Neuron, Vol. 14, 671-680, March, 1995, Copyright © 1995 by Cell Press
Gene Expression and Cellular Content of Cathepsin D
in Alzheimer's Disease Brain: Evidence for Early
Up-Regulation of the EndosomaI-Lysosomal System
Anne M. Cataldo,* Jody L. Barnett,*
Stephen A. Berman,* Jinhe Li,*
Shelley Quarless,* Sherry Bursztajn,*
Carol Lippa,t and Ralph A. Nixon*
Harvard Medical School
Belmont, Massachusetts 02178
tUniversity of Massachusetts Medical Center
Worcester, Massachusetts 01605
In Alzheimer's disease brains, more than 90% of pyra-
midal neurons in lamina V and 70% in lamina III dis-
played 2- to 5-fold elevated levels of cathepsin D (Cat
D) mRNA by in situ hybridization compared with neuro-
logically normal controls. Most of these cells appeared
histologically normal. The less vulnerable nonpyrami-
dal neuron population in lamina IV had relatively nor-
mal message levels. Neuronal populations expressing
more Cat D mRNA also displayed quantitatively in-
creased Cat D immunoreactive protein. Cat D mRNA
expression was only moderately increased in astro-
cytes. Degenerating neurons exhibited intense immu-
noreactivity but lowered Cat D mRNA levels. The up-
regulation of Cat D synthesis and accumulation of
hydrolase-laden lysosomes indicate an early activa-
tion of the endosomal-lysosomal system invulnerable
neuronal populations, possibly reflecting early regen-
erative or repair processes. These abnormalities also
represent a basis for attered regulation of amyloid pre-
cursor protein processing.
The lysosomal system is composed of several distinct and
dynamically interacting vesicular compartments that are
major sites for intracellular protein turnover and the limited
proteolytic processing of certain proteins (deDuve et al.,
1955). Acid hydrolases of different enzyme classes, which
are optimally active at acidic pH, mediate these processing
events. The biosynthesis of lysosomes begins with the de
novo synthesis of lysosomal hydrolases and membrane
glycoproteins in the endoplasmic reticulum, followed by
posttranslational modification and packaging in the Golgi
apparatus. In a subsequent series of signaling and sorting
processes, lysosomal enzymes bind to high affinity man-
nose-6-phosphate receptors (MPR) and exit the Golgi ap-
paratus via coated vesicles (for review, see Kornfeld and
Mellman, 1989; Geuze et al., 1988; Gruenberg and How-
ell, 1989). These Golgi-derived vesicles containing na-
scent lysosomal hydrolase are selectively transported to
a prelysosomal compartment, where the enzyme is disso-
ciated from the receptor by acidification of the compart-
ment (Brown et al., 1986; Dahms et al., 1989; Griffiths et
The classical literature (deDuve and Wattiaux, 1966)
categorizes lysosomes into three morphologically distinct
compartments containing catalytically competent hydro-
lases. These compartments include primary lysosomes car-
rying only newly synthesized hydrolase, secondary lyso-
somes composed of enzyme and internalized material
undergoing digestion (e.g., autophagic vacuoles, multive-
sicular/multilamellar bodies, and dense bodies), and ter-
tiary lysosomes (e.g., lipofuscin) composed of residual,
undigested material. More recent studies, however, reveal
greater complexity, including the existence of distinct early
and late endosomal compartments in addition to mature
lysosomes (Dahms et al., 1989; Griffiths et al., 1990). Acid
hydrolases are abundant in late endosomes and mature
lysosomes and are also present in lower amounts and in
active form within early endosomes (Rodman et al., 1990;
Diment et al., 1988; Lemansky et al., 1987; Bowser and
Murphy, 1990). In most cells, a small percentage of hy-
drolase escapes the receptor-mediated intracellular tar-
geting to lysosomes and is routed through the secretory
pathway (Hasilik, 1992). Late endosomes undergo both
fission and fusion with other endocytic corn partments and
autophagic vacuoles, providing a link between endocytic
and autophagic pathways (Gorden and Seglen, 1988;
Deng et al., 1991).
Immunocytochemical studies (Cataldo et al., 1990,
1994; Nixon et al., 1992, 1993; Bernstein et al., 1992;
Nakamura et al., 1991) of lysosomal hydrolases have dem-
onstrated abnormalities of the lysosomal system at early
and late stages of AIzheimer's disease pathogenesis. In-
creased levels of lysosomal hydrolases of different enzyme
classes and accumulation of acid hydrolase-containing
compartments in at-risk populations of normal-appearing
neurons precede the earliest known intracellular histo-
pathological features of the disease and worsen as overt
signs of degeneration appear (Nixon et al., 1992; Cataldo
et al., 1994). Following cell lysis, the liberation of cathepsin
D (Cat D) and many other lysosomal hydrolases within
secondary and tertiary lysosomes accounts for the abnor-
mal extracellular presence of these enzymes in associa-
tion with ~-amyloid deposits (Cataldo et al., 1994). The
persistence of acid hydrolase deposits in the extracellular
space is, to our knowledge, a pathological phenomenon
selectively related to AIzheimer's pathogenesis and other
conditions associated with I~-amyloidogenesis (Nixon and
Cataldo, 1991; Nixon et al., 1992; Villanova et al., 1993).
