Age-Related Increase in Activity of Specific Lysosomal Enzymes in the Human Retinal Pigment Epithelium

Exp. Eye Res. (1997) 65, 231–240

Age-Related Increase in Activity of Specific Lysosomal Enzymes in the Human Retinal Pigment Epithelium M A R I A E. V E R D U G O    J H A R N A R A Y* James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. (Received Cleveland 7 January 1997 and accepted in revised form 26 March 1997) Age related changes in the activity of lysosomal enzymes have been studied in the cultured human retinal pigment epithelium cells collected from 26–85 year old donors. Among four such enzymes studied, activities of cathepsin D and β-glucuronidase increased with the age of the donors while no notable change in activity of arylsulfatase B and α-mannosidase was observed. Kinetic parameters of βglucuronidase was measured in retinal pigment epithelium cells isolated from donors of different ages. Similar kinetic parameters for β-glucuronidase at different ages suggest that the observed increase in the activity of the enzyme with age is not due to post-translational modification of the enzyme. Western blot analysis provides evidence for increased synthesis of β-glucuronidase with aging. Relative proportions of glycosaminoglycans, the natural substrates of β-glucuronidase and arylsulfatase B, in the retinal pigment epithelium altered with the age of the donors. A significant decrease of dermatan sulfate levels with aging correlates well with the observed increase in the level of β-glucuronidase activity. # 1997 Academic Press Limited Key words : human, retinal pigment epithelium (RPE) ; lysosomal enzymes ; β-glucuronidase ; αmannosidase ; cathepsin D ; arylsulfatase B ; glycosaminoglycans ; aging.

1. Introduction The retinal pigment epithelium (RPE) is one of the most metabolically active cell layers of the body (Bok, 1979). It is strategically located between the photoreceptor cells and the extensive choroidal vascular network, and participates in several activities essential for visual function and the maintenance of the photoreceptor cell viability. These include remodeling of extracellular matrices (ECMs) surrounding the RPE such as interphotoreceptor matrix (IPM) on its apical site and Bruch’s membrane (BM) on its basal site, phagocytosis and degradation of shed outer segments (Young and Bok, 1969), and regulation of ion and metabolite transport (Steinberg and Miller, 1979). Disruption of these functions may result in metabolic malfunction and}or accumulation of metabolic waste products with devastating consequences to the neuroretina. Lysosomal acid hydrolases are responsible for the degradation of a variety of macromolecules such as proteins, lipids, complex carbohydrates, and nucleic acids (Berman, 1994) and are much more active in RPE than in many other tissues of the body (Hayasaka, 1974 ; Zimmerman, Godchaux and Belkin, 1983 ; Ray, Wu and Aguirre, 1997). In the RPE, these enzymes are responsible for the degradation of ingested photoreceptor outer segment materials (Ishikawa and * Address correspondence to : Jharna Ray, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A.

0014-4835}97}080231­10 $25.00}0}ey970325

Yamada, 1970 ; Young and Bok, 1969). The importance of the catabolic functions of these enzymes in the RPE is evident from their deficiency in the inherited diseases of animals which presumably also occurs in man. For example, the deficiencies of arylsulfatase B in the mucopolysaccharidosis (MPS)-VI cat, α-iduronidase in the MPS I cat, and β-glucuronidase (GUSB) in the MPS VII dog, cause a primary failure of RPE function accompanied by secondary damage to photoreceptor (PR) cells (see Aguirre and Stramm, 1991 for review). Age-related macular degeneration (ARMD) is the leading cause of visual impairment in the aged population of developed countries. ARMD represents a complex group of diseases of unknown etiology in which both genetic and environmental factors play important roles. The pathology in ARMD centers in the RPE and its surrounding ECMs, particularly Bruch’s membrane (Feeney-Burns and Ellersieck, 1985). It appears that in ARMD there is a primary failure in the RPE function which causes a progressive accumulation of basal laminar deposit between BM and the RPE. This is followed by drusen formation, neovascularization and leakage in the basal RPE zone and later, in the subretinal space (Hogan, 1972 ; Bressler, Bressler and Fine, 1988 ; Klagsbrun and D’Amore, 1991). The association between abnormalities in RPE lysosomal function and ARMD is not clear, although its seems reasonable to speculate that a malfunction in lysosomal degradative enzymes, particularly in the macular region, could be involved in the pathogenesis # 1997 Academic Press Limited

