Changes in oxidative stress parameters and neurodegeneration markers

in the brain of the senescence-accelerated mice SAMP-8

Francesc X. Sureda a,*, Javier Gutierrez-Cuesta b, Marta Romeu a, Miquel Mulero a,

Anna Maria Canudas b, Antoni Camins b, Jordi Mallol a, Merce Pallas b

a Unitat de Farmacologia, Facultat de Medicina i Ciencies de la Salut, Universitat Rovira i Virgili, c./St. Llorenc 21, E-43201 Reus, Tarragona, Spainb Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Universitat de Barcelona, Nucli Universitari de Pedralbes, E-08028 Barcelona, Spain

Received 28 November 2005; received in revised form 26 January 2006; accepted 31 January 2006

Available online 20 March 2006

Abstract

The senescence-accelerated strains of mice (SAMP) are well-characterized animal models of senescence. Senescence may be related to

enhanced production or defective control of reactive oxygen species, which lead to neuronal damage. Therefore, the activity of various oxidative-

stress related enzymes was determined in the cortex of 5 months-old senescence-accelerated mice prone-8 (SAMP-8) of both sexes and compared

with senescence-accelerated mice-resistant-1 (SAMR-1). Gluthatione reductase and peroxidase activities in SAMP-8 male mice were lower than

in male SAMR-1, and a decreased catalase activity was found in both male and female SAMP-8 mice, which correlates with the lower catalase

expression found byWestern blotting. Nissl staining showed marked loss of neuronal cells in the cerebral cortex of five months-old SAMP-8 mice.

SAMP-8 mice also had marked astrogliosis and microgliosis. We also found an increase in caspase-3 and calpain activity in the cortex. In addition,

we observed morphological changes in the immunostaining of tau protein in SAMP-8, indicative of a loss of their structural function. Altogether,

these results show that, at as early as 5 months of age, SAMP-8 mice have cytological and molecular alterations indicative of neurodegeneration in

the cerebral cortex and suggestive of altered control of the production of oxidative species and hyper-activation of calcium-dependent enzymes.

q 2006 Elsevier Inc. All rights reserved.

Keywords: Senescence; Oxidative stress; Catalase; Calpain; Tau; Neurodegeneration

1. Introduction

The study of the ageing process is an important area of

research. There is growing interest in establishing the

molecular targets that contribute to the neurobiology of ageing

and, ultimately, in developing drugs that could modulate the

activity of such targets and mitigate the multi-organic

dysfunction that is the common feature of ageing. To better

understand the ageing process, several animal models have

been developed. Among them, the senescence-accelerated

mice (SAM) model is widely used in experimental gerontol-

ogy. The SAM strain was generated in the mid-1970s by

Takeda and colleagues at the University of Kyoto, and is

characterized by a reduced life span and the presence of early

signs of senescence (lordokyphosis, loss of activity, neuro-

logical signs, among others). The senescence-accelerated

0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2006.01.015

* Corresponding author. Tel.: C34 977 75 93 57; fax: C34 977 75 93 78.

E-mail address: francesc.sureda@urv.net (F.X. Sureda).

mice-prone (SAMP) model and its equivalent with normal

ageing, the senescence-accelerated mice-resistant (SAMR)

model, are both established animal models for studying ageing

and age-associated pathologies.

A number of studies using different strains of SAM mice

indicate that alterations in the control of oxidative stress are

responsible for this accelerated ageing (for a review, see

Hosokawa, 2002). For instance, parameters indicative of lipid

or protein oxidation are greater in SAMP-8 than in SAMR-1

(Kim et al., 2002), and it has been suggested that changes in the

activity of several key enzymes account for this enhanced

oxidative stress (Liu and Mori, 1993). Based on these

observations, it has been postulated that antioxidant com-

pounds would have beneficial effects as anti-ageing com-

pounds, and several studies have been carried out to ascertain

this. In fact, melatonin reduces oxidative damage in the brain of

SAMP-8 mice (Okatani et al., 2002). Alpha-lipoic acid or

N-acetylcisteine have also shown to reverse memory impair-

ment in aged SAMP-8 (Farr et al., 2003), and Boldyrev and

coworkers found an enhanced life span in SAMP-1 treated with

the antioxidant carnosine (Stvolinskii et al., 2003).

