Download Neuronal and microglial cathepsins in aging and age

Ageing Research Reviews
2 (2003) 367–381
Neuronal and microglial cathepsins in
aging and age-related diseases
Hiroshi Nakanishi∗
Laboratory of Oral Aging Science, Faculty of Dental Sciences, Kyushu University,
Fukuoka 812-8582, Japan
Received 20 March 2003; accepted 25 March 2003
Abstract
It has been long believed that cathepsins compensate for each other because of their overlapping
substrate specificities. However, there is increasing evidence that disturbance of the normal balance
of their enzymatic activities is the first insult in brain aging and age-related diseases. The imbalance
of cathepsins may further cause age-related neuropathological changes such as accumulation of
autophagic vacuoles and the formation of ceroid-lipofuscin leading to neuronal dysfunction and
damage. Leakage of cathepsins due to the fragility of lysosomal membranes during aging also
contributes to neurodegeneration. Furthermore, the deficiency of cathepsin D has been recently
revealed to provoke a novel type of lysosomal storage disease associated with massive neurodegeneration. In these animals, microglia are activated to initiate inflammatory and cytotoxic responses
by binding and phagocytosis of storage neurons. Activated microglia also release some members of
cathepsins to induce neuronal death by degrading extracellular matrix proteins. Thus the microglial
activation possibly through sensing neuronal storage may also be an important causative factor
for neurodegeneration in lysosomal storage diseases and age-related diseases such as Alzheimer’s
disease. This review describes the pathological roles of neuronal and microglial cathepsins in brain
aging and age-related diseases.
© 2003 Published by Elsevier Ireland Ltd.
Keywords: Cathepsin; Neuron; Microglia; Endosomal/lysosomal system; Ceroid-lipofuscin; Aging; Alzheimer’s
disease; Lysosomal storage disease
1. Introduction
A group of proteases in the endosomal/lysosomal proteolytic system is called cathepsin
which is derived from the Greek term meaning “to digest” (Willstätter and Bamann, 1929).
∗
Tel.: +81-92-642-6413; fax: +81-92-642-6415.
E-mail address: [email protected] (H. Nakanishi).
1568-1637/$ – see front matter © 2003 Published by Elsevier Ireland Ltd.
doi:10.1016/S1568-1637(03)00027-8
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H. Nakanishi / Ageing Research Reviews 2 (2003) 367–381
Cathepsins are synthesized on membrane-bound ribosomes as N-glycosylated precursors
and are transferred into the endoplasmic reticulum and later into the Golgi complex. During
transport to the Golgi complex, pro-cathepsins acquire modification of their carbohydrate
moieties, which includes the formation of the mannose 6-phosphate (M6P) residues. Following the binding to M6P-specific receptors (MPRs), the enzyme–receptor complexes exit
the trans-Golgi network in clathrin-coated vesicles and are transported to the late endosomes
(Kornfeld, 1992). Upon fusion with the late endosomes, the dissociation of ligands occurs.
The delivery of pro-cathepsins to lysosomes is accompanied by a series of proteolytic cleavages into their mature forms. In addition to a M6P-dependent targeting system, cathepsins
can be also targeted to the lysosomes in a M6P-independent mechanism (Glickman and
Kornfeld, 1993).
Although the primary function of cathepsins is to degrade proteins by bulk proteolysis in
lysosomes, recent results from cathepsin gene knockouts have revealed that cathepsins carry
out their specific functions by limited proteolysis of proteins (Saftig et al., 1995; Turk et al.,
2000; Reinheckel et al., 2001). It is likely that limited proteolysis is exerted by cathepsins in
less acidic intracellular compartments such as early and late endosomes. There is increasing
evidence that disturbance of normal balance and extralysosomal localization of cathepsins
contribute to neurodegeneration in Alzheimer’s disease, stroke, and lysosomal storage diseases (Cataldo et al., 1990, 1991; Nakanishi et al., 1994, 1997; Nixon, 2000; Koike et al.,
2000; Yamashima, 2000). Furthermore, the dysfunction of endosomal/lysosomal system in
neurons is closely associated with an activation of microglia which could initiate an inflammatory response to provoke a neurodegeneration (Nakanishi, 2003b). Activated microglia
also release some members of cathepsins to induce neuronal death through degradation of
extracellular matrix proteins (Nakanishi, 2003a, 2003b). Thus, this review will focus on
the pathological roles of neuronal and microglial cathepsins in brain aging and age-related
diseases.
