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The Journal of Neuroscience, April 1, 2003, 23(7):2557
Neuronal Cell Death Is Preceded by Cell Cycle Events at All
Stages of Alzheimer's Disease
Yan
Yang1,
Elliott J.
Mufson2, and
Karl
Herrup1
1 Alzheimer Research Laboratory, University Hospitals
of Cleveland and Department of Neurosciences, Case Western Reserve
University, School of Medicine, Cleveland, Ohio 44106, and
2 Department of Neurological Sciences and Alzheimer's
Disease Center, Rush Presbyterian Medical Center, Chicago, Illinois
60612
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ABSTRACT |
Cell cycle events play a major role in the loss of neurons in
advanced Alzheimer's disease (AD). It is currently unknown, however,
whether the same is true for the neuronal losses in early disease
stages. To explore this issue we analyzed brain autopsy material from
individuals clinically categorized with mild cognitive impairment
(MCI), many if not most of whom will progress to AD. Immunocytochemistry for three cell cycle-related proteins,
proliferating cell nuclear antigen, cyclin D, and cyclin B, was
performed on sections from hippocampus, basal nucleus of Meynert, and
entorhinal cortex. The results obtained from MCI cases were compared
with material from individuals diagnosed with AD and those without cognitive impairment. In both hippocampus and basal nucleus, there was
a significant percentage of cell cycle immunopositive neurons in the
MCI cases. These percentages were similar to those found in the AD
cases but significantly higher than non-cognitively impaired controls.
In entorhinal cortex, the density of cell cycle-positive neurons was
greater in MCI than in AD. However, we observed large variations in the
percentages of immunopositive neurons from individual to individual.
These findings lend support to the hypothesis that both the mechanism
of cell loss (a cell cycle-induced death) and the rate of cell loss (a
slow atrophy over several months) are identical at all stages of the AD
disease process. The implication of the findings for human clinical
trials is discussed.
Key words:
PCNA; cyclin D; cyclin B; mild cognitive
impairment;  -amyloid; tangles
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Introduction |
Mild cognitive impairment (MCI) is a
term used to describe a subtle age-associated decline of human mental
ability. It refers to a cognitive state in which minor problems with
memory and complex mental tasks become clinically significant although
the individual may still function well in his or her daily routine. The
exact prognosis of individuals with MCI is not certain, but several studies have shown that a high percentage of them will progress to
Alzheimer's disease (AD) within 3-5 years of diagnosis [for recent
analysis, see Bennett et al. (2002) ]. Although the idea of a strict
linear progression is still debated, many researchers view MCI as a
prodromal stage of AD (Petersen, 2000a,b; Morris et al., 2001).
In MCI, as in many other conditions that involve loss of neuronal
function, there is pathological evidence of cell loss that accompanies
the mental decline (Gomez-Isla et al., 1996; Mufson et al., 2000, 2002;
Kordower et al., 2001). This raises an important question: if MCI
represents a prodromal stage of AD and is accompanied by cell loss,
what causes this first cohort of neurons to die? A clue to the answer
comes from the persistent correlation of tau pathology and the
resulting neurofibrillary tangles (NFTs) with the extent of dementia
and cell loss (Mitchell et al., 2002; Rossler et al., 2002). One source
for tau hyperphosphorylation is believed to be the deregulation of
cell-cycle kinases, in particular Cdc2 (Vincent et al., 1996, 1997).
Indeed, in end-stage AD, a number of laboratories have documented a
correlation between nerve cell loss and the appearance of cell
cycle-related proteins (Baumann et al., 1993; Lew and Wang, 1995;
Arendt et al., 1996, 1998; Vincent et al., 1996, 1997; McShea et al.,
1997; Nagy et al., 1997; Busser et al., 1998; Hoozemans et al., 2002).
These proteins are not simply mis-expressed; rather they must work
coordinately because the genomic DNA can be shown to be nearly
completely replicated (Yang et al., 2001). We have shown that the
percentage of these "cycling" neurons ranges from an average of 4%
in fluorescent in situ hybridization preparations
(Yang et al., 2001) to 9% in immunostained material (Busser et al.,
1998). These are relatively high percentages because, if cell death
were rapid, very few dying cells would be visible at any one moment
(~0.01%). We propose that these percentages are evidence that the
rate of cell death after cell cycle reentry must be slow, requiring
months to complete (Busser et al., 1998; Yang et al., 2001).