Additional changes in hydrotase expression may occur
secondarily in astrocytes in Alzheimer's disease (Diedrich
et al., 1991 ; Nakamura et al., 1991); however, histochemi-
cal, immunologic, and ultrastructural evidence indicates
that degenerating neurons (Cataldo et al., 1990, 1994;
Nixon and Cataldo, 1991; Nixon et al., 1992) and their
processes are the principal sources of extracellular hy-
drolase in plaques of Alzheimer's brain parenchyma. Most
recently, we have found that Cat D levels in ventricular cere-
brospinal fluid averaged 4-fold higher in Alzheimer's pa-
tients than in a control group of patients with Huntington's
disease and other neurodegenerative diseases (Schwagerl
and Nixon, unpublished data).
Figure 1. Increased Expression of Cat D
mRNA in At-Risk Neocortical Pyramidal Neu-
rons from Alzheimer's Brains
(A and B) In the prefrontal cortex of control
brains, pyramidal neurons in laminae III and V
(arrows) displayed modestly high levels of Cat
D mRNA within the soma and proximal den-
drites. The sense Cat D RNA probe, which
served as a negative control, yielded negligible
hybridization signal (inset in [B]).
(C and D) In Alzheimer's brains, the majority
of neurons in the same populations exhibited
increased levels of specific Cat D mRNA signal
(arrows). Signal levels were the densest in the
cell body and proximal dendrites with lower lev-
els in the basal dendrites.
(E and F) Several of the same at-risk neurons
(arrows) in AIzheimer's brain containing dense
Cat D mRNA signal expression (E) appear mor-
phologically normal in a serial adjacent Nissl-
stained section (F).
Bars, 50 p~m (A and C); 10 p.m (B and D); 20
~m (E and F).
Beyond the abnormal phosphorylation of tau protein and
formation of neurofibrillary tangles in some affected neu-
rons, little is known about the early intraceliular events
associated with Alzheimer's disease. In addition to provid-
ing information about these events, the possibility that the
endosomal-lysosomal pathway in vulnerable neurons
may be activated in Alzheimer's disease may have other
implications for the pathogenesis of the disease. A major
route of amyloid precursor protein (APP) processing and
degradation is through the endosomal-lysosomal path-
way (Estus et al., 1992; Golde et al., 1992; Siman et al.,
1993; Caporaso et al., 1992; Haass et ai., 1992; Cole et
al., 1992). Although the site of normal 13-amyloid peptide
(AI3) generation is still unresolved, endosomal (Koo and
Squazzo, 1994; Haass et al., 1993; Haass and Selkoe,
1993; Shoji et al., 1992) or late Golgi compartments (Bus-
caglio et al., 1993; Haass et al., 1993) have been proposed
on the basis of indirect evidence. Although the responsible
proteases are unknown, several cathepsins, some of
which are known to be present in each of these compart-
ments (Hasilik, 1992; Diment et al., 1988), are among the
candidates (Abraham et al., 1992; Tagawa et al., 1992;
Ishiura, 1991; Ishiura et al., 1990; Schonlein et al., 1993;
Ladror et al., 1994; Shi et al., 1994). APP is also metabo-
lized in lysosomes to smaller fragments containing the
complete AI3 domain (Estus et al., 1992; Caporaso et al.,
1992), which could be neurotoxic (Yankner et al., 1989;
Neve et ai., 1992) or be an additional potential source of
AI~, especially if lysosomal system function were disturbed
(Cataldo et al., 1990, 1994; Nixon et al., 1992; Tagawa et
al., 1992). Finally, the physiologic function and metabolism
of apolipoprotein E, a major risk factor in Alzheimer's dis-
Cat D Gene Expression in Alzheimers Brain
" L/ ,/
Figure 2. Cat D rnRNA in Nonpyramidal Neu-
Nonpyramidai neurons from lamina IV (aster-
isks) of the prefrontal cortex of age-matched
control (A) and Alzheimer's brains (B) display
similar Cat D mRNA levels (arrows in insets).
Normal message levels in these neurons con-
trast with the higher levels in nearby pyramidal
neurons in laminae III and V in Alzheimer's
brains (arrowheads in [B]). Bars, 50 ~m (A and
B); 20 ~m (insets).
ease (Corder et al., 1993; Strittmatter et al., 1993), de-
pends on the endosomal-lysosomal system (Ye et al.,
To test the hypothesis that t~e endosomal-lysosomal
system is up-regulated early in Alzheimer's pathogenesis
and to examine whether previous observations of abnor-
mal lysosome hydrolase accumulation in neurons reflect
decreased turnover or increased synthesis of lysosomal
Ilydrolases, we measured the levels of expression of Cat
D mRNA by in situ hybridization and Cat D protein levels by
immunocytochemistry. Our results show that substantial
increases in Cat D content detected immunocytochemi-
cally within at-risk populations of neurons in Alzheimer's
brains are associated with marked up-regulation of Cat D
gene expression in these same cell groups.
