Electron microscopic localization of cholinesterase activity in Alzheimer brain tissue

Brain Research, 540 i 1991) 204- 20~ t Iscvicr

204 BRES 16288

Electron microscopic localization of cholinesterase activity in Alzheimer brain tissue Keith A. Carson 1'2, Changiz Geula 3 and M.-Marsel Mesulam 3 ZLaboratory of Electron Microscopy, Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529-0266 (U.S.A .), 2Department of Anatomy and Neurobiology, Eastern Virginia Medical School, Norfolk, VA (U.S.A.) and 3Division of Neuroscience and Behavioral Neurology, Harvard Department of Neurology, Charles A. Dana Research Institute of Beth Israel Hospital, Boston, MA (U. S.A. (Accepted 21 August 1990)

Key words: Cholinesterase; Alzheimer's disease; Electron microscopy; Neurofibrillary tangle; Neuritic plaque

Acetylcholinesterase (ACHE) and butyrylcholinesterase (BChE) activity was localized by electron microscopic enzyme cytochemistry m cortex from Alzheimer brains and brains from non-demented cases. In the tangle-rich medial temporal cortex of the Alzheimer brain, most of the neuronal AChE was associated with neurofibrillary tangles. These structures also contained BChE activity. In normal neurons AChE activity was found in the rough endoplasmic reticulum, nuclear envelope and Golgi apparatus. Little BChE activity was noted in normal cortex. In neuritic plaques, AChE and BChE activity was associated mostly with the amyloid, but also with the neuritic component. INTRODUCTION In light microscopic studies on Alzheimer brain tissue, cholinesterase (ChE) activity has been reported in neuritic plaques (NP) 3'4'8'9"12"16 as well as in n e u r o n s with neurofibrillary tangles (NET) 4'9. However, the specific subcellular localization of ChE activity associated with NP or N F T has yet to be determined. We report here that both acetylcholinesterase (ACHE) and butyrylcholinesterase (BChE) activity is specifically associated with masses of tangled paired helical filaments in n e u r o n s with NFT. This enzyme localization is distinctly different from that observed in normal AChE-positive neurons. Both enzyme activities are also associated with various components of NP. Details of the localization of these enzymes may help to u n d e r s t a n d the mechanisms by which NP and N F T form during Alzheimer's disease (AD). MATERIALS AND METHODS Brain tissue from the temporal lobes of 13 individuals was used in this study. Eight brains were from patients with a clinical history of AD. Five brains were from age-matched individuals without AD. Thioflavin-S histofluorescence verified the presence of many NP and NFF confirming the diagnosis of AD. Tissue was processed through fixation and staining steps previously described9. Appropriate inhibitors including tetraisopropylpyrophosphoramide (BChE inhibitor) and 1,5-bis(allyl dimethylammoniumphenyl)-pentan-3-one-dibromide (ACHE inhibitor) were used to give selective, specific staining for AChE and BChE 4. Control incubations included both inhibitors. Following staining for ChE activity, tissues were rinsed

in 0.1 M cacodylate buffer, osmicated with 2% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, for 2 h in the refrigerator, then rinsed in buffer, and dehydrated through an ethanol series to acetone in 10 minute steps. Infiltration with epoxy resin was followed by fiat-embedding between Teflon-coated coverglasses in Polybed 812 epoxy resin. The wafers were polymerized for 48 h at 65 °C. Wafers were separated from the coverglasses, viewed by light microscopy to select areas rich in NP and NFT. Selected areas were cut out of the wafers using a scalpel and glued to the ends of blank blocks for sectioning with an ultramicrotome. Silver sections were picked up on 200 mesh copper grids, stained with uranyl acetate and lead citrate and viewed with a Hitachi HU-11B or JEOL 100 CX II transmission electron microscope. RESULTS Control sections incubated in the presence of specific inhibitors were examined both by light and electron microscopy. This tissue did not reveal staining i n the areas of interest and verified that the cytochemical reaction product observed by electron microscopy was due to enzyme activity rather than nonspecific cleavage of substrate. The morphological preservation of the tissue was much less than optimal due to the post-mortem delay prior to fixation, the fixative used, and the fact that the tissue was frozen prior to sectioning and cytochemical incubation. The tissue had to be frozen since unfrozen fixed tissue cut on the vibratome stained significantly less than fixed frozen sections. The addition of glutaraldehyde to the fixative improved the morphology of the tissue some-

