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Cholinesterases within neurofibrillary tangles related to age and Alzheimer's disease.

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Lhohnesterases withm
Neurofibdary Tangles Related to Age
and Alzheimer’s Disease
M-Marsel Mesulam, M D , and M. Asuncion Moriin, PhD”
Enzyme histochemistry showed that a substantial number of cortical neurofibrillary tangles contain acetylcholinesterase and butyrylcholinesterase activities in patients with Alzheimer’s disease and in nondemented aged individuals. In
some areas of the brain, tangles containing each of these two enzymes were differentially distributed. In other areas,
there was a very close overlap between the two types of cholinesterase.
Mesulam M-M, Moritn MA: Cholinesterases within neurofibrillary tangles related to age
and Alzheimer’s disease. Ann Neurol 22:223-228, 1987
Large numbers of neuritic plaques and neurofibrillary
tangles are widely distributed among cortical, limbic,
and subcortical structures in the brains of patients with
Alzheimer’s disease (AD). Neuritic plaques contain
degenerated axons and dendrites, while neurofibrillary
tangles seem to represent a pathological transformation of perikaryal cytoskeleton. Plaques and tangles are
also present in brains of nondemented aged individuals
and in those with some degenerative diseases other
than AD. When compared to the brains of patients
with AD, however, the plaques and tangles in nondemented aged persons are fewer and have a more limited distribution.
Prior research has indicated that neuritic plaques are
cholinesterase positive f3, 151. We report here that a
substantial number of neurofibrillary tangles also show
cholinesterase activity. This information may be useful
in understanding the composition of the tangles and
perhaps help identify those neurons that are vulnerable to this degenerative process.
The temporal lobes from six brains were used. The interval
from death to autopsy varied from 5 to 18 hours. The tissue
(fresh in 4 cases, previously frozen in 2) was fixed for 24 to
36 hours in 496 paraformaldehyde-0.1 M phosphate buffer
at pH 7.4. Fixation was followed by immersion in buffered
sucrose (10 to 40%). Sectioning was done at a thickness of
4 0 p. by a freezing microtome. Matching series of sections
through the temporal lobes were stained with thionin and
From the Bullard and Denny-Brown Laboratories, Division of
Neuroscience and Behavioral Neurology, Harvard Neurology Department and the Dana Research Institute of the Beth Israel Hospital, Boston, MA 02215.
hematoxylin-eosin for cytological detail. Bielschowsky’s silver stain and the thioflavin-S histofluorescent methods were
used to identify plaques and tangles f20J
Cholinesterase staining was obtained by reacting the tissue, first in a diluted (10%) Karnovsky-Roots [9] solution
and then intensifying the reaction product with diaminobenzidine as described by Tag0 and co-workers (181. However,
the procedure was modified by using cobalt chloride for the
intensification El]. Furthermore, we also added ethopropazine (7.2 rnglliter) as a relatively specific butyrylcholinesterase inhibitor to increase the selectivity of the histochemical reaction { 161. Four matching sets of cholinesterase
sections were prepared. Set 1: To obtain selective acetylcholinesterase (AChE) staining, acetylthiocholine and ethopropazine were both added to the Karnovsky-Roots medium.
Set 2: The Karnovsky-Roots medium for this set contained
butyrylthiocholine (in molar equivalents) instead of acetylthiocholine. The absence of a reaction product in Set 2 (because of the inhibition by ethopropazine) established that
the staining in Set 1 was due to true AChE activity. Set 3:
An additional set of sections was prepared with butyrylthiocholine as the substrate but without the addition of
ethoproprazine. The structures with staining in these sections, but not in immediately adjacent sections of Set 2, can
be considered to contain butyrylcholinesterase (BChE) activity. Set 4: Sections stained for AChE (Set 1) were then
counterstained with thioflavin-S to demonstrate concurrent
AChE and thioflavin binding within plaques and tangles.
Extensive clinical histories were available on all six patients
in the study. Five brains came from persons (70-year-old
man, 78-year-old man, 83-year-old man, and 2 88-year-old
women) with a history of progressive dementia of the Alz-
Received Nov 4 , 1986, and in revised form Dec 17. Accepted for
publication Dec 17, 1986.