Expression of Cat D-Specific mRNA in Neocortical
Neurons from Normal Human Brain
Light microscopic inspection of hybridized tissue sections
from the prefrontal cortices of control brains displayed a
distribution of specific Cat D mRNA expression (Figure 1)
consistent with that of immunoreactive Cat D seen in our
previous cytochemical studies (Cataldo et al., 1994; Ca-
taldo and Nixon, 1990; Nixon et al., 1992). Cat D mRNA
was most prominently localized in pyramidal neurons
throughout all layers of the cortical mantle and varied little
among neurons of different sizes (Figure I ; also see Figure
3). Within these neurons, Cat D signal was high in neuronal
cell bodies, low in the proximal dendrites (Figure 1), and
undetectable in axons. Cat D m RNA levels were relatively
low in nonpyramidal neurons of the prefrontal cortex (see
below). Somewhat higher levels were detected in astrocytes
identified by their nuclear size, shape, and chromatin pat-
tern; however, compared with pyramidal neurons, astro-
cytes contained much lower Cat D message levels. Cat
D signal was negligible to absent in sections from the same
brain regions hybridized with the sense Cat D cRNA probe
(Figure 1). No differences were observed between the
quality and quantity of mRNA expression detected using
biotin and digoxigenin-labeled Cat D RNA probes.
Markedly Increased Cat D mRNA Levels in At-Risk
Neuronal Populations from Alzheimer's Brains
Hybridized tissue sections from AIzheimer's brains exhib-
ited prominent increases in specific Cat D mRNA signal
in nearly all of the pyramidal neurons within layers III and
V (Figure 1). The signal was exclusively intracellular and
densest within the cell body and proximal dendrites, but
also appeared in low levels within the basal dendrites.
Most neurons expressing very high levels of signal expres-
sion displayed no evidence of atrophy or chromatolysis.
In serial adjacent sections from Alzheimer's brains stained
with Nissl (Figure 1), silver, or thioflavin S stains, nonpyra-
midal neurons in cortical lamina IV had levels of Cat D
mRNA that, by light microscopic inspection, were equiva-
lent to the levels observed in age-matched control brains
(Figure 2). Consistent with other investigators (Diedrich et
al., 1991; Nakamura et al., 1991; Bernstein et al., 1989,
1992), we detected an increased expression in the levei
of Cat D mRNA within activated astrocytes by qualitative
estimation. Modest increases in the level of glial Cat D
signal were exhibited in both white and gray matter; how-
ever, Cat D mRNA levels in pyramidal neurons consider-
ably exceeded those in astrocytes, in accord with previous
immunocytochemical studies (Cataldo et al., 1990).
To estimate the magnitude of the message increase and
the proportion of the pyramidal cell population affected,
we used computer-assisted microdensitometry to mea-
sure the density of Cat D mRNA signal in 125 individual,
at-risk pyramidal neurons in neocortical laminae layer Ill
and separately in lamina V from five AIzheimer's brains
and five control brains matched with respective postmor-
tem interval and age of the individual. Greater than 94%
of the pyramidal neurons in layer V and greater than 70%
in layer III of the Alzheimer's brains displayed Cat D mRNA
message densities that were 2 SD higher than the mean
values measured in neurons of comparable size in control
brains (Figure 3A). Small, medium, and large size pyrami-
dal neurons displayed mRNA increases of similar magni-
tude. Elevations of Cat D mRNA levels ranged from 1.5-
to 5-fold and for the whole population, averaged 2.5-fold
higher than the mean value for the population of neurons
analyzed in control cases (p < .001).
e°,~°5,,°l ,,'~ '~o •
O m 0~ ~Oo o ° _e~oO o •
p, Cat D Immunoreactivity
..'.,.,:.'.. " .
• z~ ee • oo •
- ~e ,,~-~'~.-
Z~gq~ L~oZ~ • •
~-~ . . . . . . .
AREA ( #m 2)
loo 2oo 30o ,oo 300 0~'o
Figure 3. Densitometry of Cat D mRNA and
Protein in Control and Alzheimer's Brains
(A) Microdensitometric measurements of
mRNA in hybridized tissue sections showed an
average 2.5-fold increase in the magnitude of
mRNA levels in neurons of Alzheimer's (closed
circles) brains compared with the same popula-
tions of neurons from control brains (open tri-
angles). Increases in mRNA message densities
greater than 2 SD over the mean of control
neurons (dotted line) were displayed in 70O/o -
94% of at-risk pyramidal neocortical laminae III
and V of Alzheimer's brains with no significant
differences in cross sectional areas. Northern
blot analyses (inset) performed on homoge-
nates of the prefrontal cortices from Alzhei-
mer's (designated A in inset) and age-matched
control (designated C in inset) brains showed
an increase in the relative levels of Cat D m RNA
from AIzheimer's brains that coincided with the
levels of Cat D mRNA expression detected by
in situ hybridization.