In the current work we show with immunocytochemistry of MCI autopsy
material that the percentage of neurons engaged in a lethal cell cycle
early in the disease process is similar to that found in individuals
who die with frank AD. The implication of these findings from the
standpoint of disease mechanisms and the conduct of human clinical
trials is discussed.
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Materials and Methods |
Subjects. Human autopsy brain tissue was obtained
from the two National Institute on Aging-funded AD centers. Brain
tissue was routinely Formalin fixed, paraffin embedded, and sectioned at 10 µm. Tissue from all MCI and AD cases were obtained from the
Religious Orders Study of the Rush Alzheimer's Disease Center (Chicago, IL). All MCI and AD cases were confirmed both clinically and
pathologically as described previously (Gilmor et al., 1999; Mufson et
al., 1999, 2000, 2002; Bennett et al., 2002; Mitchell et al., 2002).
Mini-mental status examination (MMSE) scores were obtained for
all cases (Table 1); the scores ranged
from 25 to 30 (27.7 ± 1.89) for MCI and 3 to 25 (18.2 ± 7.7) for AD. The average postmortem interval was of ~5 hr (5.02 ± 2.13) for AD and 4.7 hr (4.73 ± 1.89) for MCI tissue, ranging
from 2 to 9 hr (Table 1). The six age-matched cognitively intact
controls were obtained from the Memory and Aging Center, University
Hospitals and Case Western Reserve University. Control brains were
Formalin fixed, embedded in paraffin, and sectioned at 10 µm.
Immunocytochemistry. The proliferating cell nuclear antigen
(PCNA) mouse monoclonal antibody recognizes the PCNA p36 protein (SC-56; Santa Cruz Biotechnology, Santa Cruz, CA) and was
diluted 1:100 before use. The cyclin D mouse monoclonal antibody
(SC-246; Santa Cruz Biotechnology) was raised against
human cyclin D1 (p34) and used at a 1:100 dilution. The cyclin B1 mouse
monoclonal IgG2b (Upstate Biotechnology, Lake Placid, NY)
was also raised against the human protein and applied at a dilution of
1:50. A monoclonal mouse antibody, 6E10 (Signet,
MA) was diluted 1:1000 and used to detect -amyloid deposits.
Paired helical filaments and neurofibrillary tangles were detected by a
mouse monoclonal antibody against hypophosphorylated tau, AT-8
(Autogen-Bioclear, UK). This antibody was used at a 1:400 dilution.
All sections were deparaffinized in xylene and rehydrated through
graded ethanol to water. The sections were soaked in 0.3% hydrogen
peroxide in methanol for 20 min to remove endogenous peroxidase
activity, rinsed in Tris-buffered saline (TBS), and pretreated in a
solution of 0.1 M citrate buffer heated to 90-95°C for
10 min. Sections were cooled and rinsed in TBS. Slides were incubated
in a blocking solution consisting of 0.1% blocking reagent (Boehringer Mannheim, Mannheim, Germany) and 10% goat
serum in PBS at room temperature for 1 hr. After overnight
incubation with the primary antibody (4°C), the sections were washed
three times in TBS before applying the secondary antibody, which was
diluted in blocking solution at 1:200. The secondary antibody was left on the section for 1 hr at room temperature; afterward, sections were
rinsed in TBS. Rinsed sections were then incubated in Vectastain ABC
Elite reagent for 1 hr, followed by three successive washes. The
sections were then incubated in diaminobenzidine (DAB) as a substrate
for visualization of the chromagen, according to the manufacturer's specification (Vector Peroxidase Substrate DAB kit; Vector Laboratories, Burlingame, CA). Some sections were counterstained with hematoxylin to aid in cell count studies, and all
sections were covered with Permount. Immunohistochemical control
sections (data not shown) were put through the identical staining
procedure except for the omission of the primary antibody.
Cell counts. In hippocampus, all of the pyramidal neurons in
the CA1-CA4 fields of single sections were counted by profile counting
methods (Guillery and Herrup, 1997) in material from MCI, AD, and
control. A cell was scored only if it contained a clear, visible
nucleus. The total number of immunopositive cells was also counted
within all subfields of the hippocampus. The cell cycle-immunoreactive
cells were counted only where the immunostaining was above background
staining seen in other regions of the same section. To determine the
percentage of positive cells in the hippocampus, the counts of
immunopositive cells were divided by the total pyramidal cell count.