2. Cathepsins and brain aging
2.1. Cathepsin alterations during brain aging
Alterations in the concentration and localization of cathepsins in the central nervous system (CNS) have been reported in normal aged brain (Nakamura et al., 1989). We have reported that the levels of cathepsins D and E were significantly increased during normal aging
process (Fig. 1A and B) (Nakanishi et al., 1994, 1997). The enzymatic activity of cathepsin B
was also increased significantly in the neostriatum of the aged rat but is relatively unchanged
in other regions from 2 to 28 months. By contrast, the enzymatic activity of cathepsin L was
decreased significantly (approximately 90%) in all brain regions of aged rats (Nakanishi
et al., 1994). There were also marked age-related changes in cellular localization and enzymatic activities of cathepsins in peripheral neurons. Cathepsins D, B and L were widely and
evenly distributed throughout the cytoplasm as coarse intracytoplasmic granules, whereas
they were localized at focal cytoplasmic sites in trigeminal ganglion neurons of aged rats
(Fig. 1C and D). The enzymatic activities of cathepsins B and L were decreased significantly
(approximately 50%) in aged rats (Amano et al., 1995). Lynch’s group has been motivated
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Fig. 1. Age-related changes of cathepsins in CNS and trigeminal ganglion neurons. Staining of cathepsin D in the hippocampal CA1 neurons of 2-month-old (A)
and 30-month-old (B) rats, respectively. Staining of cathepsin D in the trigeminal ganglion neuron of 2-month-old (C) and 30-month-old (D) rats, respectively. Immunoreactivities of cathepsin D (E) and their colocalization with autofluorescent lipopigments (F) in cortical neurons of 30-month-old rat. Colocalization of cathepsin
D-immunoreactivities and autofluorescent lipopigments was indicated by arrowheads. Double-staining immunohistochemistry of cathepsin E (G) and carboxy-terminal
fragments of APP (APP643–695 ) (H) in the brainstem neurons of 30-month-old rat. Colocalization of immunoreactivities of cathepsin E and APP643–695 was indicated by
arrowheads. Scale bars: 10 ␮m (A–D), 50 ␮m (E and F), 100 ␮m (G and H).
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by these observations and examined effects of long-term treatment of cysteine protease inhibitors on the hippocampal slice cultures. N-CBZ-l-phenylalanyl-l-alanine-diazomethyl
ketone, a selective inhibitor of cathepsins B and L, induced increased cathepsin D levels, the
accumulation of lysosome-related dense bodies and hyperphospholylated ␶ fragments, and
the formation of meganeurites in the perykarya of CA1 neurons in cultured hippocampal
slices (Bednarski and Lynch, 1996; Bednarski et al., 1997). These observations strongly
suggest that disturbance of the normal balance of enzymatic activity of cathepsins causes
age-related neuropathological changes. However, lysosome-related dense bodies induced
by N-CBZ-l-phenylalanyl-l-alanine-diazomethyl ketone did not emit autofluorescence, indicating that they do not contain ceroid-lipofuscin, a major type of residual body observed
in various types of neurons during the aging process (Bednarski et al., 1997). This contrasts with the observation that intraventricular application of leupeptin, a cystein protease
inhibitor, or chloroquine, a general lysosomal inhibitor, in young rats induced the formation of lysosme-associated granular aggregates that closely resemble ceroid-lipofuscin (Ivy
et al., 1984).