(B) The majority of neocortical pyramids in lami-
nae III and V from Alzheimer's brains (closed
circles) exhibited increased levels of Cat D im-
munoreactivity per cross-sectional area corn-
pared with age-matched controls (open triangies) in sections examined by semiquantitative microdensitometric analyses. Increases in the cytochemi-
cal density of Cat D parallel the increases in Cat D mRNA levels within the same neuronal populations. Dotted line represents control mean
Northern blot analyses on brain homogenates from the
prefrontal cortices of five Alzheimer's and five control
brains confirmed the presence of increased levels of Cat
D mRNA in Alzheimer's brains compared with their age-
matched counterparts (Figure 3A). Relative Cat D levels
were elevated an average of 35% in Alzheimer's cases
(p < .05). Because of the extensive fallout of pyramidal
neurons in the Alzheimer's cases and the substantial con-
tribution of nonneuronal cells and unaffected neurons to
the baseline Cat D mRNA levels, it was not unexpected
that Northern blot analysis would underestimate the Cat
D mRNA increase exhibited by the remaining pyramidal
neurons and detected by in situ hybridization. Ribosomal
Ioadings were identical. Hybridization with probes for other
hydrolases (i.e., cathepsin B and hexcsaminidase A [data
not shown]) did not reveal cross-hybridization with the 18S
ribosomal species; however, if it did occur, because ribo-
somal loading was identical, cross hybridization would
have added an equal amount to the signals obtained from
Alzheimefs and control brains and would contribute to
an underestimation of the differences between the two
groups. The use of Northern blot analyses in this study
was not intended to supply major quantitative evidence
but to confirm that the Cat D probe hybridized well to a
band of the expected size.
Abnormally High Cat D mRNA Expression Is
Associated with Increased Cat D Content in
Pyramidal Neurons from Alzheimer's Brains
Neocortical sections serially adjacent to those used in the
in situ hybridization studies were immunolabeled with an
antiserum against human brain Cat D. As in previous stud-
ies, Cat D immunoreactivity in he•cortical pyramids from
layers III and V was increased to varying extents in lyso-
somal compartments that accumulate markedly within the
soma, dendrites, and axon hillock (Figure 4 and see Figure
1; for further details, see Cataldo et al., 1994; Nixon et
al., 1992). Semiquantitative microdensitometric analyses
of immunocytochemical reaction product indicated that
the majority of pyramidal neurons in either laminae III or V
in Alzheimer's neocortex displayed abnormally increased
levels of immunoreactivity per cross-sectional perikaryal
area compared with the same groups of neurons from
the control brains. The mean density of immunoreaction
product in neurons in the Alzheimer group averaged 1.5-
fold higher than the control value (p < .001). The mean
cross-sectional area of pyramidal neuron perikarya in AIz-
heimer's brains did not differ significantly from that in con-
trol brains (see Figure 3B; data not shown). Increases in
the intensity of Cat D immunostaining paralleled the in-
crease in Cat D mRNA signal in pyramidal neurons from
either layers III or V in Alzheimer brains (Figure 4) with one
exception. Neurons in advanced stages of degeneration
contained massive amounts of Cat D immunoreactivity
but in many instances, exhibited lower than normal Cat
D mRNA signals. Nonpyramidal neurons in neocortical
lamina IV displayed immunoreactive Cat D content similar
to their control counterparts. Although we have not as yet
analyzed Cat D mRNA levels in other neurodegenerative
disorders such as Pick's disease and Huntington's dis-
ease, we have found that neurons containing Pick's bodies
or at-risk neuronal populations in stage IV Huntington's
disease do not display elevated levels of Cat D immunore-
activity until the very end stages of cell death (Figure 4).
Cat D mRNA Expression in Relation to
Silver staining of adjacent tissue sections from the Alzhei-
Cat D Gene Expression in AIzheime~"s Brain
Figure 4. Cat D mRNA Expression Correlates with Cat D Content
Tissue sections from the prefrontal cortex of Alzheimer's brains immunolabeled with anti-Cat D antiserum (A) show an increase in Cat D-positive
lysosomal compartments within layer III pyramids (arrow). Serial adjacent tissue sections displayed a parallel increase in specific Cat D signal
expression within the same neurons and/or population of neurons (arrow in [B]). In this group of neurons, both Cat D immunoreactivity and signal
expression accumulated in the same intracellular regions. Early alterations in Cat D immunoreactivitywere not prominent in intact neurons containing
Pick's bodies (left inset) and at-risk neuronal populations from late-stage Huntington's disease (right inset) brains. Bars, 10 I~m.
mer's cases showed that neu rofibrillary tangles were pres-
ent in only 5%-18% of the neurons in layers III and V that
contained elevated levels of both Cat D mRNA expression
and immunoreactivity (data not shown). Degenerating
neurons and their processes are a major source of extra-
cellular Cat D associated within senile plaques (Cataldo
et al., 1990, 1994; Nixon et al., 1992). We were unable,
however, to detect the presence of Cat D-specific mRNA
within most dying neurons associated with senile plaques
or in Cat D-positive senile plaques identified by silver
staining (data not shown). Intact pyramidal neurons dis-
playing elevated Cat D mRNA levels were frequently in
close proximity with, but were not part of, the senile
plaques. Reactive astrocytes containing high Cat D mRNA
levels were observed at the crown of senile plaques periph-
eral to the location of most of the Cat D immunoreactivity
present in the plaque.