For each case a minimum of 300 total cells was counted in a single
section for each antibody examined (Table
2). The results are presented as a
percentage of neurons rather than absolute numbers; thus no correction
factors were deemed necessary. An investigator who was blinded to the condition of the sample performed all analysis.
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Results |
Although the clinical and neurological status of the MCI cases
examined was similar and relatively uncompromised, the degree of
Alzheimer-related pathology in their brains differed significantly. The
pathologically least impacted MCI cases displayed minor accumulations of -amyloid plaque deposits and NFTs (Fig.
1A,B).
The more pathologically involved MCI cases were heavily invested with
plaques and NFTs (Fig. 1C,D). The AD cases
displayed extensive plaque and tangle pathology in the hippocampus
(data not shown). Their clinical and pathological diagnoses are
summarized in Table 1.
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Figure 1.
Histopathology of the hippocampus varies among the
different MCI cases. In the hippocampus of the mildest MCI case, the
6E11 antibody recognizes only an occasional -amyloid plaque
(A). Similarly, the mouse monoclonal AT-8
antibody that recognizes phosphorylated tau protein detects relatively
few tangle-bearing neurons (B). In other MCI
cases the extent of pathology was more significant. In some cases the
level of 6E11-positive plaques (C) and
AT-8-positive tangles (D) approached levels that
might be expected in advanced Alzheimer's disease. Scale bar, 25 µm.
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Hippocampus: cell cycle immunostaining
Our most detailed analysis was performed on the large pyramidal
neurons of the hippocampus. Sections were first stained with antibodies
to cyclin D, a Cdk4 regulatory subunit that is expressed as cells leave
the G0 phase of the cell cycle, pass the
restriction point, and proceed through the end of
G1 (Baldin et al., 1993). Immunohistochemical
processing revealed significant numbers of cyclin D-expressing neurons
in MCI and AD (Fig.
2A-C). The
number of these cells is near zero in the cognitively nonimpaired
age-matched subjects (Fig. 2C). As a marker of S-phase, we
used the PCNA antigen. The epitope recognized by the PCNA antibody
resides on a subunit of the DNA polymerase holoenzyme,
which is elevated primarily during S-phase (Prosperi, 1997).
PCNA-immunopositive cells were easily visualized in both our MCI and AD
subjects (Fig. 2D,E). Finally, as a
marker of the G2-phase of the cell cycle, we
stained additional sections with antibodies to cyclin B,
the regulatory subunit of the Cdc2 kinase. This marker also
showed comparable levels of staining in both MCI and AD (Fig.
3A,B)
but not in controls (Fig. 3C). In this study, as in previous
work, there was no evidence of any neuron proceeding into M-phase.
Condensed chromosomes were not seen in any neuronal nucleus, nor was
there any evidence for the formation of a spindle apparatus.
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Figure 2.
Immunocytochemistry reveals the expression of
various cell cycle proteins in pyramidal neurons of the hippocampus. In
both MCI (A) and AD (B),
immunostaining for cyclin D reveals that a fraction of the hippocampal
pyramidal cells express proteins that are normally found only in
actively dividing cells. In contrast, age-matched controls
(C) show little if any evidence for cyclin D
expression. A second mitotic marker, the DNA polymerase subunit PCNA,
is also found in neurons of the hippocampus in MCI cases
(D) as well as in material from subjects who died
with mild to moderate AD (E). Little
evidence for reexpression of PCNA is detected in control material
(F). Arrows indicate neurons
stained positive for the two cell cycle markers. Scale bars, 25 µm.
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Figure 3.
Hippocampal pyramidal cells stained for the cell
cycle marker cyclin B1. Cells shown are from MCI
(A), AD (B), and a
representative field from a normal, nondemented individual
(C); the latter illustrates the lack of cyclin B1
staining. Arrows point to the cyclin B1-positive neurons
in MCI and AD. Scale bar, 25 µm.