Experimental Gerontology 41 (2006) 360–367

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F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367 361

However, the search for a particular key step in the

oxidative stress defence machinery has been inconclusive.

Although several authors point to an alteration of a particular

enzyme that might be responsible for increased oxidative

stress, others found transient changes or no change at all.

Apart from this, no studies have been carried out to ascertain

gender differences in enzyme activity or expression in these

strains. Whatever the cause of increased oxidative stress, a

number of publications have described changes indicative of

neuronal cell death in the brain of these strains of mice.

Extensive literature describes the involvement of caspases

(cysteine proteases) in neuronal cell death. In addition, several

studies performed in post-mortem human brain tissues suggest

the participation of these enzymes in neurodegenerative

processes and in ageing (Maccioni et al., 2001). Another

class of cysteine proteases involved in neuronal cell death is

the calcium-activated protease, calpain, with up to 12

isoenzyme forms (Nixon, 2003). The two most widely

distributed isoforms, calpain I (m-calpain) and calpain II (m-

calpain), have nearly identical substrate specificities and, so

far, have been shown to differ mainly in their calcium

requirements for activation in vitro and their tissue distri-

butions. By making selective and limited proteolytic

cleavages, they modulate the activity of enzymes, including

key signalling molecules, and induce specific cytoskeletal

rearrangements, which accounts for their roles in cell motility,

signal transduction, vesicular trafficking and structural

stabilization. Enhanced calpain activation is involved in

several ageing phenomena and diseases of late life, including

cataract formation, erythrocyte senescence, type-2 Diabetes

mellitus, hypertension, arthritis, and neurodegenerative dis-

orders. Increased calpain activation could be part of the

neurodegenerative pathway process in several neuro-

degenerative disorders such as Parkinson’s, Alzheimer’s and

Huntington’s diseases (Nixon et al., 1994). Calpains directly

or indirectly activate cdk5 and ERK/MAP kinase (Nath et al.,

2000; Patrick et al., 1999), which phosphorylate tau and

potentially promote tau-related cytoskeletal disorganization,

as well as reducing neuronal survival by other mechanisms

(Delacourte et al., 2002; Zhu et al., 2004). The overactivation

of calpains, in particular, has figured prominently in

hypotheses on cellular ageing beginning with the observation

that levels of calpain activity in the brain correlate inversely

with lifespan in across several orders of mammals (Nixon,

2003). Consistent with observations of increased intracellular

or calcium influx in ageing tissues, calpain activities also rise

during ageing (Benuck et al., 1996) The basis for high calpain

activity in ageing is, most commonly, increased activation

without any change in overall expression (Averna et al., 2003;

Nixon, 2003).

The aim of this study was to establish differences in several

key enzymes in the regulation of oxidative stress between

SAMP-8 and SAMR-1 in 5 months-old mice. Moreover, we

have established new features that are indicative of neuronal

damage and that shed light on the underlying causes of

accelerated ageing in this animal model.

2. Materials and methods

2.1. Animals

Senescence-accelerated mice prone-8 (SAMP-8) and senes-

cence-accelerated mice resistant-1 (SAMR-1) were provided

by Harlan Interfauna Iberica (Barcelona, Spain). All mice were

housed in a room equipped with automatic light cycles (12 h

light/dark), and maintained at 22G2 8C and relative humidity

of 40–60%. Food (Panlab rat chow, Panlab, Barcelona) and tap

water were offered ad libitum throughout the study.

2.2. Sample processing

For enzyme activity assays, 5 months-old mice were

anesthetized with a mixture of xylazine and ketamine (i.p. 10

and 100 mg/kg, respectively) dissolved in 0.2 M phosphate

buffered saline. Immediately after death, the brain was

removed and cerebral cortices were dissected on an ice–cold

surface. After homogenization in 0.2 M cold sodium

phosphate buffer (1:4) at pH 6.25 in a Potter–Elvehjem

homogenizer fitted with a teflon pestle, the crude soluble

fraction was obtained (centrifugation at 1,05,000!g for

60 min) and the pellet was discarded. The fresh crude soluble

fraction was divided to determine the parameters described

below. An aliquot was reserved for the glutathione-S-

transferase (GST) activity assay, which was conducted the

same day as the sample processing. The remainder was

distributed in different aliquots and maintained at K80 8C

until the day of assay.