The EndosomaI-Lysosomal System Is
Up-Regulated in Alzheimer's Disease
Our results show that nearly all pyramidal neurons from
layers III and V of Alzheimer's prefrontal cortex exhibit
abnormally elevated Cat D mRNA levels, which average
2- to 3-fold higher than their counterparts in age-matched
normal control neocortex. By contrast, the less vulnerable
population of nonpyramidal neurons in lamina IV exhibited
relatively normal Cat D expression and immunoreactive
content. Increased hydrolase synthesis, therefore, at least
partly explains the marked accumulation of Cat D and
probably the increased content of many other acid hy-
drolases previously observed in pyramidal neurons in AIz-
heimer's brains (Cataldo et al., 1990, 1994; Nixon and
Cataldo, 1991 ; Nakamura et al., 1991). In this regard, addi-
tional Northern blot analyses for Cat B, which is present
by immunocytochemistry in neurons to a much greater
extent than astrocytes, showed increases of greater
magnitude in Alzheimer's brain compared with controls
(A. M. C. and R. A. N., unpublished data). In the same
neuron populations, hydrolase-laden vesicular compart-
ments accumulate and have been shown by double-label
confocal imaging to be primarily Cat D immunoreactive
secondary lysosomes or prelysosomal compartments,
rather than lipofuscin (A. M. C. and R. A. N., unpublished
data). This agrees with other findings that lipofuscin, a
relatively nonspecific concomitant of neurodegeneration,
is only modestly increased in Alzheimer's disease (Mann
et ai., 1984a, 1984b). Taken together, these findings are
strong evidence that the endosomal-lysosomal system is
abnormally activated in neuronal populations at risk to de-
generate in Alzheimers disease. The observation that acti-
vation is evident in an extremely high percentage of cell
populations and develops before neurofibrillary tangles
or other signs of degeneration appear implies that these
changes are an early stage of metabolic compromise in
vulnerable neuronal populations.
Early Endosomal Up-Regulation in Relation to
Changes in acid hydrolase expression reflect the function-
ing of several different compartments of the endosomal-
lysosomal system. Cat D and other hydrolases are abun-
dant not only in lysosomes but in prelysosomal compart-
ments, including early and late endosomes, where many
hydrolases may exist in both proenzyme and mature forms
(Lemansky et al., 1987; Riederer et al., 1994; Erickson
and Blobel, 1983). Although these various acidic vesicular
compartments have certain distinguishing biochemical
and morphotogic features, acid hydrolases cleave proteins
within each of these compartments (Diment et al., 1988;
Bowser and Murphy, 1990; Rodman et al., 1990; Leman-
sky et al., 1987). Acid hydrolases, like Cat D, are also
present to some extent in the secretory pathway, although
their activity is not known (Hasilik, 1992). Studies of non-
neuronal cells indicate that late endosomes and lyso-
somes are sites of convergence of the endocytic (hetero-
phagic) and autophagic pathways (Gordon et al., 1992;
Deng et al., 1991). Therefore, up-regulation of hydrolase
synthesis and lysosome biogenesis may reflect acceler-
ated endocytosis, autophagy, or both. Each of these re-
sponses has been reported in relation to states of nutri-
tional deprivation and a few neurodegenerative states (for
review, see Nixon and Cataldo, 1993); however, endoso-
mal-lysosomal system activation, manifested by second-
ary lysosome accumulation, is not an essential concomi-
tant of either apoptotic or necrotic cell death (Nixon and
Cataldo, 1993). The nature and magnitude of the lyso-
somal response in Alzheimer's disease and Down's syn-
drome are, in fact, uncommonly seen as features of neu-
ronal cell death in many neuropathologic conditions (Nixon
and Cataldo, 1993). In this regard, corresponding immuno-
cytochemical studies of other neurodegenerative dis-
eases, including Huntington's disease, multiple sclerosis,
amyotrophic lateral sclerosis, and progressive supranu-
clear palsy (Nixon et al., 1992; li et al., 1993; A. M. C.
and R. A. N., unpublished data) indicate relatively minor
lysosomal accumulation in affected neuronal populations,
even as these neurons are degenerating. Even less com-
monly described is the persistence of lysosomal compart-
ments in the extracellular space as hydrolase-laden neu-
rons degenerate (Cataldo et al., 1994). To our knowledge,
this feature has not been observed in conditions other than
those in which 13-amyloid is deposited, and it therefore may
be a useful diagnostic feature of the degenerative process
in Alzheimer's disease and related ~-amyloidoses.
Although their triggering mechanisms are not well un-
derstood, autophagy and endocytosis are responses
expected in cells chronically attempting to regenerate,
catabolize injured membranes, and synthesize new mem-
branes. Membrane dysfunction in Alzheimer's disease is
suggested by alterations in membrane fluidity (Zubenko
et al., 1987; Hajimohammadreza and Brammer, 1990),
lipids (Farooqui et al., 1988; Pettegrew, 1989), lipolytic
enzymes, and membrane and membrane-associated pro-
teins (Bosman et al., 1991), including mutations of APP
(Castano et al., 1991; Goate et al., 1991). Moreover, the
extensive axon terminal degeneration and dendritic dys-
trophy in Alzheimer's disease, which may precede mor-
phologic changes in neuronal perikarya, implies the break-
down and replacement of large areas of membrane
surface. Accelerated membrane trafficking to and from the
periphery may account for the easily detected endosomal-
lysosomal system up-regulation in perikarya in a very high
percentage of otherwise normal-appearing pyramidal neu-
rons that display prominent accumulations of lysosomal
compartments at the perikaryal level. The suspected im-
portance of the apolipoprotein E (Apo E) genotype as a
risk factor in Alzheimer's disease (Corder et al., 1993;
Strittmatter et al., 1993) could also relate to its function
in cholesterol transport and membrane synthesis (Ser-
ougne et al., 1976; Mahley, 1988), which requires endoso-
mal-lysosomal system activity (Ye et al., 1993). Apo E
synthesis in astrocytes and Apo E uptake by neurons are
markedly up-regulated in response to neuronal injury (Die-
drich et al., 1991; Muller and Minwegan, 1985; Poirer et
al., 1993), presumably to provide cholesterol for new mem-
brane synthesis and regenerative processes (Mahley,
1988). Finally, aging, a major risk factor in AIzheimer's
disease, is associated with an additional modest up-
regulation of the lysosomal system (Matus and Green,
1987; Lajtha et al., 1992; Nakamura et al., 1989), which
would superimpose on the disease-related alterations of
this system observed in our studies.