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Nucleus basalis: cell cycle immunostaining
The pyramidal cells of the hippocampus are not the only neurons to
die during the progress of AD. Adrenergic and serotonergic neurons of
the brain stem are lost (Zweig et al., 1988) as are the large
cholinergic neurons of the nucleus basalis of Meynert (Whitehouse et
al., 1982). At late stages of AD, we have shown previously that each of
these neuronal populations has significant numbers of neurons that show
evidence of having entered a cell cycle (Busser et al., 1998; Yang et
al., 2001). We wished to determine whether in MCI the cycle-induced
loss of cells was restricted to the hippocampus. Therefore, we examined
the neurons of the nucleus basalis. Figure
4 shows that despite the higher overall density of cells, the basal nucleus neurons have significant cell cycle
involvement in MCI cases (Fig. 4A); indeed, the
levels of involvement are quite similar to those found in AD (Fig.
4B). As in hippocampus, nondemented age-matched
controls show little or no evidence of cell cycle activity (Fig.
4C).
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Figure 4.
Nucleus basalis of Meynert immunostained for the
cell cycle marker PCNA. In this second region where substantial
neurodegeneration is known to occur in Alzheimer's disease, there is
significant evidence for ectopic expression of cell cycle proteins in
neurons that should be permanently postmitotic. Regions shown are from
a representative MCI case (A), an AD case
(B), and an age-matched control
(C). Arrows indicate
immunopositive cells. Scale bar, 25 µm.
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Entorhinal cortex: cell cycle immunostaining
Various evidence has suggested that the earliest neurodegenerative
changes that accompany the progression of AD can be found in the
entorhinal cortex. This is evident both in the extent of neuronal loss
and in the appearance of neurofibrillary tangles (Hyman et al., 1984,
1986; Arnold et al., 1991; Braak and Braak, 1991; Price et al., 1991;
Kordower et al., 2001; Mitchell et al., 2002). To compare the timing of
appearance of cell cycle enzymes with these structural markers, we
examined the presence of PCNA, cyclin D, and cyclin B in the entorhinal
cortex. The present findings are consistent with previous analyses of
the distribution of NFTs. The MCI cases currently examined showed early
involvement of the large neurons of entorhinal cortex, specifically
layers II, III, and V (Fig.
5A-C). Cell cycle
markers were a prominent feature of the cells in these layers in all
MCI cases examined; control tissue showed few or no immunopositive
neurons (Fig. 5F). There were also substantial
numbers of neurons that were strongly positive for cell cycle markers
in the AD material (data not shown). Significantly, the
incidence of neurons expressing such markers dropped to near zero at
the boundary between entorhinal cortex and the adjacent temporal
neocortex.
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Figure 5.
Expression of cell cycle proteins in neurons of
the entorhinal cortex in MCI cases. All of the same markers found in
the hippocampus are also observed in this region of temporal cortex.
PCNA is found in some neuronal nuclei of Layer V of the entorhinal
cortex (A). As illustrated here for neurons in
layer II, neurons were also found that expressed cyclin D
(B) and cyclin B1 (C).
Among the different MCI cases, there was variation in the levels of
cell cycle protein expression. In some cases, positive cells appeared
in clusters consisting of many neurons (D),
whereas in other cases only isolated immunopositive neurons were found
(E). As in hippocampus, little or no PCNA
staining is found in age-matched controls (F).
Scale bars, 25 µm.
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Of note is the fact that there was substantial variability in the
density of cell cycle-positive neurons from one case to the next. For
example, the density of PCNA-positive cells was found to range from
large clusters of cell cycle-positive cells (Fig. 5D) to a
few isolated cells in a region of immunonegative neurons (Fig.
5E). These differences are real and representative of each
case, because we found that the different cell cycle markers each gave
comparable results if applied to nearby sections (data not shown).
Because access to the MCI tissue is limited, it was not possible to
determine whether this variability was caused by regional variations
within a single individual or by global differences in cell cycle
involvement among the different cases. Others have seen similarly large
ranges of pathological involvement in MCI (Mitchell et al., 2002).
The similarity of these findings to those reported previously for
neurofibrillary pathology raises the question of whether the location
and density of the cell cycle changes that we observe bear a
relationship to the presence of neurofibrillary changes. We stained
sections from the same region as those used for the cell cycle studies
with the AT-8 monoclonal antibody that recognizes phosphorylated tau
protein. We performed this analysis on 10 MCI cases in which we had
observed cell cycle changes. In every case, the regions that showed the
highest levels of reactivity for cell cycle proteins (Fig.
6B,D)
were the same regions that (by AT-8 antigen) were the highest in
hyperphosphorylated tau (Fig.
