Mechanisms of Ageing and Development
83 (1995) 133-141
ELSE:VIER
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A,ge-related changes in cellular localization and
enzymatic activities of cathepsins B, L
and D in the rat trigeminal ganglion neuron
Tutomu Amano”,b, Hiroshi Nakanishi”“, Teruyoshi Kondo”,
Teruo Tanakac, Masuichiro Okab, Kenji Yamamoto”
aL)epartment of Pharmacology, Kyushu University, Faculty of Dentistry, Fukuoka 812, Japan
bDepartment of Oral Surgery, Kyushu University, Faculty of Dentistry, Fukuoka 812, Japan
‘Department of Oral Anatomy, Kyushu University, Faculty of Dentistry, Fukuoka 812, Japan
Received 21 February 1995; revision received 30 May 1995; accepted 13 June 1995
Abstract
Altered localization and cellular level of three distinct lysosomal proteinases, cathepsins B
(CB), L (CL), and D (CD), with aging were investigated in the rat trigeminal ganglion (TG)
by immunohistochemical
and quantitative analyses. At the light microscopic level, the
intrac$oplasmic distribution of these three enzymes was found to change with aging: These
lysosomal proteinases in the TG of young rats (2-3 months of age) were widely and evenly
distributed throughout the cytoplasm as coarse intracytoplasmic granules, whereas they were
localized at focal cytoplasmic sites of the TG neurons of aged rats (28-31 months of age) as
coarse aggregates. A similar distribution was observed with a major lysosomal membrane
sialoglycoprotein having an apparent molecular mass of 107 kDa (LGP107). The cellular
distribution of the three cathepsins as well as LGP107 in the TG neurons of aged rats
corresponded well with that of autofluorescent lipofuscin. At the electron microscopic level,
the age-related redistribution of these cathepsins in the TG neurons was found to be due to
their great accumulation in autolysosomes localized at the focal perinuclear sites. The cellular
levels of CB and CL determined by activity measurement in the TG of the young rats were
1.8 and 1.7 times as much as those of the aged rats respectively. In contrast, no significant
difference was observed between the CD activities in the two age groups. These results
* Corresponding
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Author, Fax: + 81 92 6321226.
0 1995 Elsevier Science Ireland Ltd. All rights reserved
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134
T. Amuno et (11.1Mechunisms of’ Ageing crnd Development 83 (1995) 133-141
strongly suggest that age related changes in localization and cellular level of CB, CL, and CD
in TG neurons are closely linked with the increased formation
of autolysosomes
and
lipofuscins, which is the most ubiquitous age-related cytological alteration.
Kevwords:
Aging; Trigeminal
ganglion;
Cathepsins;
Lysosome;
Phagocytosis
1. Introduction
Cathepsins B (CB), L (CL), and D (CD) are typical and well-characterized
lysosomal proteinases that are widely but unevenly distributed in a variety of
mammalian cell types including neuronal cells [3,4,25]. Besides their intralysosomal
proteolysis for intracellular proteir. turnover, these enzymes have been suggested to
be involved in a variety of pathological conditions such as inflammation [12,20] and
neoplastic transformation [5,21]. More recently, there is increasing evidence that the
disorder or breakdown of lysosomal systems has pathological significance in
neurodegenerative diseases such as Alzheimer’s disease. In Alzheimer’s disease,
enzymatically active CB and CD have been found extracellularly at high levels in
the senile plaques [7] and there was a marked accumulation of secondary lysosomes
and residual bodies in affected neurons [6,18]. In rat brain, it has been reported that
enzymatic activities of CD [10,14,15,17] and cathepsin E (CE), a non-lysosomal
aspartic proteinase, were significantly increased with advancing age [ 171. Lysosomal
leakage in the aged rat brain has also been demonstrated to result in increased
cytosolic CD activity [14, 151. Furthermore, these cathepsins are known to exhibit
the ability to degrade physiologically significant neuronal constituents such as
neurofilaments, microtuble-associated proteins and myelin basic proteins [2, 14, 19,
241. The abnormality of lysosomal systems is considered to have pathological
significance in the ageing process of the central nervous system.
In contrast to the central nervous system, little or no information is available
about age-related alterations of the lysosomal system in the peripheral nervous
system. The present study was thus undertaken to elucidate age-related changes in
localization and cellular level of CB, CL, and CD as well as CE in the rat
trigeminal ganglion (TG) neurons.