EndosomaI-Lysosoma! System Activation
and APP Processing
Given the known localization of acid hydrolases in early
and late endosomes as well as lysosomal compartments
(Bowser and Murphy, 1990; Rodman et al., 1990; Leman-
sky et al., 1987), up-regulation of the endosomal-lyso-
somal system is likely to influence the processing of APP.
Lysosomes are a major site for the intracellular turnover
of APP (Cole et al., 1992; Golde et al., 1992; Caporaso
et al., 1992; Haass et al., 1992), and regulated sorting of
APP to lysosomes may determine functional levels of APP
in cells as well as influence levels of secreted APP (Sam-
bamurti et al., 1992; Golde et al., 1992; Caporaso et al.,
1992). Soluble forms of A~ 1-40, and possibly AJ~ 1-42
are normally secreted from certain cells (Shoji et al., 1992;
Haass et al., 1992), and indirect evidence exists that A~
1-40 may be generated normal!y in a late-Golgi or endoso-
real compartment by noncysteine proteases (Buscaglio et
al., 1993; Haass et al., 1993; Koo and Squazzo, 1994).
Although the responsible proteases have not been defini-
tively identified, certain cathepsins cleave model peptide
sequences of APP at sites relevant to this process. Cat
D was found recently to generate the N terminus in AI~
from a model peptide substrate (Ladror et al., 1994), and
this process was accelerated more than 70-fold when the
two amino acid residues corresponding to the double mu-
tation in a Swedish family with early-onset familiar Alzhei-
mer's disease (Almquist et al., 1993; Lilius and Lannfelt,
1994) were substituted. Cathepsin S has also been impli-
cated indirectly in A[3 formation (Shi et al., 1994). APP
processing within post-Golgi or mature lysosomal com-
partments also represents a route to the generation of
potentially amyloidogenic fragments of APP containing
the entire A~ domain (Caporaso et al., 1992; Siman et al.,
1993; Estus et al., 1992), some of which may be neurotoxic
when expressed in high amounts (Yankner et al., 1989;
Neve et al., 1992; Hayashi et al., 1992). It should also
be considered that the trafficking and processing of APP
could change when ceils are stressed (Baskin et al., 1991)
or injured. Up-regulated hydrolase synthesis, for example,
might be expected to increase levels of certain enzymes
in vesicular compartments that normally contain negligible
or minimal levels, thereby greatly accelerating some nor-
mal or alternative processing events. Finally, in AIzhei-
mer's disease, enzymatically competent lysosomal hy-
drolases are found in abnormal extracellular locations
within both diffuse and classical amyloid-containing senile
Cat D Gene Expression in Alzheimer's Brain
plaques (Cataldo et al., 1990, 1994). Dysregulation of nor-
mal processing pathways in degenerating neurons has
been proposed as an additional route that may be respon-
sible for the generation of AI3 or amyloidogenic fragments
in these lesions (Cataldo et al., 1994; Nixon and Cataldo,
Relationship of Cat D Expression to Endstage
Degenerating neurons or their processes are a major
source of extracellular Cat D and the various other ly-
sosomal hydrolases and hydrolase activities in senile
plaques (Cataldo etal., 1990; 1994). However, the most
intensely Cat D immunoreactive degenerated neurons in
the plaques exhibit lowered Cat D mRNA levels, owing to
depletion or breakdown of RNA accompanying the degen-
erative process and to the scavenging responses of infiltra-
tive cells. Reactive gliosis consisting of increased num-
bers of hypertrophic astrocytes and the presence of
reactive astrocytes at the crown of both cored and uncored
senile plaques is a characteristic feature of Alzheimer's
neuropathology (Beach et al., ~989; Dully et al., 1982).
Our results are consistent with earlier findings (Diedrich
et al., 1991) that some astrocytes express higher levels
of Cat D mRNA in Alzheimer's brain. Increased lysosomal
hydrolase expression in these cells is observed in various
degenerative states, suggesting that lysosomal system
up-regulation may accompany the hyperplastic/hypertro-
phic response of astrocytes (Massacesi etal., 1988; Bever
et al., 1989; Bernstein et al., 1992; Nakanishi et al., 1993).
Several lines of evidence indicate, however, that astrocytes
are an additional but minor source of the extracellular hy-
drolases present in senile plaques compared with neu-
rons. In cytochemical experiments involving double-
labeling techniques with glial fibrillary acidic protein and
Cat D antisera, astrocyte cell bodies were most abundant
at the crown of amyloid-containing senile plaques but were
not present in the central portions of the plaque where
Cat D immunoreactivity was highest (Cataldo et al., 1990).