Upregulation of the endosomal/lysosomal system has also been demonstrated in affected
neurons of Alzheimer’s disease brain (Nixon and Cataldo, 1993, 1995; Nixon, 2000). In
the senile plaques, the enzymatically active cathepsin D, as well as cathepsins B and L,
are present extracellularly at high levels (Bernstein et al., 1989; Cataldo and Nixom, 1990;
Cataldo et al., 1991). Furthermore, secondary lysosomes and residual bodies are markedly
increased in affected neurons of Alzheimer’s disease brain (Cataldo et al., 1990; Nixon and
Cataldo, 1993). The activation of the endosomal/lysosomal system has been suggested to
be closely associated with increased production of amyloid ␤ (A␤) peptides in sporadic
Alzheimer’s disease (Nixon, 2000).
2.2. Amyloid precursor protein (APP) metabolism
It is well known that APP is metabolized through two different pathways, secretory
and endosomal/lysosomal pathways. In the secretory pathway, ␤-site APP cleavage enzyme (BACE), a novel transmembrane aspartic protease, has been recently identified as
␤-secretase (Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). On the other hand,
cathepsins D and E are considered to influence A␤ peptides generation within the endosomal/lysosomal system because they have ␤-secretase activity (Grüninger-Leitch et
al., 2000). BACE was also found in early endosomes (Vassar et al., 1999). We have
found that cathepsins D and E are colocalized with ceroid-lipofuscin (Fig. 1E and F)
and carboxy-terminal fragments of APP (Fig. 1G and H) in CNS neurons of aged rats
(Nakanishi et al., 1997). Immunoblot analysis revealed that the molecular sizes of accumulated carboxy-terminal APP fragments were 19 and 22 kDa, suggesting that they might
contain an intact A␤ peptide domain and therefore be amyloidogenic. Mathews et al. (2002)
have recently reported that overexpression of MPRs which selectively redirect lysosomal
hydrolases to early endosomes increases the generation of A␤ peptides. It is also known
that MRPs are more highly expressed in Alzheimer’s disease brain (Nixon, 2000). These
observations strongly suggest that the upregulation of early endosomes as a consequence of
increased expression of MRPs are closely associated with enhanced A␤ peptides production
and secretion in Alzheimer’s disease. Although Saftig et al. (1996) excluded the possible
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involvement of cathepsin D in amyloidogenic processing of APP by utilizing cathepsin
D-deficient mice, age-associated factors such as increased expression and endosomal localization may be required for cathepsin D to work as ␤-secretase (Mathews et al., 2002).
2.3. Lysosomal membrane impermeability
Besides functions inside the endosomal/lysosomal system, there is evidence that some
members of cathepsins are also involved in extracellular proteolysis resulting in pathological
conditions. Leakage of cathepsins into the cytoplasm is often achieved by the endocytosis of
oxidizable substrates that destabilize the lysosomal membranes through lipid peroxidation.
It has been proposed that the increased level of cytosolic cathepsin D in the aged rat brain
is due to the age-dependent increase in the fragility of the lysosomal membrane (Matsu
and Green, 1987; Nakamura et al., 1989). We have previously reported that subcellular
fractionation showed that the amounts of cathepsins D and E in the soluble fraction of
the aged rat brain were markedly increased as compared with those of the young rat brain,
suggesting that these enzymes leaked into the cytoplasm. More recently, leakage of cathepsin
D into the cytoplasm of neurons in aged rats is demonstrated by immunoelectron microscopy
(Jung et al., 1999). In senile plauques of Alzheimer’s disease brain, lysosomal hydrolases
such as cathepsin D distribute in an abnormal extracellular location (Cataldo et al., 1991). In
vitro, cathepsin D and ␤-hexosaminidase are increased in the soluble cytosolic compartment
of the cells following treatment with A␤ peptides (Yang et al., 1998).