6A,C). This spatial correlation
supports our contention that the cell cycle changes observed are
related to the neurodegenerative events.
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Figure 6.
Spatial association of hyperphosphorylated tau
protein and cell cycle markers in the MCI entorhinal cortex. In this
series of near-neighbor sections, the monoclonal AT-8 antibody reveals
the location of hyperphosphorylated tau protein in intraneuronal
tangles in layer V (A) and layer II
(C). In nearby sections, immunostaining for
cyclin B1 (B) and cyclin D
(D) shows that the cell cycle-positive neurons
are closely associated with these NFT-bearing neurons. Scale bar, 25 µm.
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Cell counts
The presence of cell cycle-positive neurons in MCI temporal cortex
and the correlation between the location of these cells and other
observed AD pathologies is strong evidence for the early involvement of
cell cycle events in the neuronal death that occurs during the
progression to AD. To obtain a more precise comparison between the
situations in MCI and AD, we determined the percentages of
immunopositive neurons in the hippocampal region containing cell cycle
markers. As shown in Figure 7, the
percentages of cell cycle-positive neurons were similar in AD and MCI
(Fig. 7B,D). This is in contrast
with the considerably lower percentage of immunopositive cells seen in
the nondemented controls. To achieve a higher level of detail, we
tabulated each of the CA regions separately. This exercise is likely to
contain a certain level of imprecision, because we had a single series
of sections from each case and the anatomical location of our section
sample varied from case to case. The representation of the individual
CA fields was not uniform, and thus regional variability might well
distort our numbers in any one sample. Despite these caveats, there
appear to be few differences among the percentages of immunopositive neurons in the CA fields of either AD cases or MCI. One consistent exception is a higher level of cell cycle involvement in area CA1 (Fig.
7A,C). These differences are
modest, and additional counts would be needed to verify their
significance.
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Figure 7.
Percentages of immunopositive neurons
stained with cell cycle markers in the hippocampus of MCI, AD, and
control. Cells were scored for the presence of cell cycle proteins as
described in Materials and Methods. Because the anatomical position of
the sections scored varied from case to case, all results were
expressed as "percent positive" neurons. The results for PCNA
(A, B) and cyclin D (C, D) are shown
separately. For each case an attempt was made to count percentages in
four CA fields separately (A, C). There will be
inaccuracies in these estimates as discussed in Results. The
percentages of the entire hippocampal pyramidal cell population are
also reported (B, D). In A and
C, the percentage for CA1 is shown in
black, CA2 is shown in dark gray, CA3 is
shown in light gray, and CA4 is shown in
white. Values shown are means of the percentages
calculated in each case; the error bars represent SEs.
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Discussion |
The results presented here document that the involvement of cell
cycle processes occurs early in the progression of AD. In every case
clinically categorized as MCI, a small but important fraction of
neurons in the hippocampal complex is immunopositive for several
proteins that should only be present in cells undergoing mitosis. This
finding applies to the pyramidal neurons of the hippocampus as well as
to cell bodies within the entorhinal cortex and the magnocellular
cholinergic neurons of the basal nucleus of Meynert. Counts in the
hippocampal region suggest that in both AD and MCI, the percentage of
neurons that are positive for cell cycle markers is between 5 and 10%.
These findings have considerable importance because a significant
amount of experimental evidence both in vivo (al-Ubaidi et
al., 1992; Jacks et al., 1992; Lee et al., 1992) and in
vitro (Park et al., 1997, 1998; Wu et al., 2000) has shown that
once a CNS neuron leaves the ventricular zone it can never reenter a
cell cycle. If it does so, whether by force (al-Ubaidi et al., 1992;
Feddersen et al., 1992, 1995) or by natural processes (Herrup and
Busser, 1995), it will die rather than divide.