Address correspondence to Dr Mesdam, Neurology Depanment,
Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215.
*Present address: Departamento de Morfologia, Facultad de
Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo,
sln, 28029 Madrid, Spain.
Fig I. Acetylcboltnesterase (AChEI reaction in layer 5 of entorhinal cortex zn a 78-year-old man with Alzhetmeri disease.
Structures with the morpbologicalj2atures of tangles are strongly
AChE-positive. I x 200 befure 30% reduction.)
heimer type [ I l l . The sixth brain came from a 91-year-old
man who had been normal mentally until he died from a
sudden cerebellar hemorrhage.
The thionin, hematoxylin-eosin, Bielschowsky, and
thioflavin-S stains confirmed the diagnosis of A D in
four of the demented subjects. In one of the demented
patients (83-year-old man), plaques and tangles were
present, but their number and distribution were not
different from age-matched control values and a preliminary diagnosis of nonspecific dementia was made.
In the brain of the 91-year-old normal control subject,
widespread plaques and tangles confined to limbic
structures were seen but remained within the ageappropriate values for nondemented persons { 111. In
addition to the terminal cerebellar hemorrhage, rare
cortical microinfarcts were also seen in this brain.
The cholinesterase staining in each of the six brains
confirmed previous reports that neuritic plaques in aging and demented persons contain AChE activity 13,
151. The focus of the present study was on the
cholinesterase content of neurofibrillary tangles. All
six brains contained AChE-positive structures with the
typical morphological appearance of neurofibrillary
tangles (Fig 1). The distribution of AChE-positive
tangles closely followed the distribution of tangles
demonstrated with the Bielschowsky and thioflavin-S
stains. Tangle counts on matching tissue sections in the
entorhinal cortex (in all six specimens) and in the supratemporal neocortex (in the four A D brains) showed
that the thioflavin-S stain demonstrated about twice as
many tangles per square millimeter as the AChE his224
Annals of Neurology
Vol 22 No 2
August 1087
Fig 2 . Entorbinal cortex of a 91-year-old nondemented man. The
tissue was processed for acetylcholinesterase (AChE)activity and
then counterstained with t,bio$azin-S. Ultraviolet ep?$uorescence
was bark-illuminated. Tht,bright reaction product (single arrow) indicates the sites of thiojazin-S binding: the black reaction product (double arrow) indziztes sites of AChE activity.
i n this neuron, the neurofibrillary tangle contains both reaction
products. ( x 400.1
tochemical stain. This may indicate that approximately
50% of tangles are AChE positive. Alternatively, it is
conceivable that the AChE procedure we used is less
sensitive than the thioflavin-S stain for visualizing tangles and that the proportion of AChE-positive neurofibrillary tangles is even greater than 50%.
The combined thioflavin- AChE stain demonstrated
many tangles that were only thioflavin positive and
many others that were only AChE positive. Few tangles were positive for both markers. However, tangle
counts in sections stained with this double procedure
indicated that each marker interfered with the sensitivity of the other. For example, the combined stain
showed a 40% reduction of thioflavin-positive tangles
when compared to the thioflavin-S stained specimens,
and a 50% reduction of AChE-positive tangles when
compared to the AChE-stained sections. One iniplication of these observations is that the AChE reaction
product interferes with the subsequent binding of
thioflavin-S. Therefore, the combined stain was not
useful in determining the number of AChE-positive
tangles. However, the existence of at least some double staining proved that the AChE positivity of tangles
is a real phenomenon (Fig 2).