For Western blot, caspase-3 and calpain activity determi-

nations the cortices were quickly excised and weighted.

Afterwards, they were homogenized using a Polytron

(Kinematica) in 10 volumes of cold buffer (0.32 sucrose and

5 mM tris–HCl, pH 7.4). Homogenates were centrifuged twice

at 15,000!g for 30 min. Finally, the pellet was dissolved in

50 mM Tris–HCl buffer, pH 7.4, with a Potter–Elvejhem

homogenizer.

Protein was determined with Bradford method, with using

bovine serum albumin used as standard (Merck, Germany).

For immunohistochemistry studies, mice were anesthetized

with sodium pentobarbital (75 mg/kg, i.p.) and exsanguinated

with 25 ml cold 0.1 M phosphate-buffered saline, pH 7.4 (PBS)

followed by fixation with 25 ml 4% paraformaldehyde in 0.1 M

(PBS), pH 7.4. Brains were removed and postfixed overnight in

4% paraformaldehyde and then cryoprotected in 20% sucrose

in 0.1 M PBS. The fixed brains were coronally cut into 30-mmthick sections on a cryostate and stored in 4% paraformalde-

hyde plus sodium azide (0.2%) at 4 8C.

2.3. Enzyme activity assays

Glutathione-S-transferase (GST) activity was determined

immediately after centrifugation, using the method of Habig et

al. (1975) and with CDNB (1-chloro-2, 4-dinitrobenzene) as a

substrate. As CDNB is a non-specific substrate, total GST

activity was determined.

F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367362

Glutathione reductase (GR) and glutathione peroxidase

(GPx) activities were determined by the Wheeler et al. (1990)

method in a COBAS–MIRA autoanalyzer (Roche Diagnostics)

and expressed in units/g of mg protein (one unit, UZ1 mmol of

NADPH transformed/min).

Catalase (CAT) activity was determined by the Cohen et al.

(1970) method, by monitoring the rate of disappearance of

hydrogen peroxide (15 mM) in a Lambda 2 PerkinElmer

spectrophotometer. Values are expressed in terms of units/mg

of protein (one unit, UZ1 mmol of hydrogen peroxide

transformed/min).

Caspase-3 activity was determined by using the colorimetric

substrate Ac-DEVD-p-nitroaniline, a synthetic substrate of

caspase-3. Briefly, the protein aliquots were incubated with

200 mM of Ac-DEVD-p-nitroaniline in assay buffer in 96-well

plates at 37 8C for 24 h. Absorbance of the cleaved product was

measured at 405 nm in a microplate reader (BioRad). Results

are expressed as arbitrary units of absorbance.

Calpain activity was measured by using a commercial kit

(MBL International). In brief, fluorimetric calpain substrate

Ac-LLY-AFC was used as follows: cortex samples were

collected in a lysis buffer and 0.5 mg/ml of protein was

incubated with 200 mM of fluorometric substrate in assay

buffer in 96-well plates at 37 8C for 24 h. Negative control was

through using the calpain inhibitor Z-LLY-FMK. The

fluorescence intensity of the liberated AFC was monitored by

using a microplate spectrofluorometer (PerkinElmer Victor 3)

at an excitation wavelength of 400 nm and an emission

wavelength of 505 nm. Calpain activity is expressed as

Relative Fluorescence Units (RFU) per milligram of protein.

2.4. Western-blot analysis

Aliquots of tissue homogenate, containing 30 mg (catalase

and cdk5) or 5 mg (spectrin) of protein per sample, were placed

in sample buffer (0.5 M Tris–HCl pH 6.8, 10% glycerol, 2%

(W/V) SDS, 5% (V/V) 2-b-mercaptoethanol, 0.05% bromo-

phenol blue) and denaturised by boiling at 95–100 8C for

5 min. Samples were separated by electrophoresis on 10%

acrylamide gels. Subsequently, proteins were transferred to

polyvinylidene fluoride (PVDF) sheets (Immobilone-P,

Millipore Corp.) by means of a transblot apparatus (BioRad).