Correspondence: K.A. Carson, Laboratory of Electron Microscopy, Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529-0266, U.S.A. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

205 what, but could not be used because it also resulted in significant inhibition of the A C h E staining in N F T and NP. These studies focused primarily on entorhinal cortex and inferior temporal gyrus. By light microscopy, the staining patterns of neurons and fibers in the normal brain tissue closely resembled those previously reported in other species 13'15. In normal human cortex ACHEpositive structures included a dense plexus of stained fibers and many stained pyramidal neurons (Fig. 1). In addition, endothelial cells and erythrocytes had enzyme activity. In A D cortex, the distribution of A C h E activity was quite different (Fig. 2). In keeping with our previous experience, there were few, if any, AChE-positive fibers in the medial temporal lobe areas that we examined. In addition, perikaryal staining of normal cells was rare. Most of the A C h E activity was observed in neurons with N F T and in NP 4"8'9'16. NFT-containing neurons could be recognized in most cases due to their abnormal morphological characteristics. The subcellular localization of A C h E reaction product as observed by electron microscopy was quite different in the normal human brain compared to that in A D brain tissue. In cortical neurons of n o n - A D brain tissue, the enzyme activity was concentrated in the nuclear envelope, the rough endoplasmic reticulum, and occasionally in the Golgi apparatus (Fig. 3). In the neuropil, A C h E reaction product was found in the extracellular space associated with axonal and dendritic membranes, especially in the vicinity of synaptic junctions. This pattern of staining is very similar to that observed in various areas of rat brain by other investigators 5"13.

Fig. 1. AChE localization in an area of medial temporal cortex from a non-demented 72-year-old male. A dense plexus of AChE-positive fibers occurs throughout the cortex while AChE-containing pyramidal neurons (arrowheads) are especially concentrated in layer 3. Bar = 100 pm.

Fig. 2. AChE localization in medial temporal cortex from a 76-year-old female diagnosed with Alzheimer's disease. ACHEpositive plaques (double arrowhead) and neurons with neurofibrillaw tangles (single arrowheads ) are common. Neurbns with neurofibrillary tangles typically exhibited distorted morphology. Bar = 100 gm. In A D cortex, the distribution of A C h E reaction product was distinctly different. The AChE-positive neurons in A D cortex had variable, but usually large

Fig. 3. In n'6urons of medial temporal cortex of a non-demented male, AChE reaction product was localized to nuclear envelope

(double arrowhead) and endoplasmic reticulum (single arrowheads). Enzyme activity was not associated with lipofuscin granules (L). Bar = 2 #m.

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Fig. 4. This AChE-positive cortical neuron in entorhinal cortex from an AD brain had a pyramidal shape and was filled with paircd helical filaments (double arrowheads) that exhibited heavy deposits of AChE reaction product (single arrowheads). Bar = 2 tim.

amounts of reaction product in the cytoplasm closely associated with the masses of tangled filaments corresponding to the paired helical filaments of N F T (Fig. 4 and 5). Virtually all of the perikaryal A C h E in the electron microscopic studies was associated with NFT. However, not all n e u r o n s with N F T contained A C h E reaction product. A C h E reaction product was absent in about 20% of the NFT-containing n e u r o n s observed by electron microscopy. Counts were done on tissue from 3 A D cases. Fifty N F T - c o n t a i n i n g neurons were counted in each case.