Some lysosomal cysteine proteases such as cathepsins B, H and L are relatively unstable
after leakage from lysosomes, whereas others such as cathepsins D, E, and S are stable even
at neutral pH (Bednarski and Lynch, 1996; Turk et al., 2000). Therefore, these enzymes
are capable of degrading cytoskeletal proteins and bioactive peptides in the cytoplasm
of neurons. Furthermore, there is increasing evidence that some members of cathepsins
are directly involved in apoptosis. Cathepsin D has been first reported to play as a final
executioner of apoptosis induced by interferon-␥, Fas/APO-1, TNF-␣ (Deiss et al., 1996),
chemotherapeutic agents such as adriamycin and etoposide (Wu et al., 1998), and serum
deprivation (Shibata et al., 1998; Isahara et al., 1999). Cathepsin B has been also implicated
in the activation of the proinflammatory caspases 1 and 11 (Vancompernolle et al., 1998;
Schotte et al., 1998), and the cleavage of Bcl-2 family member Bid (Stoka et al., 2001)
which may lead to cytochrome c release from the mitochondria and subsequent caspase
activation (Guicciardi et al., 2000). Taken together, the abnormal localization of cathepsins
resulting from the disintegration of lysosomal as well as plasma membranes may contribute
to the pathogenesis of age-related neuronal dysfunction and Alzheimer’s disease.
2.4. Upregulation of endosomal/lysosomal system in microglia
In aged rat and Alzheimer’s disease brains, microglia are known to express activation
markers such as major histocompatibility complex (MHC) class II molecules, lysosomal
membrane marker ED1, and the leucocyte common antigen and CD4 (Perry et al., 1993;
Togo et al., 2000). Although the microglial reaction has long been considered as a secondary
event following neuronal damage and death, there is increasing evidence that the activation
of microglia is closely associated with brain aging and the pathogenesis of Alzheimer’s
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disease. A␤ peptides have been reported to induce neuronal death indirectly by activating
microglia to produce inflammatory mediators such as nitric oxide (NO), cytokines, and
reactive oxygen intermediates (Meda et al., 1995; El Khoury et al., 1996).
Several lines of evidence suggest that activated microglia secrete cathepsins to induce
neuronal death. In response to lipopolysaccharide (LPS), there was a substantial increase in
cathepsin S activity secreted from both macrophages and microglia (Petanceska et al., 1996).
This may suggest that cathepsin S plays a role in degenerative disorders because cathepsin
S degrades components of extracellular matrix proteins even at neutral pH. Cathepsin B
was also secreted from immortalized murine microglial cell line, BV-2 cells, as the heavy
chain form in addition to the proform upon stimulation with LPS (Ryan et al., 1995). More
recently, it has been demonstrated that secreted cathepsin B is a major causative factor of
microglia-induced neuronal apoptosis (Kingham and Pocock, 2001). We have shown that
some cathepsins are closely linked with the proteolytic processing of exogenous antigens
and invariant chain of MHC class II molecules through the endosomal/lysosomal system of
microglia (Nishioku et al., 2002). Although the precise implication of exogenous antigen
presentation in the CNS is not fully understood, inflammatory cytokines secreted from
activated helper T cells and microglia activated through their interaction may contribute
to the tissue damage and/or repair. Therefore, increased levels of cathepsins in activated
microglia distributed especially in the white matter of aged rat (Nakanishi et al., 1994) and
Alzheimer’s disease brain (Bernstein and Wiederanders, 1994; Yoshiyama et al., 2000) may
have some pathological significance.
2.5. Degradation of Aβ peptides in lysosomes
Cathepsin D has been suggested to be responsible for the intracellular clearance of A␤
peptides in human and rat brains (Hamazaki, 1996; McDermott and Gibson, 1996). A␤
peptides are taken up predominantly by microglia via class A scavenger receptors and class
B scavenger receptor type I (Paresce et al., 1996; Husemann et al., 2001). Then A␤ peptides are accumulated and degraded in the lysosomes of microglia (Paresce et al., 1997).