Fig. 1. Determinations of the activities of glutathione-S-transferase (GST), glutath

cerebral cortex of male or female SAMR-1 (open bars) and SAMP-8 mice (black bar

gender, different strain. **p!0.01 vs. same gender, different strain. ##p!0.01 vs.

Membranes were blocked overnight with 5% non-fat milk

dissolved in TBS-T buffer (Tris 50 mM; NaCl 1.5%; Tween 20,

0.05%, pH 7.5). Membranes were then incubated with primary

mouse monoclonal antibodies against catalase, cdk5 and

a-spectrin (1:1000, Santa Cruz Biotechnology), After 90 min,

blots were washed thoroughly in TBS-T buffer and incubated

for 1 h with a peroxidase-conjugated anti-mouse IgG antibody

(Amersham Corp., Arlington Heights, IL). Immunoreactive

protein was viewed by a chemiluminiscence-based detection

kit, according to the manufacturer’s protocol (ECL kit;

Amersham Corp.). Digital images were taken with a Chemidoc

XRS system (Biorad), which permits semiquantitation of band

intensity.

2.5. Immunohistochemistry

Free-floating Immunohistochemistry was performed with

the following antibodies, at the concentration indicated in

parentheses: OX-6 (1:1000) (Serotec) a mouse monoclonal

antibody recognizing MHC class II antigens that is used as a

marker of reactive microglia/macrophages, a monoclonal

antibody against GFAP (1:500) (Sigma-Aldrich), a monoclonal

antibody against NeuN (1:400) (Dako) and anti Tau [pSer422]

(1:500) (Biosource). Briefly, each section was preincubated

with 3% normal goat serum followed by treatment with 0.3%

hydrogen peroxide in phosphate-buffered saline, each for

30 min at room temperature. Then, sections were washed in

T-PBS (phosphate buffered saline containing 0.5% Triton-

X100) and incubated overnight at 4 8C with the primary

antibody. After rinsing, the sections were incubated with

biotinylated goat anti-rabbit IgG (Vector) for 2 h, followed by

ABC-peroxidase complex (Vector) for 2 h at room tempera-

ture. After rinsing, the immunolabel could be seen with 0.015%

diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and

0.003% hydrogen peroxide in 50 mM Tris/HCl buffer (pH

7.6). To assess tissue architecture, sections from each fixed

brain were stained following the Nissl method.

Double immunohistochemistry was carried out by incu-

bation of the sections with a second primary antibody, as

described previously (Planas et al., 1998). The first immunor-

eaction was designed to label astroglia with GFAP; and the

second reaction was for neurons with the monoclonal antibody

ione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) in the

s). Each value represents meanGSD from 3–8 experiments. *p!0.05 vs. same

same strain, different gender.

F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367 363

against NeuN. After extensive washing following the first

immunoreaction, sections were incubated with 3% normal

horse serum for 2 h to block nonspecific binding sites for NeuN

immunohistochemistry. Sections were incubated overnight at

4 8C with the corresponding primary antibody, followed by

treatment with biotinylated secondary antibody (1:200), and

the ABC complex. Finally, sections were washed in 0.01 M

sodium phosphate buffer, pH 6, preincubated in 0.01%

benzidine dihydrochloride and 0.025% sodium nitroferricya-

nide in 0.01 M sodium phosphate buffer for 10 min and then

developed in the same solution containing 0.005% of H2O2.

3. Results

3.1. Changes in activity and expression of oxidative-stress

related enzymes

GST activity does not differ between SAMP-8 mice and

SAMR-1 mice. However, we found a statistically significant

Fig. 2. Representative images of 3–4 experiments of Nissl staining, NeuN, GFAP, an

Arrows indicate representative positive cells in each experiment.

difference in GST activity between males and females in

SAMR-1 mice. Specifically, GST activity in male SAMR-1

mice was approximately half than that of females. However, no

other differences were found for this enzyme. Male SAMP-8

mice had lower GR (60% less) and GPx activity (74% less)

than SAMR-1, but this difference was not found in females.

CAT activity was the only enzyme that showed a consistently

reduced activity in both SAMP-8 male (35% decrease) and

female (29%) mice than in SAMR-1 (Fig.1).

To ascertain whether so little activity was due to less enzyme

expression, aWestern blot analysis was performed. As shown in

Fig. 3A a significantly reduced CAT expression was found in

both male and female SAMP-8 mice than in SAMR-1 mice.