Our analysis of the regional variation in cell cycling in hippocampus
should be considered in the context of earlier work on the relative
extent of cell loss in the various hippocampal subfields. It is
generally considered that the cell loss in the CA1 region is greater
than in the other CA fields (West et al., 1994). Although we find no
dramatic regional differences in the levels of cell cycle-positive
neurons, there is a trend for the CA1 field to have a higher percentage
of PCNA-positive neurons in both MCI and AD. The absence of a large
difference among the CA fields is noteworthy because it differs from
the generally held idea that the disease reveals a different
vulnerability among the CA neurons. One explanation of this apparent
discrepancy might lie in the fact that the neuronal cell counts
referred to in the earlier studies represent an absolute level of
cumulative neuron loss, whereas our cell counts reflect the percentage
of cells in the process of dying. Thus one factor contributing to the
unexpectedly small differences among the fields might be that the rate
of cell loss is regionally variable. For example, if the death of CA4 neurons after entrance into the cell cycle were slow while that of CA1
were fast, then one would predict much less cell death in the former
region than in the latter. Other factors affecting the comparison of
our data with earlier studies might include the possibility that not
all neurons are capable of forming NFTs, whereas others may form them
at different rates. If this is correct, cell cycle reentry might be
activated simultaneously in different parts of the brain, but its
progression and subsequent death mechanism might proceed at variable
rates. It is interesting in this regard that we found cell cycle
markers within the magnocelluar/cholinergic neurons of the nucleus
basalis, which are not affected in MCI and early AD (Gilmor et al.,
1999). Further studies of this neuronal population as well as others
resistant to AD pathology (i.e., basal ganglia, thalamus, and occipital
cortex) would lend support to the hypothesis that rate of neuronal loss
in response to cell cycle changes is regionally and pathologically
variable. At present there is no obvious way to test these ideas,
although the analysis of additional cases would be of considerable benefit.
Our finding of large numbers of cell cycle stained neurons in the
entorhinal cortex is in agreement with earlier work showing the early
involvement of this region in the expression of AD pathology. For
example, several studies have shown an early loss of neurons in this
region (Gomez-Isla et al., 1996; Kordower et al., 2001), a finding that
is consistent with the early appearance of neurofibrillary tangles
(Braak and Braak, 1991). By the time the disease progresses to severe
AD, previous studies report a 75-90% neuronal loss (Lippa et al.,
1992; Fukutani et al., 1995). The material that we have sampled,
although less extensive than these earlier studies, is in substantial
qualitative agreement.
The present findings suggest that, throughout the course of the
disease, neuronal death in AD has as its root cause an ectopic reentrance into the cell cycle. This is an important finding because it
offers a single unified mechanism of cell loss for this highly prevalent dementia and suggests that a single disease process is at
work throughout. The probability of an alternative hypothesis, that
cell cycle involvement is a peculiarity of the end stage of the
disease, is diminished by the present findings. Rather than being a
rare but stable event that collects in the brain or an agonal
process that occurs only in the final stages of disease, our findings
showing that cell cycle proteins are found within neurons of people
with MCI suggest that cell cycle-induced death is a central mechanistic
feature of the disease. A further implication of this line of reasoning
is that cell cycle antigens are perhaps one of the best markers for
dying neurons at all disease stages.
Individuals with a diagnosis of MCI have minor but significant symptoms
of the early stages of AD. Although some researchers would be hesitant
to portray MCI as the obligate first stage of AD, the fact is that a
relatively high percentage of these individuals will become
progressively impaired and will eventually deteriorate to the point
where a diagnosis of AD is appropriate (Petersen, 2000a,b; Bennett et
al., 2002). For the purposes of our study, it is assumed that the cases
obtained from individuals classified as MCI are at the very least
highly enriched for a prodromal stage of AD. However, we recognize that
many would argue that, had they lived, up to 30% of our cases might
have gone on to other neurological conditions (Petersen, 2000b). This
raises an important consideration. Because we used clinical-behavioral
criteria to group our subjects, a priori one might have expected to
find on the order of 30% of our MCI cases to be without significant
cell cycle involvement. That 10 of 10 of our MCI cases were cell cycle
positive (range, 2.2-15.4% of cells) suggests that nearly all forms
of dementia that evolve from MCI are accompanied by cell
cycle-triggered cellular dysfunction. This conclusion is implicit in
earlier studies (Mufson et al., 2000, 2002); an alternative
explanation, that all MCI goes on to AD, is contradicted by the
literature on the topic [see as only one example Meyer et al.
(2002)]. We are currently pursuing these predictions by examining
later stages of various non-Alzheimer dementias.