2. Materials and methods
2.1. Materials
Antibodies against rat spleen CB, CD, and CE were raised in rabbits and purified
by affinity chromatography as described previously [16,17]. Antibodies specific for
CL and the 107 kDa lysosomal membrane sialoglycoprotein (LGP107) were
provided from Drs. Y. Nishimura and Y. Tanaka (Department of Physiological Chemistry, Kyushu University Faculty of Pharmaceutical Sciences, Japan),
respectively. N-(L-3-trans-propyl-carbamoyloxirane-2-carbonyl)L-isoleucyl-L-proline (CA-074) was kindly supplied by Taisho Pharmaceutical Co. Ltd (Tokyo,
Japan). Two age groups of Fischer 344 male rats (young: 2-3 months of age; aged:
28-31 months of age) were used in the present study.
T. Amano
et al. / Mechanisms
2.2. Immunocytochemical
oJ Ageing
and Development
83 (1995)
133-141
135
procedures
Deta.iled immunohistochemical
procedures were described previously [ 16,171.
Briefly., young (2-3 months, n = 5) and aged (28-31 months, n = 5) rats anaesthetized with sodium pentobarbital were perfused with chilled isotonic saline and
then with chilled fixative consisting of 4% paraformaldehyde in 0.2 M phosphate
buffered saline (PBS, pH 7.4). After perfusion, the TG was removed, fixed in the
same fi.xative for 24 h at 4”C, and then immersed in 20% sucrose (pH 7.4). Floating
parasagittal sections (30-40 pm thick) were prepared by a cryostat and stained by
anti-CB (6.5-8.0 mg/ml), CL (OS-o.8 mg/ml), CD (lo-20 pgglml), LGP107 (50
pg/ml), IgG at dilution of 1: 800-2400 in PBS with the avidin-biotin-peroxidase
complex method using a Vecstatin Kit (Vector Laboratories, Burlingame, CA,
USA) followed by the diaminobenzidine reaction. For double immunostaining of
CB and LGP107, aminoethylcarbazole (AEC) was used as a chromogen yielding a
xylene,-soluble orange precipitate. After immunostaining for CB, AEC precipitates
for CEI labeling, were removed by incubating the sections with xylene and then the
sections were further stained by anti-LGP107 IgG. For immunoelectron microscopic examination, aged rats (31 months, n = 3) were perfused with chilled isotonic
saline and then with chilled fixative (4% paraformaldehyde and 0.5% glutalaldehyde
in 0.2 M PBS). After perfusion, the TG was removed, fixed in the same fixative for
3 h at 4°C. Sections (20-25 pm thick) were cut by a Microslicer (DTKlOOO,
Dosaka EM, Kyoto, Japan) and processed similarly to those for light microscopy,
except that all procedures were performed at 4°C and they were not treated with
Triton X-100. After immunostaining as described above, the sections were further
postfixed with 0.5% osmium tetroxide, dehydrated through graded ethanol
and embedded in Epon 812. Ultrathin sections were examined with a JEOL200CX
electron microscope. Control sections were treated in the same way except that
the primary antibodies were removed or replaced by a non-immune rabbit
IgG.
2.3. Determination
of cathepsins activities
Detailed methods for determination of enzymatic activities were also described
previously [16,17]. Shortly, the TG extracts from young (2-3 months, n = 6-7) and
aged rats (28-31 months, n = 4-5) were prepared by ultrasonication of the
homo.genate in the presence of 0.1% Triton X-100 followed by centrifugation. CB
and CL activities in the extract were measured with benzyloxycarbonyl-phenylalanyl-arginine-4-methyl-7-coumarylamide
(Z Phe-Arg-MCA) at pH 6.0. Knowing
the total activity against Z-Phe-Arg-MCA, the levels of CB and CL were determined. by measuring the activity sensitive and insensitive to the CB specific inhibitor
CA-O’74 [9], respectively. One unit for CB and CL was defined as the amount of
each <enzyme required to release 1 nmol of 7-amino-4-methylcoumarin
liberated
from the substrate per min. CD and CE activities were measured with acid-denatured hemoglobin (Hb) as a substrate at pH 3.5. The levels of CD and CE were
deterrnined from the total activity by immunoprecipitation utilizing discriminative
antibodies specific for each enzyme. One unit for CD and CE was defined as the
amount of each enzyme required to release 1 pug of tyrosine per min. Protein was
136
T. Amano et al. / Mechanisms of’ Ageing and Development 83 (199.5) 133- 141
determined by the method of Lowry et al. [13] using bovine serum albumin as a
standard.
3. Results
3.1. Age-related changes in cellular localization of cathepsins B, L and D
At the light microscopic level, three distinct lysosomal proteinases CB, CL, and
CD were abundantly and evenly distributed through all the TG neurons of young
rats (Fig. lA-C). These enzymes were found in the cells as coarse intracytoplasmic
granules. The neuronal nuclei were devoid of immunoreactive products for each
enzyme. No significant difference in staining pattern of the respective cathepsins
was observed among different sized TG neurons with a diameter ranging lo-60
pm. In aged rats, the relative amount of the medium-sized TG neurons was
increased and the mean diameter was shifted from 27 to 32 pm (n = 100). More
than 60% of the TG neurons of aged rats showed intense and local immunostaining
for CB, CL, and CD at the focal cytoplasmic sites as large coarse aggregates (Fig.