3.2. Immunohistochemistry and histological study with Nissl

staining

To assess the presence of neurodegenerative features, Nissl

staining and immunostaining with neuronal and glial markers

d OX6 immunohistochemistry in cerebral cortex of male SAMR-1 and SAMP-8.

F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367364

were performed. By Nissl staining, consistent neuronal

degeneration with loss of cerebral layers was observed in

SAMP-8 cerebral cortex, but not in the SAMR-1 mice tissues

(Fig. 2A). In the immunohistochemical study, the SAMR-1

group showed very little or no presence of astroglial and

microgial cells, whereas the SAMP-8 group showed obvious

astrogliosis, with astrocytes surrounding vessels (Fig. 2B and

C), and also microgliosis (Fig. 2D). In addition, neurofibrilar

tangles in cortical neurons immunostained with anti-Tau

[pSer422] are more clearly seen in SAMP-8 than in SAMR-1,

which indicates the activation of the enzymatic pathway

involved in the hyperphosphorylation of tau protein (Fig. 4 A

and B).

3.3. Activation of cysteine proteases

Obvious calpain-induced proteolysis of a-spectrin (SBPD

150 kDa fragment) in the cerebral cortex of SAMP-8 was

observed by Western blot, apparently accompanied by a

slightly greater caspase-3 activity (SBPD 120 kD) than in

SAMR-1 (Fig. 3B). An increase in calpain activity was also

Fig. 3. (A) Left panel: Representative Western blot experiment of the expression of

Right panel: Bar chart showing semi-quantitative optical density (arbitrary units of b

SBDP, characteristic of calpain activation and the indetectable band of 120 kDa

fluorimetrically as indicated inMethods (RFU: relative fluorescence units), and caspas

cortex of male or female SAMR-1 (open bars) and SAMP-8mice (black bars). Each p

gender, different strain.

observed in a biochemical assay. A slightly greater amount, but

statistically significant, was noted in both genders of SAMP-8

mice than in SAMR-1. However, no differences were found

when the caspase-3 activity was measured. (Fig. 3C).

3.4. Cdk5 expression increase and phosphorylation of tau

protein

According to previous results of our laboratory, an increase

in cdk5 expression was observed in both male and female

SAMP-8 comparing to SAMR-1 (Fig. 4A). Moreover, a change

in the cellular localization of tau was observed in immunohis-

tochemical studies (Fig. 4B). In SAMR-1 cerebral cortex this

protein is mainly localized in the axons of neurons, whereas in

SAMP-8 the staining is localized in neuronal bodies.

4. Discussion

The SAMP strains (Takeda et al., 1981, 1997) are unique

and useful animal models for ageing studies, since they show

‘accelerated senescence’ as an inherited phenotype, unlike the

catalase in the cerebral cortex of male and female SAMR-1 and SAMP-8 mice.

lot bands). (B) Protein immunoblot studies of a-spectrin breakdown in 145 kDa

SBDP characteristic of caspase-3 activation. (C) Calpain activity measured

e 3 activitymeasured colorimetrically (absorbance arbitrary units) in the cerebral

oint is the meanGSEM of 3–5 animals, tested in duplicate. ** p!0.01 vs. same

Fig. 4. (A) Left panel: Western blot showing the increase in cdk5 expression in the cerebral cortex of male and female SAMP-8 vs. SAMR-1 mice. Right panel: Bar

chart showing semi-quantitative optical density (arbitrary units of blot bands). (B) Representative images of 3–4 experiments of Tau [pS422] immunohistochemistry

in cerebral cortex of SAMR-1 (mainly axonal staining) and SAMP-8 (neuronal body staining) males. Arrows indicate the axonal staining in SAMR-1 and the

neuronal cell body staining in SAMP-8.

F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367 365

SAMR strains, which do not show accelerated senescence.