Importantly, pepstatin A has been reported to inhibit the degradation of A␤ peptides in
microglia (Kakimura et al., 2002). These observations strongly suggest that phagocytosed
A␤ peptides are mainly degraded by cathepsin D in lysosomes of microglia. It is also noteworthy that immunization with A␤ peptides has been demonstrated to reduce A␤ peptides
in transgenic mice with A␤ plaques (Schenk et al., 1999). Thus, the phagocytosis and subsequent degradation of A␤ peptides by microglia may play a pivotal role in a strategy for
the immunotherapy of Alzheimer’s disease.
3. Cathepsins and lysosomal storage diseases
3.1. Cathepsin D-deficient mice
To gain more insight into functions of cathepsin D, mice deficient in cathepsin D were
generated by gene targeting (Saftig et al., 1995). The homozygous mutant mice developed a
progressive atrophy of the intestinal mucosa and a profound destruction of lymphoid tissues.
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Although the mutant mice could develop normally during the first 2 weeks, they stopped
thriving in the third week, decreased in weight, and then died between 25 and 27 days.
At the cellular level, however, lysosomal bulk proteolysis was maintained in these mice.
It was concluded that the essential functions of cathepsin D depend on limited proteolysis
of biologically active proteins such as growth factors rather than on bulk degradation of
proteins in lysosomes.
During the course of our studies on morphological as well as functional changes in CNS
neurons of cathepsin D-deficient mice, we noticed that these animals showed neurological
phenotypes such as seizures and blindness near the terminal stage. The most striking feature
found in the CNS was a profound storage of autophagosome/autolysosome-like bodies with
part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles (Koike et al.,
2000). Almost all neurons especially in the cerebral cortex and thalamus contained large
autofluorescent bodies, indicating the accumulation of ceroid-lipofuscin in the lysosomal
structures (Fig. 2A and B). These ceroid-lipofuscin loaded lysosome contained subunit
c of mitochondrial F1 F0 -ATP synthase, a common storage material of neuronal ceroid
lipofusinosis (NCL) except for the infantile form of NCL. Interestingly, however, the protein
and activity levels of tripeptidyl peptidase I, whose deficiency causes late-infantile NCL,
were increased in cathepsin D-deficient mouse brain (Koike et al., 2000). These observations
strongly suggest that the loss of cathepsin D activity causes a novel type of lysosomal storage
disease associated with massive neurodegeneration.
3.2. Activated microglia-induced inflammation in lysosomal storage diseases
We have observed a marked accumulation of morphologically transformed microglia exhibiting expanded and round cell bodies with a few thick processes especially in the cerebral
cortex and thalamus near terminal stage of cathepsin D-deficient mice (Fig. 2C). The morphological transformation of microglia may be caused by binding and uptake of neurons
laden with large autofluorescent bodies (Fig. 2D and E). Furthermore, there is a prominent
expression of inducible nitric oxide synthase (iNOS) in both morphologically transformed
microglia and peripheral macrophages in cathepsin D-deficient mice (Nakanishi et al.,
2001). NO and the superoxide anion, which is generated in mitochondria, react rapidly to
form a peroxinitrite anion. This, in turn, generates highly toxic hydroxyl radicals and hydrogen peroxide. Although NO is synthesized from l-arginine by NOS, iNOS is thought to be
the isoform that produces the large quantities of NO that can result in tissue damage or death.