Ninety-six percent of the AChE-positive

neurons in A D cortex contained NFT. The reaction product was associated with the N F T in every instance. Detailed examination of the paired helical filaments in AChE-positive neurons revealed that the A C h E reaction product was closely associated with the filaments (Fig. 6),

Fig. 5. In this AChE-positive neuron in AD cortex the paired helical filaments show a more restricted distribution in the perikaryal cytoplasm (single arrowheads) and have associated electron-dense particles of AChE reaction product. Bar = 2/~m.

Fig. 6. In this medial temporal area of AD cortex, AChE reaction product crystals (double arrowhead) were closely associated with paired helical filaments (single arrowhead) in this longitudinal section of a proximal dendrite. The surrounding neuropil components had very little enzyme activity. Bar = 0.5 um.

207

Fig. 7. This neuritic plaque contained scattered dark granules of AChE reaction product (double arrowhead) in the region of the core and had densely stained areas at the periphery (single arrowheads). Bar = 2/~m.

Fig. 8. This plaque is from tissue incubated in control medium with specific AChE and BChE inhibitors. The amyloid and neuritic material of the plaque has no ChE reaction product (arrowheads). Bar = 1/~m.

but due to the large size of the reaction product crystals it was not possible to determine definitively whether the reaction-product was attached to the paired helical filaments. In some instances NFT that did not appear to be intraneuronal also exhibited AChE reaction product. However, the morphological preservation was not always sufficient to determine whether or not a NF-F was intracellular or a 'ghost' tangle remaining after degeneration of the neuron that originally contained it. The localization of BChE at the subcellular level was identical to that of AChE in the A D brain tissue, and was associated with the paired helical filaments of tangles. BChE activity was very low in normal cortex, being restricted to endothelial cells, erythrocytes, and a very few widely scattered neurons and fibers. The localization of AChE and BChE in plaques was more difficult to interpret from electron micrographs. In several instances, plaques were observed to have light to moderate patchy AChE or BChE activity (Fig. 7). In some plaques, the AChE or BChE reaction product overlapped with the amyloid material (Fig. 7) at the periphery of the NP. Plaques in tissue from control incubations did not have any specific reaction product (Fig. 8).

DIscussION The results of this study confirm, at the ultrastructural level, the association of ChE activity with NFT and NP. Most of the ChE has a perikaryal and neuropil location in the normal temporal cortex whereas in A D this enzyme activity is primarily associated with NP and NFT. Furthermore, perikaryal ChE in the normal brain is located in the nuclear envelope, rough endoplasmic reticulum and Golgi apparatus, whereas it is found mostly in association with the paired helical filaments of tangle-bearing neurons in AD. The heterogeneous distribution of the ChE reaction product in the NP reflects the morphological complexity of these structures. Most of the ChE activity appeared to be associated with amyloid, especially in the periphery of the NP. However, some ChE activity was also seen in what appeared to be the neuritic part of plaques. The loss of the cholinergic innervation in neocortical areas has been well documented and the disappearance of the AChE-rich, putatively cholinergic, fibers is consistent with these reports e~6'1°. The depletion of ACHErich neurons in cortex has been described previously 7. These cortical pyramidal neurons are not thought to be

208 cholinergic since they lack choline acetyltransferase. If these cortical pyramidal n e u r o n s have A C h E because they are cholinoceptive, then loss of their cholinergic input could conceivably cause a trans-synaptic depression of A C h E production. However, in the rat, interruption of the cholinergic input to cortex does not have such a trans-synaptic effect on cortical AChE-positive neurons 11. The A C h E and B C h E activity in NP and N F T could represent the r e m n a n t s of premorbid enzymes. For example, A C h E - r i c h cholinergic axons could degenerate into A C h E - c o n t a i n i n g NP while AChE-rich cortical n e u r o n s could yield A C h E - c o n t a i n i n g NFT. This could

chemical staining also increases 100-fold in A D cortex compared to normal cortex 4. These differences indicate that the ChE in A D brain could be substantially different from the ChE in the normal brain. W h e t h e r this reflects a passive conformational shift or a f u n d a m e n t a l molecular difference remains to be seen. Conceivably, the A C h E and B C h E activity in NP and N F T could represent the synthesis of new enzymes. Evidence suggests the existence of proteolytic activity in ChE 1"14. Whatever their molecular origin may be, the ChE in A D could therefore contribute to the putative imbalance of protein processing and could participate in the formation of NP and NFT.