While maintained as an inbred strain, several lines of mice

showed accelerated senescence such as loss of activity, hair

loss, lack of glossiness, skin coarseness, and shortened life

span. The other noteworthy feature is that each SAMP strain

has various age-associated and strain-specific pathologic

phenotypes (Takeda et al., 1997). These age-associated

diseases include senile amyloidosis in SAMP-1, 2, 7, 9, 10

and 11 (Higuchi et al., 1991); impaired immune response in

SAMP-1, 2 and 8 (Hanada et al., 1991; Hosokawa et al., 1987);

deficits in learning and memory in SAMP-8 (Yagi et al., 1988,

1989, 1998); cataracts in SAMP-9 (Hosokawa et al., 1988,

1993); and brain atrophy in SAMP-10 (Shimada et al., 1993).

Despite the general consensus among researchers pointing

to increased oxidative stress as the main mechanism

responsible for the accelerated senescence that is observed in

SAMP mice (Hosokawa, 2002; Mori et al., 1998), published

data concerning the activity of oxidative-stress related

enzymes in SAMP mice are still controversial. Although

other mechanisms could be responsible for such an increase, it

seems plausible that this increased oxidative status could be the

result of a decreased activity of a particular enzyme normally

involved in the control of oxidative stress. However, although

several groups have investigated this aspect thoroughly, not a

single enzyme has been identified as a responsible for increased

oxidation of proteins and lipids. Sato and co-workers have

found an early decrease in the activity of CAT in SAMP-8

mice, but not in GPx or superoxide dismutase (SOD) activities

(Sato et al., 1996). Boldyrev and coworkers (Stvolinskii et al.,

2003) found less Cu/Zn-SOD activity in the brain of aged

SAMP-1 mice, while other authors have found greater brain

SOD activity in the SAMP strain of mice than in ddY mice (Liu

and Mori, 1993; Okatani et al., 2002). Okatani and colleagues

also found a decrease in glutathione peroxidase (GPx) activity

in 12 months-old SAMP-8 mice, but not in younger mice (3- or

6-months old).

Apart from the discrepancies between studies by different

laboratories, which could be due to the different methods of

assay, gender may act as another confusing factor, since

researchers generally use male mice or both genders without

distinction. This is the first study of gender oxidative-stress

related enzymes in which gender differences were established.

Interestingly, GR and GPx activities in male 5 months-old

SAMP-8 mice were lower than in SAMR-1, but this difference

has not been found in female mice. Using a pooled population

of both genders (Okatani et al., 2002), there was less GPx

activity in 12 months-old SAMP-8 mice, but not in 6 months-

old ones. On CAT activity, our results are consistent with

findings published by Sato (Sato et al., 1996). Decrease in the

activity of a particular enzyme can be due to several factors,

including a constitutive lower expression and post-transcrip-

tional alterations. In fact, the activity of glutamine synthase is

used as a marker of oxidative stress, since excessive reactive

oxygen species evokes molecular changes in the enzyme that

affect its activity (Butterfield et al., 1998). As well as less CAT

activity in SAMP-8 mice, our Western blot study found less

CAT expression in male and female SAMP-8 mice than

SAMR-1 mice. Thus, we hypothesize that a decrease in CAT

expression may well be responsible for more oxidative stress

through a deficient control of the detoxification of reactive

oxygen species. At this point, it is interesting to note that other

investigators have suggested that overexpression of antioxidant

F.X. Sureda et al. / Experimental Gerontology 41 (2006) 360–367366

enzymes, such as Cu/Zn superoxide dismutase (Cu/Zn SOD)

might be responsible for the delayed ageing that is observed in

the dwarf mice (Hauck and Bartke, 2000). These researchers

also found greater hypothalamic CAT activity, but failed to

demonstrate greater CAT expression. However, other mech-

anisms cannot be ruled out: Poon et al. have suggested that the

b-amyloid peptide may increase oxidative stress in the brain of

SAMP-8 mice, since the intracerebroventricular administration

of antisense oligonucleotides of the APP gene reduces lipid

peroxidation and protein oxidation (Poon et al., 2004).

We found signs of gliosis and neurodegeneration in the brain

of SAMP-8, which was consistent with the oxidative stress

features. Immunohistochemistry techniques revealed important

neuroglial changes at as early as 5 months of age in the SAMP-8

strain of mice. There was a significant increase in reactive

astrocytes and microgliosis is demonstrated, according to other

previous reports on the SAM strain (Katoh-Semba and Kato,

1994). The loss of cellular bodies and cerebral cortex structures

are evident in the SAMP-8 at 5 months andmay be due in part to

oxidative stress and glial reactivity but other mechanisms

cannot be ruled out. Although recent reports indicate that

neuronal death in the SAMP-8 hippocampus is not mediated by

the intrinsic apoptotic pathway (Wu et al., 2005), the

participation of other proteases has not been studied till now.