When butyrylthiocholine was used instead of acetylthiocholine (and in the presence of ethopropazine) virtually no staining of tangles was seen, thus establishing
the specificity of the AChE reaction in tangles. However, when ethopropazine w a eliminated (as in Set 3),
there was strong tangle and plaque staining with butyrylthiocholine as the substrate, indicating that there are
also BChE-positive tangles and plaques. Depending on
the region and cortical layer, the number of BChEpositive tangles varied from 50 to 110% of the num-
F i g 3. Anterior entorhinal cortex from a 78-year-old man with
Alzheimer's disease. Examples of plaques (double arrows) and
tangles (single arrows) are indicated by horizontal awws. The
distribution of acetylcholinesterase (AChE) tangles (A) closely
parallels that shown by thiojavin-S (Thio-S) (B). I n this part
of the brain, there are butyylcholinesterase (BChE) tangles (C)
in the deeper parts of layer 3 and below but not in the more
superficial aspects of cortex. Note the presence of BChE-positive
plaques. ( x 40 befre 16% reduction.)
ber of AChE-positive tangles. In the four AD brains,
there were approximately equal numbers of BChE
and AChE tangles in the deeper layers of entorhinal
and neocortical regions. Conversely, layer 2 and the
superficial parts of layer 3 contained many more AChE
than BChE tangles (Fig 3). A similar differential laminar distribution was also present among the entorhinal
tangles in the brain from the 9 1-year-old nondemented
subject. One exception occurred in the posterior entorhinal cortex of one A D brain in which AChE and
Mesulam and Moran: Cholinesterases in Neurofibrillary Tangles
Annals of Neurology
Vol 2 2
No 2
August 1987
BChE tangles were distributed in equal numbers in the
superficial as well as deep layers (Fig 4).
The brain of the 91-year-old nondemented subject
also contained many AChE-positive, tangle-free cell
bodies in almost all cortical areas, especially in layers 3
and 5 of neocortex. The normal perikaryal morphology of these cells was easily distinguished from the
,jpical tangle morphology. This brain also contained
AChE-positive normal axons in widespread areas of
cortex. Except for rare cells in layer 6, there were
virtually no BChE-positive normal fibers or cells in
In all six brains, the AChE staining of plaques and
tangles was more prominent at a p H of 6.8 to 7.0 than
at a p H of 8.0, whereas the converse was observed
in the staining of AChE-positive cortical fibers and
tangle-free cells.
Our observations show that a substantial number (at
least 50%) of neurofibrillary tangles in aging persons
and those with A D contain AChE or BChE, or both.
In some parts of the brain (e.g., deeper layers of temporal cortex), the distribution of BChE-positive tangles
paralleled that of AChE-positive tangles. In other
regions of the temporal lobe (e.g., superficial layers
of most cortical areas), AChE-positive tangles predominated markedly. These results suggest that there
may be laminar and perhaps regional variations in the
biochemical properties of tangles. We do not know
whether some (or all) tangles within the areas of closest
overlap (e.g., deep layers of cortex) contained both
AChE and BChE or if each histochemical reaction
identifies a separate population of tangles.
The morphological appearance of tangles obtained
with either histochemical stain was identical to that
obtained with the Bielschowsky and thioflavin-S stains.
This suggests either that some protein constituents of
tangles (e.g., the paired helical filaments) have cholinesterase activities or that such enzymes are passively
trapped within the tangle. Studies based on ultrastructural histochemistry will be needed to resolve this
The temporal relationship between tangle formation
4 Fig 4. Layer 2 of posterior entorhinal cortexfrom the same brain
as in Figure 3. Three adjacent sections have been stained in
three different ways. (A)The acetylcholinesterase (AChE) stain.
(B) The thiojavin-S (Thio-S) stain examined uith ultraviolet
epifluorescence.(C) The butytylcholinesterase (BChE) stain.