To directly address the possible involvement of NO in tissue damage and neuronal death
in cathepsin D-deficient mice, we examined effects of l-NG -nitro-arginine methylester (lNAME), a potent competitive NOS inhibitor and S-methylisothiourea hemisulfate (SMT),
an iNOS inhibitor. Chronic treatment with l-NAME or SMT significantly decreased the total
number of terminal dUTP nick-end labeling (TUNEL)-positive cells counted in the thalamus
of cathepsin D-deficient mice (Nakanishi et al., 2001). In the course of these experiments,
we unexpectedly found that chronic treatment with l-NAME or SMT markedly ameliorated
a severe hemorrhage-necrotic appearance of the small intestine and atrophic changes of the
ileal mucosa of cathepsin D-deficient mice. Therefore, the activated microglia/macrophageinduced inflammatory response is considered to be a major causative factor for pathological
changes in the CNS and the small intestine of cathepsin D-deficient mice.
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Fig. 2. Accumulation of ceroid-lipofuscin in CNS neurons of 24-day-old cathepsin D-deficient mice and phagocytosis by microglia. Autofluorescent lipopigments
accumulated in thalamic neurons of the wild-type littermate (A) and cathepsin D-deficient mouse (B), respectively. (C) Accumulation of activated microglia stained with
F4/80 in the thalamus of cathepsin D-deficient mouse. (D) Engulfment of neurons that were laden with large autofluorescent bodies (orange) by microglia labeled by F4/80
(green) in the thalamus of cathepsin D-deficient mouse. (E) Electron micrograph of microglia (m) attached to neuron (n) laden with autophagosome–autolysosome-like
bodies in the cerebral cortex of cathepsin D-deficient mouse. Scale bars: 30 ␮m (A and B), 20 ␮m (C and D), 4.0 ␮m (E).
H. Nakanishi / Ageing Research Reviews 2 (2003) 367–381
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Our hypothesis for a mechanism underlying activation of microglia and subsequent massive neuronal death in the CNS caused by cathepsin D-deficiency is summarized in Fig. 3.
When cathepsin D activity is deficient, hydrophobic proteolipids such as subunit c of mitochondrial F1 F0 -ATP synthase are first accumulated in lysosomes. These storage materials
further facilitate the formation of ceroid-lipofuscin especially in lysosomes of CNS neurons
leading to neuronal dysfunction and damage. By binding and phagocytosis of these storage
neurons, microglia are activated to produce NO through iNOS. Microglia may be also activated and/or primed through their own accumulation of proteolipids due to cathepsin Ddeficiency. Finally, the sustained and high levels of NO released from activated microglia initiate an intensive inflammatory response in the CNS leading to secondary neuronal damage.
Bone marrow transplantation (BMT) has emerged as a potential treatment for some
types of lysosomal storage diseases. It has been shown that bone-marrow-derived monocytes/macrophages can cross the blood–brain barrier and become perivascular macrophages
and microglia (Eglitis and Mezey, 1997; Ono et al., 1999; Priller et al., 2001). The secretion
of lysosomal enzymes from microglia/macrophages derived from hematopoietic donor cells
and uptake by surrounding brain cells via M6P receptor-mediated endocytosis are proposed
as the basis for BMT therapy for lysosomal storage diseases. However, it has been recently
reported that microglia/macrophages secrete lysosomal enzymes such as arylsulphatase A
and cathepsin D, which are incompetent for M6P receptor-dependent uptake by brain cells
due to the lack of M6P residues (Muschol et al., 2002). Furthermore, BMT has been reported to extend the lifespan and ameliorate neurologic symptoms of Sandhoff disease mice
without a clear reduction of neuronal GM2 ganglyoside storage (Norflus et al., 1998; Wada
et al., 2000). Furthermore, the involvement of activated microglia in the pathogenesis of
lysosomal storage diseases has been also suggested in other mouse models; metachromatic
leukodystrophy (Hess et al., 1996), Sandhoff disease (Wada et al., 2000), Niemann-Pick
disease type C (German et al., 2002), and mucopolysaccharidoses I and IIIB (Ohmi et al.,
2003). Taken together, it is likely that the pathogenesis of these diseases is dominated by activated microglia-induced inflammation rather than lysosomal storage due to the deficiency
of lysosomal enzymes. Furthermore, infiltration of Blood-born microglial precursors after
BMT may improve neuronal pathology by a mechanism other than supplying the missing
enzymes.
