Age-related changes in rat cerebral occludin and zonula occludens-1(ZO-1)

Arshag D. Mooradian *, Michael J. Haas, Joe M. Chehade

Department of Internal Medicine, Division of Endocrinology, Diabetes and Metabolism, St. Louis University School of Medicine, 1402 South Grand

Blvd., St.Louis, MO 63104, USA

Received 7 January 2002; received in revised form 8 March 2002; accepted 11 March 2002

Abstract

The endothelial or epithelial tight junctions create a rate-limiting barrier to diffusion of solutes. A major determinant of the

barrier function is the density of tight junction proteins. Since aging is associated with significant alterations in the blood�/brain

barrier (BBB) it is possible that specific tight junction proteins may be altered in the cerebrum of aging rats. To test this hypothesis,

Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1)

proteins and their mRNA in cerebral tissue of 3-, 12- and 24-month-old rats. The cerebral occludin content in 24-month-old rats

(732.59/99.9 arbitrary units) was significantly reduced compared to 12-month-old rats (1043.49/131.8) or 3-month-old rats

(1021.49/62.8), P B/0.01. The cerebral ZO-1 protein content in 24-month-old rats (161.79/8.1 arbitrary units) and 12-month-old rats

(144.39/35.9) were not significantly reduced compared to 4-month-old rats (189.09/27.2). The occludin mRNA content relative to

G3PDH mRNA was 1.119/0.05, 1.119/0.07 and 1.009/0.05 in 3-, 12- and 24-month-old rats, respectively. The differences did not

achieve statistical significance. The ZO-1 mRNA content of cerebral tissue relative to G3PDH mRNA was significantly increased in

24-month-old rats compared to 3-month-old rats (1.2809/0.030 vs. 0.9569/0.038), P B/0.001. It is concluded that aging in rats may

alter the molecular anatomy of the BBB by altering the content of select structural proteins of tight junctions.

# 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Tight junctions; Blood�/brain barrier; Aging; Occludin

1. Introduction

Aging in experimental animals and humans is asso-

ciated with significant structural and functional altera-

tions in the blood�/brain barrier (BBB) (Mooradian,

1994, 1998; Shah and Mooradian, 1997). The precise

biochemical basis of these alterations is not known.

Previously published studies have reported age-related

changes in the lipid and protein composition of cerebral

microvessels (Mooradian and Meredith, 1992; Moora-

dian and Smith, 1992; Mooradian and Uko-eninn,

1995). The molecular anatomy of the tight junctions

that characterize the BBB has been the focus of several

studies.The endothelial tight junctions create a rate-limiting

barrier to diffusion of solutes (Mitic and Anderson,

1998). The permeability of this barrier varies depending

on the tissue. The tight junctions of the BBB have very

limited permeability compared to the endothelial cells of

non-neural tissue, where the tight junctions are prone to

leakage (Mitic and Anderson, 1998). This large differ-

ence in permeability may be in part related to the level of

expression of tight junction structural proteins such as

occludin and zonula occludens-one (ZO-1) (Hirase et al.,

1997; Fruse et al., 1993). Since aging in rats is associated

with significant alterations in BBB function (Moora-

dian, 1994, 1998; Shah and Mooradian, 1997), it is

* Corresponding author. Tel.: �/1-314-577-8458; fax: �/1-314-773-

4567.

E-mail address: mooradad@slu.edu (A.D. Mooradian).

Mechanisms of Ageing and Development 124 (2003) 143�/146

www.elsevier.com/locate/mechagedev

0047-6374/03/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 0 4 7 - 6 3 7 4 ( 0 2 ) 0 0 0 4 1 - 6

possible that specific tight junction proteins in cerebral

tissue may be altered with age. To test this hypothesis

Western and Northern blot analysis were carried out to

measure the steady-state levels of occluding and ZO-1protein and mRNA levels in cerebral tissue of rats at 3,

12 and 24 months of age.

2. Materials and methods

2.1. Animals

Male Fischer 344 rats at 3, 12 and 24 months of agewere obtained from Harlan Industries (Indiana). The

rats were kept in the animal facilities for 2 weeks before

the experiment. The rats were killed by exsanguination

through the abdominal aorta under light pentobarbital

anesthesia (45 mg/kg). The animals were inspected for

gross pathology and those bearing tumors or those with

renal failure (serum creatinine �/1.5 mg/dl) were

excluded from the study.