This was the only brain from an A D patient and the only site
among those examined where the BChE tangles were almost as
numerous as the AChE tangles in a superficial cortical layer. In
the other parts of this brain the superficial cortical layers contained a greater density of AChE than BChE tangles. ( X 100.1
and the emergence of AChE activity remains poorly
understood. The AChE reaction of a tangle could conceivably act as a marker, indicating that the tanglebearing neuron had been AChE positive in the premorbid period. In this case, the normal brain should
contain AChE-positive neurons at sites that in AD
usually contain AChE-positive tangles. The existence
of many neocortical AChE-positive neurons within
layers 3 and 5 in the brain of the 91-year-old nondemented subject substantiates this expectation, as these
cortical layers also contain the greatest density of tangles in AD. This line of reasoning raises the possibility
that at least 50% of the cortical tangles could be
formed within the population of normally AChEpositive neurons. It is therefore interesting to note that
the subcortical neuronal groups most vulnerable to
AD (nucleus basalis, raphe nuclei, nucleus locus8
ceruleus, the lateral and tuberoinfundibular nuclei of
the hypothalamus, the intralaminar thalamic nuclei,
and the substantia nigra) normally also have strong
perikaryal AChE activity 14, 7 , 8, 10, 12, 131. These
nuclei share the additional feature of sending widespread projections to the cerebral cortex. This feature
is also characteristic of cortical neurons in layers 3 and
5 , as these neurons participate in the formation of corticocortical interconnections. Thus, the presence of
perikaryal AChE activity and also of widespread corticopetal projections may constitute two important factors that in AD collectively increase the vulnerability
of both cortical and subcortical neurons to tangle formation.
However, the hypothesis that cholinesterase-positive tangles form within previously cholinesterase-positive neurons meets with several objections. First, the
tangle-free L2 neurons of the entorhinal cortex in the
brain of the 91-year-old nondemented subject were
not particularly AChE positive, even though these
cells are highly vulnerable to tangle formation {6,lo]
and contained strongly AChE-positive tangles in all
four AD brains we examined. Second, the AChE activity of tangles was much more prominent at p H 6.8
than at p H 8, whereas this was reversed in the case of
the AChE in normal fibers and many of the tangle-free
cortical cells. Although this difference in optimal pH
could merely reflect conformational changes imposed
by the relatively rigid structure of the tangle, another
implication is that the AChE in tangles is different
from the AChE normally present in neurons. Third,
the brain of the 91-year-old subject showed that very
few, if any, BChE-positive cells normally exist in
neocortex. Thus, the BChE activity in tangles could
not be associated with the premorbid existence of
BChE in the pertinent neurons.
It is therefore necessary to consider the alternate
possibility that the AChE and BChE reaction in tangles
(and perhaps also in plaques) represents a de novo
Mesulam and Moran: Cholinesterases in Neurofibrillary Tangles 227
pathological process, perhaps reflecting increased hydrolytic and catabolic activity in the affected neurons.
Whether this occurs in response to the tangle or
whether it contributes to its formation remains to be
Since normal brain tissue contains very little cortical
BChE, our demonstration that this enzyme exists in
plaques and tangles suggests that the activity of BChE
is likely to be increased in the brains of patients with
AD. In fact, such an increase has previously been reported by neurochemical investigations { 141. From a
practical point of view, it is conceivable that BChE
activity in cortical biopsies or perhaps in cerebrospinal
fluid can provide an index of the disease process [ 5 ] .
Neurochemical determinations have generally shown a
net decrease of cortical AChE in patients with A D {Z,
143. However, this most likely represents the loss
of AChE-positive cholinergic s o n s coming from the
nucleus basalis. Therefore, a net decrease of cortical
AChE could still be compatible with the emergence of
new AChE activity within plaques and tangles.
From a therapeutic point of view, it is conceivable
that agents which inhibit the cholinesterase activity of
plaques and tangles may have a salutary effect. Perhaps
the beneficial effect of physostigmine and T H A (tetrahydroaminoacridine) in patients with AD, reported
in a few studies [17, 193, reflects the ability of these
agents to inhibit cholinesterase activity in plaques and
tangles in addition to their more widely recognized
effects in enhancing cholinergic transmission.
Supported in part by the McKnight Foundation, Javits Neuroscience
Investigator Award (NS20285), and an Alzheimer’s Disease Research Center Grant (AGO5134).
The authors are grateful to Drs Catharine Joachim, Deborah Mash,
Elliott Mufson, Bruce Pnce, Dennis Selkoe, and Ana Sorrel for their
help in the acquisition of the specimens. We thank Leah Christie and
Terry Martin for expert secretarial and technical assistance.
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