3.3. White Swedish Landscape sheep
Tyynelä et al. (2000) have shown a novel form of NCL, congenital ovine NCL (CONCL)
arising from a naturally occurring cathepsin D mutation. CONCL was observed in a flock of
White Swedish Landscape sheep maintained on an experimental farm in Northern Sweden.
In CONCL lambs, a single nucleotide mutation in the cathepsin D gene is substituted by
a conserved active-site aspartate by asparagines at position 295 (Asp → Asn) resulting
in a catalytically inactive protein. These animals show some phenotypes similar to those
of cathepsin D-deficient mice such as neuronal death in the cerebral cortex and neuronal
storage of autofluorescent granules. The activity of tripeptidyl peptidase I was also elevated.
However, there are several differences between CONCL lambs and cathepsin D-deficient
mice. Unlike cathepsin D-deficient mice, the newborn CONCL lambs do not exhibit any
pathological changes in the lymphoid tissues and ileal mucosa. On the other hand, CONCL
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Fig. 3. Microglial activation and neuronal death in CNS neurons of cathepsin D-deficient mice. Neuronal storage of hydrophobic storage materials such as subunit c
of mitochondrial F1 F0 -ATP synthase is the primary insult due to cathepsin D-deficiency. The accumulation of these proteolipids facilitate the formation of neuronal
ceroid-lipofuscin leading to neuronal dysfunction and damage. Microglia are activated by opposing these storage neurons and/or their own accumulation of proteolipids
to produce NO through iNOS. Microglial NO may initiate an intensive inflammatory response in the CNS leading to secondary massive neurodegeneration.
H. Nakanishi / Ageing Research Reviews 2 (2003) 367–381
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lambs have a severe brain atrophy which is never observed in cathepsin D-deficient mice.
Theses discrepancies may be due to the species difference, the maturity of the CNS, and/or
the gestation periods.
3.4. Phenotypes of cathepsin B- and cathepsin L-deficient mice
Cathepsin B-deficient mice show no obvious phenotype (Deussing et al., 1998), but
a reduction in premature intrapancreatic trypsinogen activation (Halangk et al., 2000) and
increased resistance to TNF-␣-mediated hepatocyte apoptosis (Guicciardi et al., 2000) were
observed under experimental conditions. Cathepsin L-deficient mice also present with a
reduction in CD4+ T cells (Nakagawa et al., 1998) and recurrent hair loss (Roth et al., 2000).
More recently, however, it has been reported that combined deficiency of cathepsins B and L
is lethal during the second to fourth week of life. These double-mutant mice reveal massive
apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell
layers resulting in a brain atrophy (Felbor et al., 2002), suggesting that cathepsins B and L
are essential for maturation and integrity of the postnatal CNS and that they compensate for
each other. In the double-mutant mice, neurodegeneration is preceded by an accumulation of
inclusions characterized by electron-dense, often membrane-bound bodies of varying size
and shape occasionally containing lamellae. Furthermore, the double-mutant mice show
normal activities of both pamitoyl protein thioesterase and tripeptidyl peptidase I, and no
clear accumulation of subunit c and ceroid-lipofuscin. Thus, phenotypes of mice deficient
for both cathepsins B and L is ultrastructurally and biochemically distinct from those seen
in cathepsin D-deficient mice and classical NCLs.
4. Conclusions
Cathepsins are now recognized to have more complex functions than simply being
garbage disposers, and their imbalance during aging and age-related diseases may provoke deleterious effects on CNS neurons. The growing understanding of consequences of
their age-related changes in neurons and microglia could contribute to the development of
therapeutic interventions in massive neurodegeneration associated with age-related diseases
such as Alzheimer’s disease and NCLs.
Acknowledgements
This work was supported by a Grants-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports and Culture of Japan.
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