The molecular layer of the prefrontal cortex, for example,
contains numerous nodules of strongly Cat D-positive
astrocytes, which are not associated with extracellular hy-
drolase deposits (Cataldo et al., ~ 990). Moreover, cathep-
sins H and G, proteases abundant in gila but not neurons,
were not detectable extracellularty in senile plaques. Con-
versely, acid hydrolases that were abundant in neurons
but absent or at much lower levels in glia (e.g., Hex A and
cathepsin B) were well represented in plaques extracel-
Postmortem brain tissue from the prefrontal cortex (Brodmann area
10) of ten individuals with the probable clinical diagnosis of AIzheimer's
disease and nine age-matched (62-78 years) neurologically normal
controls were used in this study. Tissue was procured from the Brain
Tissue Resource Centers at McLean Hospital (Belmont, MA) and the
University of Massachusetts Medical School (Worcester, MA) and the
Neuropathology Core Facility of the Massachusetts Alzheimer's Dis-
ease Research Center (Massachusetts General Hospital, Boston, MA).
Control brains ranged from 1200 to 1350 g in weight and exhibited
no gross anatomical pathology and only minimal histopathological
changes (0-3 neuritic plaques per low power field; 0-6 neurofibrillary
tangles per low power field).
The presence and magnitude of neurodegeneration and neurofibril-
lary histopathology were confirmed on adjacent, serial paraffin sec-
tions from all brains analyzed using Nissl and Bielschowsky silver
stains, and the diagnosis of Alzheimer's disease was established by
the Consortium to Establish a Registry of Alzheimer's Disease criteria
(Mirra et al., 1991). The presence of the 13-amyloid protein was detected
histologically using thioflavin S staining. Postmortem interval for all
brain tissue employed was 0-6 hr, with a total fixation time of 2 weeks
Immunocytochemical studies were performed on paraffin or vibratome
sections using an antiserum directed against human brain CAT D.
This antibody was raised in sheep, and its immunospecificity was con-
firmed as described in previous studies (Nixon and Marotta, 1984;
Cataldo et al., 1990).
Hydrolase immunoreactivity was demonstrated using a modification
of the avidin-biotin technique of Hsu et al. (1981) and Vectastain kits
(Vector Laboratories, Inc., Burlingame, CA), with diaminebenzidine as
the chromagen. Negative controls consisted of tissue sections incu-
bated in preimmune antisera or in the absence of primary antiserum.
Source and Labeling of Probes
cDNA for human Cat D was generously provided by Dr. P. Faust (1985).
Isolation of the plasmid DNA was performed by the general method
outlined by Maniatis et al. (1989). The Cat D clone, CDpSP64, which
contained a 2.2 kb cDNA, was inserted into a pSP64 vector and ori-
ented so that the SP6 promotor produced antisense Cat D cRNA. For
the purpose of obtaining an RNA control probe, the Cat D insert was
subcloned in the PGEM2 vector system (Promega) oriented so that
transcription by T7 polymerase yielded a sense RNA probe.
The CDpSp64 was linearized with Kpnl so that the SP6 promotor
would produce approximately 400 bp antisense cRNA. The labeled
RNA was produced in a 100 p.I solution containing 40 mM Tris-HCI
(pH 7.5), 6 mM MgCI2, 2 mM spermidine, 10 mM NaCI, 20 mM DTT,
1 mM ATP, 1 mM GTP, 1 mM CTP, and 1 mM either biotinylated or
digoxigenin conjugated UTP, together with 0.1 mM UTP, 100 U of
RNAsin ribonuclease inhibitor, 2 v.g linearized plasmid DNA, and 200
U of SP64 RNA polymerase and incubated for 90 min at 37°C. After
incubation, 0.38 v 10 N ammonium acetate, 2.5 v ethanol, and 1 ~l of
concentrated glycogen (Boehringer-Mannheim) was added, and after
incubation at 4°C for 30 rain, the precipitate was recovered by centri-
fuging for 10 min at 10,000 x g. The RNA was redissolved in 100 p.I
of 1 M ammonium acetate and reprecipitated as described above. In
some cases, the DpSP64 was cut with EcoR1, and the resultant 2.2
kb RNA product was digested to an average size of 400 bp by limited
alkaline hydrolysis (Cox et al., 1984).
In Situ Hybridization and Detection
The protocol employed for in situ hybridization and detection employed
a modified hapten affinity cytochemical technique using biotin-
streptavidin and digoxigenin antibodies (Boehringer-Mannheim Bio-
chemicals, Mannheim, Germany) and the kit detection system of Gibco
BRL (Life Technologies, Inc., Gaithersburg, ME)). Deparaffinized sec-
tions were washed in phosphate-buffered saline (PBS; pH 7.4) twice
for 5 min each and transferred to 0.2 M Tris-HCL plus glycine (pH
7.4) for 10 min. After a brief rinse in PBS, slides were incubated in
predigested (40 rain) proteinase K (40 p~g/ml) for 10 min at 37°C. Sec-
tions were immersed for 1 min in 4% paraformaldehyde/PBS (pH 7.4)
at room temperature, rinsed for 5 min in PBS, and dehydrated through
a series of alcohols. Tissue sections selected as negative controls
were incubated for 45 min at 37°C in RNAase (20 ~g/ml) or were
hybridized with a sense RNA probe. Positive control slides for the
biotinylated RNA probes were included in the system and consisted
of paraffin-embedded HeLa cells infected with adenovirus type 2. The
slides were hybridized in sealed humidity chambers for 4-5 hr at 42°C.