Here we demonstrate the participation of other proteases,

calpains, in the neuronal death involved in senescence in

SAMP-8. Once activated, calpains and caspases degrade the

cytosketal protein a-spectrin (280 kD) into three main

fragments of 145 kDa (calpains), 120 kDa (caspase-3) and

150 kDa (calpains-caspases). Both the presence of a-spectrinbreakdown products (SBDP 150 kDa) and the increased activity

of this enzyme demonstrate a solid relationship between

neuronal death and ageing in this model of senescence.

Calpain is activated by increased intracellular calcium

concentrations (Nixon et al., 1994). Our results indicate that in

SAMP-8 there are certain biochemical changes, probably

through a deficient control of ROS production causing a rise in

the intracellular calcium concentration, thus increasing the

calpain activation in the cortex. Several mechanisms for

explaining the intracellular calcium concentration that would

lead to calpain activation have been put forward. For example,

it has been reported that increased cytosolic ROS causes the

calcium concentration in the mitochondrial matrix to increase,

which could interfere with the respiratory chain and in the

tricarboxylic acid cycle. This would promote the opening of the

mitochondrial permeability transition pore, thus releasing

proapoptotic factors into the cytosol (Wu et al., 2005).

Moreover, the neuronal calcium buffering capacity might

also be impaired due to an age-dependent decrease in calbindin

(Armbrecht et al., 1999).

Recent findings from our laboratory demonstrate that there

is a hyperphosphorylation of the microtubule-associated

protein tau in SAMP-8 at 5 months-old (Canudas et al.,

2005). Calpains are activated in neuronal processes before

abnormalities in the protein tau are detected. In a latter phase,

the activated protease associates prominently with abnormal

neuronal tau aggregates (neuropil threads and neurofibrillary

tangles), a hallmark of Alzheimer’s disease neuropathology

(Avila et al., 2004). It has been suggested that calpain breaks

cdk5/p35 down into cdk5/p25, which is followed by an

increase in cdk5/Gsk3ß activity, thus inducing post-transla-

tional modifications in tau (Baumann et al., 1993; Canudas

et al., 2005; Cho and Johnson, 2003) and neuronal cell death in

the cerebral cortex. The activation of cdk5/p25 activity was

demonstrated in 5 months-old SAMP-8 accompanied by tau

modifications (Canudas et al., 2005). Here we report an

alteration in an upstream step (increased calpain activity), that

would lead to cdk5 activation and, consequently, to tau protein

alterations. As it is illustrated by the immunohistochemical

studies against anti-Tau [pSer422], the typical staining of as

drop depositions can be seen, thus indicating a loss of the

original structure. Moreover, other age-related alterations in

several tissues have been attributed to increased calpain

activity, including cataract formation or characteristic

reductions in the major erythrocyte transmembrane and ion

transport protein in red cells (Hosokawa et al., 1984, 1988;

Nixon et al., 1994; Takeda et al., 1981; Takeda et al., 1991).

Ageing-induced cell death may be caused through altera-

tions of mitochondrial function, and in fact, our results

corroborate the hypothesis by which a defective expression

of CAT or other oxidative stress-related enzymes (namely

superoxide dismutase) in the cerebral cortex, may lead to

mitochondrial alterations an greater activity of proteases that

would enhance cdk5/Gsk3b thus induces an increase in tau

phosphorylation, neurofibrilar tangles formation and gliosis,

cell loss in cerebral cortex and the functional decline during

ageing in SAMP-8.

Acknowledgements

This study was made possible by a grant from the Spanish

‘Instituto de Salud Carlos III (ISCIII)’, through the G03/137

project, by grants SAF2005-05179-C02-01 from Ministerio de

Educacion y Ciencia, European Funds. We are grateful to

Vanessa Sanchez–Martos and Ana Isabel Dıez for their

excellent technical assistance. We thank Almirall–Prodesfarma

for Pharmacology Award 2005.

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