2.2. Materials

The polyclonal rabbit antioccludin and anti ZO-1

antisera were obtained from Zymed Laboratories Inc.

(South San Francisco, CA., Cat. # 71-1500 and # 61-

7300, respectively). The mouse occludin cDNA was a

gift from Dr Shoichiro Tsukita, of the Kyoto University,Kyoto, Japan (Ando-Akatsuka et al., 1996). The cDNA

insert was isolated subsequent to digestion of the

plasmid pSK-MOC (Ando-Akatsuka et al., 1996) with

the restriction endonuclease NotI. The full-length hu-

man ZO-1 cDNA was obtained from the American

Type Culture Collection (ATCC, Manassas, VA). The

probes were labeled to high-specific activity with the

Rapid-Prime Oligolabeling kit (Amersham-PharmaciaBiotech, Arlington Heights, IL). All other reagent grade

chemicals were purchased from either Sigma Chemical

Company or Fisher Scientific Company (Pittsburgh,

PA).

2.3. Western blot analysis

Cerebral tissue (500 mg) was homogenized in 9.5 mlof phosphate buffered saline. The protein concentration

was determined with the method described by Lowry et

al. (1951). Cerebral protein samples (10 mg) were

electrophoresed in a 10% sodium dodecyl sulfate

(SDS)-polyacrylamide gel (Laemmli, 1970), and trans-

ferred electrophoretically to a nitrocellulose membrane

(Towbin et al., 1979). The membrane was incubated

with anti-occludin or anti ZO-1 primary antibody at afinal concentration of 1 mg/ml for 2 h at room

temperature. Horseradish peroxidase-linked goat anti -

rabbit IgG was used at a final dilution of 1:10,000 at

room temperature. Blots were developed using the

enhanced chemiluminescence (ECL) reagents (Amer-

sham-Pharmacia Biotech, Chicago, IL) as described by

the manufacturer. The tissue content of occludin andZO-1 was determined by densitometry using the perso-

nal densitometer purchased from Molecular Dynamics

(Sunnyvale, CA).

2.4. Northern blot analysis

Total RNA was isolated from the cerebral tissue using

a single-step acid guanidium phenol�/chloroform extrac-

tion procedure (Chirgwin et al., 1979). Aliquots of totalRNA (10�/15 mg) were separated electrophoretically on

a denaturing 1% agarose gel containing 2.2 M formal-

dehyde (Sambrook et al., 1989). The 18S- and 28S-

ribosomal RNA bands were visualized by ethidium

bromide staining to ensure equivalent loading of total

RNA in each lane. The fractionated RNA was trans-

ferred to a nylon membrane (Hybond, Amersham-

Pharmacia Biotech.) and either simultaneously or se-quentially probed with a 32P-labelled occludin or ZO-1

cDNA probe with a control cDNA probe for the

glyceraldehyde-3-phosphate dehydrogenase (G3PDH)

mRNA. The membrane was incubated with these probes

in Rapid Hyb (Amersham-Pharmacia Biotech) for 2 h at

65 8C. After washing the membranes under high

stringency conditions (0.1% SDS. 0.1�/ standard saline

citrate. 65 8C for 30 min), they were exposed to filmsfor 4�/6 h. To detect the occludin mRNA, the incubation

and washing of the mebrane was done at 55 8C. These

adjustments in hybridization conditions were necessary

to enhance occludin signal. The amount of hybridization

signal was quantified with a scanning laser densitometer,

(Molecular Dynamics, Sunnyvale, CA).

2.5. Statistical analysis

All the results are reported as mean9/SEM. Analysis

of variance (ANOVA) followed by the Student’s t-test

for paired variables was used to evaluate the statistical

significance of the differences. A P B/0.05 was consid-

ered the limit for statistical significance.

3. Results and discussion

Occludin and ZO-1 protein expression was examined

by Western blot analysis. A representative immunoblot

of cerebral proteins is shown in Fig. 1A. The expected 65

kDa occludin appears as a single band. The studies of

occludin concentrations measured in cerebral tissue

from 3-, 12- and 24-month-old rats are summarized inFig. 1B. The occludin content of cerebral tissue from 24-

month-old rats (732.59/99.9 arbitrary units) was sig-

nificantly reduced compared to 12-month-old rats

A.D. Mooradian et al. / Mechanisms of Ageing and Development 124 (2003) 143�/146144

(1043.49/131.8), and 3-month-old rats (1021.49/62.8)

(P B/0.01). In contrast, as shown in Fig. 2 the cerebral

tissue content of ZO-1 protein was not significantly

altered with age (189.09/27.2 vs. 144.39/35.9 vs. 161.79/

8.1 arbitrary units in 3-, 12- and 24-month-old rats,

respectively). The resolution of 10% polyacrylamide gel

is not sufficient to identify the two isoforms of ZO-1.

