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
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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.,
Vonsattel, J. P., Kowall, N. W., and Nixon, R. A. (1995). Elevated levels
of the endosomal-lysosomal proteinase cathepsin D in cerebrospinal
fluid in Alzheimer disease. J. Neurochem. 64, 443-446.