I-D, E, F). When changes in the cellular localization of CB, CL and CD were also
examined in the middle age group, a similar focal distribution of these three
enzymes was found in TG neurons of 20-month-old rats, but barely detectable in
those of 12-month-old rats (data not shown), suggesting that changes in their
localization are closely associated with such senescent cytological alterations as
increased formation of autolysosomes and lipofuscins. On the other hand, CE was
not detectable in TG neurons of either young or aged rat groups. Only
macrophage-like cells in this tissue from both age groups gave diffuse staining for
CE in the cytoplasm (data not shown).
In the great majority of TG neurons from the aged rat group, intense
autofluorescent granules were found to accumulate in the focal cytoplasmic site
(Fig. 2A). These autofluorescent pigments were considered to be lipofuscins since
they exhibited yellow-brown fluorescence under ultraviolet light. The TG neurons
from the young rat group showed faint or no such autofluorescence (data not
shown). Granular immunoreactive products of both CB (Fig. 2B) and LGP107
(Fig. 2C) were also found in the focal cytoplasmic site of the TG neurons and their
distribution corresponded well with that of autofluorescent pigments. All the
CB-positive cells contained both lipofuscin and LGP107, but LGP-positive and
lipofuscin-containing cells were not always positive for CB. Similar observations
were obtained for CL and CD, although there were slight regional differences in
immunostaining patterns among these cathepsins (data not shown).
At the electron microscopic level, the TG neurons from the aged rat group were
characterized by the presence of a number of autolysosomes, which engulfed
swollen cytoplasmic organelles such as mitochondria, vacuoles and lipofuscin
pigments (Fig. 2-D,E). The immunoreactive materials for CB appeared in the form
of fine electron-dense granules were observed in lysosomes (Fig. 2-D, single arrow
heads) and autolysosomes which contained degrading cytoplasmic organelles such
as mitochondria and electron-dense lipid vacuoles (Fig. 2-D, double arrow heads).
often consisted of two membranes,
indicating
CB-positive autolysosomes
T. Amano et al. / Mechanisms of Ageing and Development 83 (1995) 133~-141
137
Fig. 1. Immunohistochemical
staining of CB, CL and CD in TG neurons of the young and aged rat. The
left and right columns are TG neurons from the young and aged rat, respectively. The top panels (A,D):
staining of CB; the middle panels: staining of CL; the bottom panels: staining of CD. Intense granular
immunoreactive
materials of each enzyme are homogeneously
distributed through the cytoplasm of the
young TG neuron. In the aged TG neuron, however. focal accumulations
of immunoreactivities
of these
enzymes are noted. Bar = 10 pm.
138
T. Amano et ul. I Mechanisms of’ Age@
and Development 83 1199.5) 133-141
Fig. 2A-C. Localization of lipopigment autofluorescent (A), CB (B) and LCP107 (C) in the same
section of the TG from the aged rat. D: immunocytochemical staining of CB in the TG neurons of the
aged rat. lmmunoreactive products of CB are observed as fine electron-dense granules in both lysosomes
(marked by arrow heads) and aurolysosomes (marked by double arrow heads). E: control staining. Both
lysosomes (marked by arrow heads) and autolysosomes (marked by double arrow heads) are devoid of
immunoreactive materials. Bar = 40 /lrn (A), 1 pm (D).
Table 1
Enzymatic activities of CB, CL and CD in the trigeminal ganglion from young and aged rats
Young
Aged
Cathepsin B
Cathepsin L
Cathepsin D
18.4 _+5.6 (n = 7)
10.4 + 0.5 (n = 4)**
9.8 k 3.6 (n = 7)
5.8 + 0.6 (n = 6)*
13.5+1.8 (n=6)
14.2 + 2.7 (n = 5)
Values are presented as mean i S.D. in units/mg protein. p is calculated according to Student’s t -test
for the defferences between young and aged groups.
*P < 0.05.
**p < 0.01.
T. Amano et al. / Mechanisms of Ageing and Development 83 (1995) 133-141
139
that they are in the early stage of autophagocytosis. No fine electron-dense granule
representing immunoreactivity was observed in any lysosomal structures when
tissues were incubated without anti-CB antibody or when anti-CB antibody was
replaced by non-immune IgG (Fig. 2-E).