These isoforms have a small molecular mass difference

of only 80 residues (Balda and Anderson, 1993; Knissel

and Hartwig, 2000). The distribution of these isoforms

does not correlate with differences in junctional resis-

tance (Balda and Anderson, 1993; Knissel and Hartwig,

2000) and therefore the relative importance of each

isoform as a determinant of barrier function is not

known.

The lack of a significant change in ZO-1 protein

between 12 and 24 months of age when occludin is

reduced by approximately 30% suggests that the number

of capillaries and possibly the number of tight junctions

did not change with age.

Occludin and ZO-1 mRNA levels were examined with

Northern blot analysis. Representative Northern blots

for occludin, ZO-1 and G3PDH mRNAs are shown in

Fig. 3. The occludin mRNA content of cerebral tissue

relative to G3PDH mRNA in 24-month-old rats (1.009/

0.14) was not significantly altered compared to 12-

month-old (1.119/0.07) and 3-month-old rats (1.119/

0.05). However, the ZO-1 mRNA content relative to

G3PDH mRNA in 24-month-old rats (1.2809/0.030)

was significantly increased compared to 3-month-old

rats (0.9569/0.038) P B/0.001.

The biological significance of the observed changes in

cerebral content of tight junction proteins is not clear.

Previously published studies have found an age-related

increase in the leakiness of the BBB (Pappolla and

Andorn, 1987). However, some studies in aging rats

have not found a significant age-related change in BBB

permeability (Rapoport et al., 1979). Differences in

animal models and experimental techniques used may

account for some of the inconsistency in the literature.

In a recent study, we had found that cerebral occludin

content is reduced in experimental diabetes (Chehade et

Fig. 1. (A) A representative western blot of cerebral proteins from 3-

month-old (1�/3), 12-month-old (4�/6) and 24-month-old rats (7�/9).

The blots were incubated with a specific polyclonal antiserum for

occludin. The expected 65 kDa occludin band is shown and is

significantly reduced in 24-month-old rats. (B) The mean9/SEM of

occludin content of cerebral tissue from 3-, (n�/10), 12- (n�/10) and

24-month-old rats (n�/10). *P B/0.001 compared to 3-month-old rats.

Fig. 2. (A) A representative western blot of cerebral proteins from 3-,

12- and 24-month-old rats. The blots were incubated with a specific

polyclonal antiserum for ZO-1 protein. (B) The mean9/SEM of ZO-1

protein content of cerebral tissue from 3- (n�/10), 12- (n�/10) and 24-

month-old rats (n�/10). The differences were not significant.

Fig. 3. A representative Northern blot of cerebral tissue RNA from 3-,

12- and 24-month-old rats. The blots were simultaneously hyberdized

with occludin and G3PDH cDNA (A), or with ZO-1 and G3PDH

cDNA (B). The quantitation of the intensity of bands from various

experiments did not show significant age-related changes in either

occludin or mRNA, although ZO-1 mRNA was increased in 24 month

old rats.

A.D. Mooradian et al. / Mechanisms of Ageing and Development 124 (2003) 143�/146 145

al., 2002). Therefore, it is possible that some of the

changes in the cerebral tight junction proteins with age

are partly the result of age-related increase in glucose

intolerance (Mooradian, 1988). The aged rats used inthese studies were not tested for glucose tolerance.

However, it is noteworthy that the changes seen in

aging rats are not identical to those found in diabetic

rats. The ZO-1 mRNA content in diabetic rats were not

altered compared to control rats whereas the aging rats

had a significant increase in ZO-1 mRNA content. The

lack of a significant age-related change in cerebral ZO-1

protein despite an increase in ZO-1 mRNA contentsuggests that there are age-related changes in either

translational or posttranslational processing of ZO-1

protein.

A potential limitation of this study is that the total

cerebral tissue extracts rather than isolated microvessels

were studied. However, since the tight junction proteins

are more concentrated in the endothelial cell membranes

compared to other cellular elements in the brain it islikely that the changes observed in the cerebral tissue

reflect the changes in the microvessels.