We find that the percentages of stained neurons are nearly equivalent
in both MCI and mild to moderate AD. As we have previously pointed out,
the relatively high percentage of positive neurons that we observe
(5-10%) is incompatible with a cell death process that is rapid and
apoptosis-like. If cell death were to take 12 hr (from cell cycle
entrance to cell body loss) as it does in vitro (S. M. Cicero, K. Herrup, unpublished observations) and in developing
mouse brain in vivo (Lee et al., 1994; Busser et al., 1998),
then ~1 of 7300 neurons (0.01%) would be in the death process at any
one moment if we assume a ~10 year disease process. We find hundreds
of times that number, suggesting that the cells are "stuck" for
many months (possibly up to 1 year) in a cycle they cannot complete.
These observations indicate that in both early and latter stages of AD,
cell loss will continue even if disease progression is arrested
completely. This has implications for the conduct of Alzheimer clinical
trials in general and drug trials in particular. An effective treatment
might stop the disease process but would do nothing to alleviate the
nuclear-cytoplasmic "imbalance" in the 5-10% of the neurons that
we have identified as cell cycle positive. These cells are still
fated to die. Thus in the period between therapy initiation and
final stabilization, neurons would continue to be lost, and, it may be
presumed, cognition would continue to decline. The prediction,
therefore, is that clinical trials that are only weeks in duration
could significantly underestimate the efficacy of a proposed new
treatment. An obvious corollary to this concept is that there is a
significant advantage to detecting and treating Alzheimer's disease at
the earliest possible stage.
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FOOTNOTES |
Received Oct. 29, 2002; revised Jan. 7, 2003; accepted Jan. 10, 2003.
This work was supported by a gift from the Blanchette Hooker
Rockefeller Fund, by grants from the National Institute on Aging (AG08012) and the National Institute of Neurological Diseases and
Stroke (NS20591) to K.H. and Y.Y., and by grants from the National
Institute of Aging (AG14449, A16668, A 09446, and AG10161) to E.J.M. We
are indebted to the altruism and support of the hundreds of nuns,
priests, and brothers from the following groups participating in the
Religious Orders Study: Archdiocesan Priests of Chicago, Dubuque, and
Milwaukee; Benedictine Monks, Lisle, IL, and Collegeville, MN;
Benedictine Sisters of Erie, Erie, PA; Benedictine Sisters of the
Sacred Heart, Lisle, IL; Capuchins, Appleton, WI; Christian Brothers,
Chicago, IL, and Memphis, TN; Diocesan priests of Gary, IN; Dominicans,
River Forest, IL; Felician Sisters, Chicago, IL; Franciscan Handmaids
of Mary, New York, NY; Franciscans, Chicago, IL; Holy Spirit Missionary
Sisters, Techny, IL; Maryknolls, Los Altos, CA, and Maryknoll, NY;
Norbertines, DePere, WI; Oblate Sisters of Providence, Baltimore, MD;
Passionists, Chicago, IL; Presentation Sisters, Dubuque, IA; Servites,
Chicago, IL; Sinsinawa Dominican Sisters, Chicago, IL, and Sinsinawa,
WI; Sisters of Charity, B.V.M., Chicago, IL, and Dubuque, IA; Sisters
of the Holy Family, New Orleans, LA; Sisters of the Holy Family of
Nazareth, Des Plaines, IL; Sisters of Mercy of the Americas, Chicago,
IL, Aurora, IL, and Erie PA; Sisters of St. Benedict, St. Cloud and St.
Joseph, MN; Sisters of St. Casimir, Chicago, IL; Sisters of St. Francis
of Mary Immaculate, Joliet, IL; Sisters of St. Joseph of LaGrange,
LaGrange Park, IL; Society of Divine Word, Techny, IL; Trappists,
Gethsemane, KY, and Peosta, IA; and Wheaton Franciscan Sisters,
Wheaton, IL. We also are indebted to the dedication and hard work of
Julie Bach, Religious Orders Study Coordinator, Beth Howard, Wayne
Longman, and Sabeena Shafaq of the Rush Brain Bank, and Greg
Klein, WenQing Fan, and Joanne Wu for data retrieval. We thank
Drs. D. Bennett and E. Cochran, heads of the Religious Orders Study
Clinical and Neuropathology Cores, respectively.
Correspondence should be addressed to Dr. Yan Yang, Alzheimer Research
Laboratory, University Hospitals of Cleveland and Department of
Neurosciences, Case Western Reserve University, School of Medicine (E504), 10900 Euclid Avenue, Cleveland, OH 44106. E-mail:
yxy33{at}po.cwru.edu.
| |
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TOP
ABSTRACT
Introduction