explain the localization of A C h E in NP and NFT, but does not account for the B C h E activity in these structures. F u r t h e r m o r e , the pH for optimal A C h E activity shifts from 8.0 in the normal perikarya and axons to 6.8-7.0 i n the N F T and NP of the A D brain s . The concentration of inhibitors required to block histo-

Acknowledgements. We are grateful to Michael Adam for technical assistance. Supported in part by NRSA Award AG05455, Alzheimer Center Grant AG05134, the Alzheimer's Disease and Related Disorders Association, a Javits Neuroscience Investigator Award NS20285 and the Department of Biological Sciences, Old Dominion University.

REFERENCES 1 Balasubramanian, A.S., Have cholinesterases more than one function?, Trends Neurosci., 7 (1984) 467-468. 2 Davies, P., Neurotransmitter related enzymes in senile dementia of the Alzheimer type, Brain Research, 171 (1979) 319-327. 3 Friede, R.L., Enzyme histochemical studies of senile plaques, J. Neuropathol. Exp. Neurol., 24 (1965) 477-491. 4 Geula, C. and Mesulam, M-M., Special properties of cholinesterases in the cerebral cortex of Alzheimer's disease, Brain Research, 498 (1989) 185-189. 5 Lewis, P.R. and Shute, C.C.D., The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope, J. Cell Sci., 1 (1966) 381-390. 6 Mesulam, M-M. and Geula, C., Nucleus basalis (Ch4) and cortical cholinergic innervation iN the human brain: observations based on the distribution of acetylcholinesterase and choline acetyltransferase, J. Comp. Neurol., 275 (1988a) 216-240. 7 Mesulam, M.-M. and Geula, C., Acetylcholinesterase-rich pyramidal neurons in the human neocortex and hippocampus: absence at birth, development during the life span, and dissolution in Alzheimer's disease, Ann. Neurol., 24 (1988b) 765-773. 8 Mesulam, M-M., Geula, C. and Moran, M.A., Anatomy of cholinesterase inhibition in Alzheimer's disease: effect of physostigmine and tetrahydroaminoacridine on plaques and tangles, Ann. Neurol., 22 (1987) 683-691. 9 Mesulam, M.-M. and Moran, M.A., Cholinesterases within

10

11

12

13 14 15

16

neurofibrillary tangles related to age and Alzheimer's disease, Ann. Neurol., 22 (1987) 223-228. Mountjoy, J.H., Rossor, M.N., Iverson, L.L. and Roth, M., Correlation of cortical cholinergic and GABA deficits with quantitative neuropathological findings in senile dementia, Brain, 107 (1984) 507-518~ Mufson, E.J., Kehr, A.D., Wainer, B.H. and Mesulam, M-M., Cortical effects of neurotoxic damage to the nucleus basalis in rats: persistent loss of extrinsic cholinergic input and lack of transsynaptic effect upon the number of somatostatin-containing, cholinesterase-positive, and cholinergic cortical neurons, Brain Research, 417 (1987) 385-388. Perry, R.H., Blessed, G., Perry, E.K. and Tomlinson, B.E., Histochemical observations on cholinesterase activities in the brains of elderly normal and demented (Alzheimer-type) patients, Age Ageing, 9 (1980) 9-16. Silver, A., The Biology of CholinestercL~es, North-Holland, Amsterdam, 1974, 596 pp. Small, D.H., Ismael, Z. and Chubb, I.W., Acetylcholinesterase exhibits trypsin-like and metaUoexoperoxidase-like activity in cleaving a model peptide, Neuroscience, 21 (1987) 991-995. Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. Tago, H., McGeer, P.L. and McGeer, E.G., Acetylcholinesterase fibers and the development of senile plaques, Brain Research, 406 (1987) 363-369.