Following hybridization, the slides were rinsed in 1 M SSC and incu-
bated in RNAse for 30 min at 37°C. Tissue sections then were rinsed
three times briefly in 0.2 M SSC and again in 0.2 M SSC for two 10
rain washes. Sections were blocked for 20 min at room temperature
and incubated for 1-2 hr in either the streptavidin-alkaline phospha-
tase conjugate or digoxigenin-alkaline phosphatase conjugate. Slides
were rinsed twice for 15 rain each and then briefly for 5 rain in alkaline
substrate buffer containing Trizma base, sodium chloride, and magne-
sium chloride (pH 9.5), followed by incubation in the reaction mixture
(200 Id nitroblue tetrazolium and 166 ~1 4-bromo-5-chloro-3-indolyl-
phosphate/50 ml alkaline substrate buffer) for 1-3 hr at 37°C. Color
development was stopped by rinsing sections in deionized water. The
tissue was dehydrated through ethanols, air dried, and coverslipped
with an aqueous mounting medium. All solutions were prepared in
diethyl pyrocarbonate-treated autoclaved water and passed through
sterile 0.22 p,m filters before use.
Northern Blot Analysis
Total cytoplasmic RNAs were isolated with TRI Reagent (Molecular
Research Center, Cincinnati, OH) from flash frozen blocks of the pre-
frontal cortices from Alzheimer's and control brains following the sug-
gested protocol of the manufacturer. The Northern blot analyses were
conducted according to the procedure described by DuPont NEN Re-
search Products (Boston, MA) for Genescreen Plus. In brief, the RNAs
(60 p.g for each) were separated on a 1% formaldehyde denaturing
agarose gel and transferred to the GeneScreen Plus nylon membrane,
and the blot was hybridized with the specific cDNA probe (109 to 2 x
101° cpm/llg, 106 cpm/p.I). The cDNA probe was labeled with ~2P-dCTP
(DuPont NEN Research Products) using the Random Primer Kit (Boeh-
ringer-Mannhein Biochemicals, indianapolis, IN). The blot was subse-
quently washed and exposed to X-ray film (Kodak).
G ross loading errors would be unlikely using 60 p.g of RNA; however,
as an additional loading control, gels were stained with ethidium bro-
mide prior to transfer to confirm that ribosomal bands were identical
in thickness and intensity. To detect cross-hybridization or comigration
of Cat D with the 18S ribosomal species, additional hybridization stud-
ies were performed using probes for cathepsin B and hexosamini-
Paraffin sections from the prefrontal cortices of Alzheimer's and age-
matched control brains were analyzed by densitometric analyses. Im-
mediate adjacent serial sections immunolabeled with anti-Cat D antise-
rum were hybridized with the specific human digoxigenin-labeled Cat
D RNA probe (above) to detect mRNA expression using in situ hybrid-
ization. The sections were coded and analyzed blindly with a user-
interactive, IBM DEK 486 computer-assisted microdensitometric tech-
nique. The prefrontal cortices were viewed with a Leitz Laboriux 12
light microscope interfaced with a Bioquant Meg IV image analysis
operating system (OS.2 Bioquant version 1.57, R and M Biometrics,
Nashville, TN) via a Dege MTI CCD-72 solid state digital camera using
a mouse/keyboard determination system to outline each neuron. De-
terminations were done in reversed polarity so that dense areas on
the slides appeared as light areas on the video monitor. A 100x oil
immersion objective (nA 1.518) was used to identify pyramidal neurons
from laminae III and V of the prefrontal cortex (Brodmann Area 10) in
both immunolabeled and hybridized tissue sections. The image sys-
tem was calibrated by placing an image box over laminae of interest
and recording the optical density (expressed as 0-255 levels of gray
scale; 0, lowest density; 255, highest density).
A total of 25 small, medium, and large neurons with visibly defined
nuclei were selected at random from layers ill and V of each brain.
With the aid of the computer imaging system, the perimeter of each
neuron was digitized and the area (in square micrometers) recorded.
For each neuron, a second tracing was made by outlining the nucleus.
The average optical density representing the total area of the neuron
and the total area density minus the optical density of the nucleus
was recorded. For each field, an image box was placed in various
background regions and the average optical density measured. Statis-
tical computations were performed using Student's t test.
The authors wish to thank Mrs. Denise McCarthy and Mrs. Sherry
Scullin for secretarial assistance in preparing the manuscript for publi-
cation and Timothy Wheelock for technical expertise. This research
was supported by grants from the National Institute on Aging (AG08278,
AG05134, and AG 10916). The Brain Tissue Research Center is sup-
ported by a grant from the National Institute on Mental Health
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 USC Section 1734
solely to indicate this fact.
Received August 1, 1994; revised November 11, 1994.
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Note Added in Proof
The data referred to as Schwaged and Nixon, unpublished data, have
now been published: Schwagerl, A. L., Mohan, P. S., Cataldo, A. M.,
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of the endosomal-lysosomal proteinase cathepsin D in cerebrospinal
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