD pathology, phosphotyrosine levels in the presumptive ameboid microglia are clearly significantly higher than in ramified microglia which are barely detectable at the dilutions of antibody used for these studies. This would indicate that microglia in AD pathology either turn on tyrosine kinase activities or turn off tyrosine phosphatase activities. This issue is somewhat difficult to address biochemically using heterogeneous brain tissue, but it is possible to measure tyrosine kinase and phosphatase activities in homogenates of AD and control tissue for comparative analysis. Another approach to address this question would be to compare substrates recognized by the antibody in cultured microglia and in AD pathology. Regardless of the mechanism for accumulating elevated levels of phosphotyrosine in the presumptive ameboid microglia in AD, the results do suggest that phosphotyrosine may be involved in the response of microglia to AD pathology, possibly through activation of receptors on the microglial cell surface. While much attention has understandably focused on the tau family of phosphoproteins in the NFT and NP, the present results suggest a potential role for tyrosine kinase substrates in these pathological structures. The exact nature of that role and the substrates involved remain to be elucidated. Supported by National Institutes of Health Grant AG 06383 and NS 27731. We thank J. Soria and S. Chaiyachati for excellent technical assistance, and we thank Dr. Jean Y.J. Wang for generous donation of the anti-phosphotyrosine antibody. l Arendt, T., Zvegintseva, H.G. and Leontovich, T.A., Dendritic changes in the basal nucleus of Meynert and the diagonal band nucleus in Alzheimer's disease---a quantitative Golgi investigation, Neuroscience, 19 (1986) 1265-1278. 2 Cotman, C.W., Synaptic plasticity vs pathology in Alzheimer's disease: implications for transplantation. In F. Gage, A. Privat, Y. Christian (Eds.), Neuronal Grafting and Alzheimer's Disease, Springer, Berlin, 1989, pp. 54-62. 3 Ferrer, I., Aymami, A., Rovira, A. and Grau Veciana, J.M., Growth of abnormal neurites in atypical Alzheimer's disease. A study with a Golgi method, Acta Neuropathol., 59 (1983) 167 170.
4 Flament, S. and Delacourte, A., Abnormal tau species are produced during Alzheimer's disease neurodegeneration process, FEBS Lett., 247 (1989) 213-216. 5 Geddes, J.W., Anderson, K.J. and Cotman, C.W., Senile plaques as aberrant sprout stimulating structures, Exp. Neurol., 94 (1986) 767 776. 6 Grundke-Iqbal, I., lqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M. and Binder, L.I. Abnormal phosphoryLation of the microtubule-associated protein tau in Alzheimer cytoskeletat pathology, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 49134917. 7 Hyman, B.T., Kromer, L.J. and van Hoesen, G., Reinnervation of the hippocampal perforant pathway zone in Alzheimer's disease, Ann. Neurol., 21 (1987) 259 267. 8 Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 40444048. 9 McKee, A.C., Kowall, N.W. and Kosik, K.S., Microtubular reorganization and dendritic growth response in Alzheimer's disease, Ann. Neurol., 26 (1989) 652-659. 10 McLean, I.W. and Nakane, P.W., Periodate-lysine paraformaldehyde fixative. A new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. 11 Paula-Barbosa, M.M., Cardosa, R.M., Guimaraes, M.L. and Cruz, C., Dendritic degeneration and regrowth in the cerebral cortex of patients with Alzheimer's disease, J. Neurol. Sci., 45 (1980) 129 134. 12 Pollock, N.J. and Wood, J.G., Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation, J. Histochem. Cytochem., 36 (1988) l 117 1121. 13 Probst, A., Basler, V., Bron, B. and Ulrich, J., Neuritic plaques in senile dementia of the Alzheimer type: a Golgi analysis in the hippocampal region, Brain Res., 268 (1983) 249-254. 14 Scheibel, A.B. and Tomiyasu, U., Dendritic sprouting in Alzheimer's presenile dementia, Exp. Neurol., 60 (1978) 1-8. 15 Streit, W.J. and Kreutzberg, G.W., Response of endogenous glial cells to motor neuron loss induced by toxic ricin, J. Comp. Neurot., 268 (1988) 248-263. 16 Tillotson, M.L. and Wood, J.G., Tyrosine phosphorylation in the postnatal rat brain: a developmental, immunohistochemical study, J. Comp. Neurol., 282 (1989) 133-141. 17 Tillotson, M.L. and Wood, J.G., Phosphotyrosine antibodies specifically label ameboid microglia in vitro and ramified microglia in vivo, Glia, 2 (1989) 412-419. 18 Wood, J.G., Mirra, S.S., Pollock, N.J. and Binder, L.I., Neurofibrillary tangles of Alzheimer's disease share antigenic determinants with the axonal microtubule-associated protein, tau, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4040-4044. 19 Wood, J.G., Zinsmeister, P., lmmunohistochemical evidence for reorganization of tau in plaques and tangles in Alzheimer's disease, Histochem. J., 21 (1989) 659-662.