These results taken together indicate that aging alters

the molecular anatomy of the tight junctions in cerebral

tissue. Since the BBB is enriched in tight junction

proteins it is likely that the age-related reduction in

cerebral occludin content has an important impact on

the functional and structural integrity of the BBB.

Acknowledgements

The authors thank Jian Ping Li for excellent technical

assistance. The work was supported in part by Harold

Braun Memorial Fund.

References

Ando-Akatsuka, Y., Saitou, M., Hirase, T., et al., 1996. Interspecies

diversity of the occludin sequence: cDNA cloning of human,

mouse, dog, and rat�/kangaroo homologues. J. Cell Biol. 133,

43�/47.

Balda, M.S., Anderson, J.M., 1993. Two classes of tight junctions are

revealed by ZO-1 isoforms. Am. J. Physiol. 264, C918�/C924.

Chehade, J.M., Haas, M.J., Mooradian, A.D., 2002. Diabetes-related

changes in rat cerebral occludin and zonula occludens-1 (ZO-1)

expression. Neurochem. Res. 27, 249�/252.

Chirgwin, J.M., Przybylz, A.E., MacDonald, R.J., Rutter, W.J., 1979.

Isolation of biologically active ribonucleic acid from sources

enriched in ribonuclease. Biochemistry 18, 5294�/5299.

Fruse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S., Tsukita,

S., Tsukita, S., 1993. Occludin: a novel integral membrane protein

localizing at tight junctions. J. Cell Biol. 123, 1631�/1633.

Hirase, T., Staddon, J.M., Saitou, M., Ando-Akatsuka, Y., Itoh, M.,

Furuse, M., Fujimoto, K., Tsukita, S., Rubin, L.L., 1997. Occludin

as a possible determanant of tight junction permeability in

endothelial cells. J. Cell Sci. 110, 1603�/1613.

Knissel, U., Hartwig, W., 2000. Tight junctions of the blood�/brain

barrier. Cell Mol. Neurobiol. 20, 57�/76.

Laemmli, U.K., 1970. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680�/685.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951.

Protein measurement with the folin phenol reagent. J. Biol. Chem.

193, 265�/275.

Mitic, L.L., Anderson, J.M., 1998. Molecular architecture of tight

junctions. Annu. Rev. Physiol. 60, 121�/142.

Mooradian, A.D., 1988. Tissue specificity of premature aging in

diabetes mellitus: the role of cellular replicative capacity. J. Am.

Geriatr. Soc. 36, 831�/839.

Mooradian, A.D., 1994. Potential mechanisms of the age-related

changes in the blood�/brain barrier. Neurobiol. Aging 15, 751�/755.

Mooradian, A.D., 1998. The effect of aging on the blood�/brain

barrier. Neurobiol. Aging 9, 31�/39.

Mooradian, A.D., Meredith, K.E., 1992. The effect of age on protein

composition of rat cerebral microvessels. Neurochem. Res. 17,

665�/670.

Mooradian, A.D., Smith, T.L., 1992. The effect of age on lipid

composition and order of rat cerebral microvessels. Neurochem.

Res. 17, 233�/237.

Mooradian, A.D., Uko-eninn, A., 1995. Age-related changes in the

antioxidative potential of cerebral microvessels. Brain Res. 67,

159�/163.

Pappolla, M.A., Andorn, A.C., 1987. Serum protein leakage in aged

human brain and inhibition of ligand binding at alpha2-adrenergic

and cholinergic binding sites. Synapse 1, 82�/89.

Rapoport, S.I., Ohno, I.K., Pettigrew, D., 1979. Blood�/brain barrier

permeability in senescent rats. J. Gerontol. 34, 162�/169.

Sambrook, J., Frisch, E.F., Maniatis, T., 1989. Molecular Cloning: A

Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory,

Cold Spring Harbor, New York.

Shah, G.N., Mooradian, A.D., 1997. Age-related changes in the blood

brain barrier. Exp. Gerontol. 32, 501�/519.

Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer

of proteins from polyacrylamide gels to nitrocellulose sheets. Proc.

Natl. Acad. Sci. (USA) 76, 4350�/4354.

A.D. Mooradian et al. / Mechanisms of Ageing and Development 124 (2003) 143�/146146