3.2. Age-related changes in enzymatic activities of cathepsins B, L and D
Table 1 shows the activity levels of CB, CL, and CD in the Triton X-100 soluble
extract of the TG from both young and aged rat groups. In either group, the
Z-Phe-Arg-MCA- and Hb-hydrolyzing activities in the TG extract were totally
inhibited by E-64 and pepstatin, respectively. The results strongly suggest that the
Z-Phe-Arg-MCA- and Hb-hydrolyzing activities were composed of the cysteine
proteinases CB and CL and the aspartic proteinases CD and CE, respectively. The
CB aml CL activities were determined by measuring the activity sensitive and
insensitive to CA-074, respectively. In both groups, about 65% of the total
Z-Phe-.4rg-MCA-hydrolyzing
activity was attributed to CB and about 35% to
cathepsin L. Thus, the ratio of the CB to CL contents in the tissue was likely to be
independent of aging. On the other hand, the Hb-hydrolyzing activities in the TG
extracts from both groups were totally immunoprecipitated by increasing amounts
of the anti-CD IgG but not the anti-CE IgG, indicating that this tissue contained
only CD activity. As shown in Table 1, the levels of CB and CL in the young rat
group were significantly higher than those in the aged rat group by 55% (P < 0.01)
and 60%, (P c O.OS),respectively. In contrast, the CD level in the tissue was not
significantly changed with aging.
4. Discussion
The present study provides evidence that the lysosomal system in the TG neurons
is clearly altered with aging. The TG neuron from the young rat group exhibited
wide and homogeneous distribution of cathepsins as well as the lysosomal membrane protein LGP107 through the cytoplasm, whereas most of the TG neurons
from the aged rat group showed their limited and focal distributions in the
perinuclear regions. In order to examine whether changes in the cellular localization
of lysosomal cathepsins occurred between young age and maturity, we examined
their expression in TG neurons of the middle aged rats. A similar limited and focal
distribution of these enzymes in TG neurons was found in rats of 20 months of age,
but barely detectable in rats of 12 months of age. Therefore, it is more likely that
changes in the expression of these enzymes are associated with senescence but not
maturity. By immunoelectron microscopic study, strong antigenecities for these
enzymes in the TG neurons from the aged rat group were mainly shown in
autolysosomes which were focally formed in the perinucler cytoplasmic sites,
indicating enhanced autophagocytosis. These autolysosomes contained engulfed
cytoplasmic organelles such as mitochondria and lipofuscin pigments. A similar
prominent accumulation of autolysosomes and lipofuscins has been reported, in
some pathological states. In Alzheimer’s disease, CB and CD positive lysosomes
containing lipofuscin pigments were markedly increased in cortical pyramidal
T. Amano et al. / Mechanisms of Ageing and Development 83 (1995) 133- 141
140
neurons in the absence of atrophic, chromatolytic or neurofibrillary changes [6,18].
CD-positive lipofuscin granules have been shown to dramatically increase in
affected spinal cord neurons of the motor neuron degeneration mouse, a mutant
strain of which motor neurons degenerate only in late adulthood [18]. In the sciatic
nerve of the rat, axonal degeneration also corresponded to sites of the accumulation
of CD and secondary lysosomes [22]. Taken together, the age-related redistribution
of cathepsins in the TG neurons is closely associated with the increased formation
of autolysosomes and lipofuscins which is the most ubiquitous age-related cytological alteration [23]. Furthermore, resultant accumulation of such autolysosomes is
likely to sterically interfere with cell functions such as intracellular vesicular
transport.
The reason for the age-related decrease in the activity levels of CB and CL but
not CD in the TG remains unknown. However, endogenous cysteine proteinase
inhibitors such as cystatins B and C may be also increased and accumulated in the
TG neurons with advancing age, since the cellular level of calpastatin, an endogenous inhibitor of calpain, is known to increase in the aged rat brain [ll].
Interestingly, injection of leupeptin, a cysteine proteinase inhibitor, or chloroquine,
a general lysosomal inhibitor, into young rat brains was found to induce the
formation of lysosome-associated granular aggregates which closely resemble the
ceroid-lipofuscin [8]. Furthermore, age-related change in CB activity was not found
in the rat brain, while CL activity was significantly decreased with advancing age
[17]. Therefore, reduced activities of cysteine proteinases are considered to be
closely related with the formation of lipofuscin. On the other hand, CD activity in
the TG showed no age-related change, whereas CD activities in both human and
rat brains have been reported to increase with aging by two-fold [l, 10, 14, 15, 171.
The major factor responsible for this discrepancy between the TG and the brain
may be due to difference in cellular constituents between the two different tissues,
since the increased activity of CD with aging in the brain is considered to be mainly
associated with glial proliferation [ 171.
Acknowledgment
This research was partially supported
Education, Science and Culture, Japan.
by Granti-in-Aid
from The Ministry of
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