232

of ARMD. An age dependent increase in acid phosphatase and cathepsin D (Boulton et al., 1994) but decrease in α-mannosidase (Cingle et al., 1996 ; Wyszynski et al., 1989) in human RPE cells have been reported. However, it is difficult to compare the results of these studies due to variations of the patient population, as well as different assays used to estimate the enzyme activities from the cultured and fresh tissues. To examine the effect of aging on the RPE lysosomal enzyme activity, we have used cultured cells from donors of different ages and examined the activities of four lysosomal enzymes—β-glucuronidase (GUSB), arylsulfatase B (ASB), α-mannosidase (α-Mann), and cathepsin D (Cat )—which are important for RPE} photoreceptor functions (Verdugo, Aguirre and Ray, 1996). GUSB and ASB are responsible for the degradation of certain glycosaminoglycans (GAGs) present in the cell layer and ECMs. For example GUSB degrades heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (CS) whereas ASB degrades DS and CS. The critical role of GUSB and ASB in normal RPE physiology is evident from the severe RPE pathology that results from the inherited deficiency of GUSB (MPS VII) and ASB (MPS VI) ; there is a progressive accumulation of undegraded GAGs in the lysosomal compartment causing MPS VII in dog and mouse (Stramm et al., 1990 ; Lazarus et al., 1993) and MPS VI in cat (Haskins et al., 1980). Cat D, the most important protease of the RPE, is involved in the regeneration of the photoreceptors by digesting the rhodopsin-rich disc membrane engulfed during phagocytosis of shed outer segment (Regan et al., 1980). αMann is responsible for the degradation of mannose, one of the major monomers of the oligosaccharide side chain of rhodopsin (Planter and Kean, 1976 ; Fukuda, Papermaster and Hargrave, 1979). Alteration of any of these enzyme activities with aging might shed some light on the mechanism involved in age-related disorder, particularly ARMD. 2. Materials and Methods Eye Collection Human eyes from 16 donors ranging from 26–85 years of age were obtained from the NDRI (National Disease Research Interchange, Philadelphia, PA, U.S.A.) and the Rochester Eye and Human Part Bank (Rochester, NY, U.S.A.). The eyes were enucleated 4–6 hours after death and transported in ice in the moist gauge soaked with saline. The RPE cells were harvested and cultured 24–48 hours after enucleation (Table I). RPE Cell Culture Human RPE cultures were established using a modified method of Stramm et al. (1983) which reduced the cell harvest time by 50 % (Ray et al.,

M. E. V E R D U G O A N D J. R A Y

1997). Briefly, the posterior segment of the eye was mounted in an aluminium support that holds the eye cup open and prevents contamination by scleral or choroidal fibroblasts. Vitreous and retina were removed by gentle aspiration and the RPE cells were released by repeated trypsinization with 0±5 % trypsin for 20 minutes (4–5 times) at 37°C. The cells were rinsed twice with media containing fetal bovine serum (FBS ; Sigma, St. Louis, MO, U.S.A.) and plated on Falcon plastic ware (Becton Dickinson Labware, Bedford, MA, U.S.A.) at a seeding density of 2–3¬10& cells}35 mm dish. Cultures were grown in Dulbecco’s modified Eagle media (DMEM) containing 15 % FBS, 2±5 % glutamine and 1 % antibiotic}antifungal solution (10 000 U ml−" penicillin G sodium salt, 10 000 U ml−" streptomycin sulfate, 25 µg ml−" amphotericin B ; Life Technologies, Gaithersburg, MD, U.S.A.) and maintained in a humidified atmosphere at 37°C in the presence of 95 % air}5 % CO . Once the # primary (P ) cultures became confluent, they were ! passaged at a ratio of 1 : 3 (P ). The passaged cells (P ) " " were grown to confluency and then maintained at FBS concentration of 5 %. Unless otherwise stated, P " cultures were used throughout our studies and analysed 7–10 days after reaching confluency. Preparation of Cell Extract and Enzyme Assays For measurement of enzyme activity, lysates from the cultured RPE cells and media were prepared as previously described (Ray et al., 1997). Briefly, cultured RPE cells were dissociated by trypsinization with 0±25 % trypsin in Pucks F solution without calcium and cell numbers were counted using a hemocytometer. The dissociated cells were rinsed twice with phosphate buffered saline (PBS) and harvested with 20 m Tris–HCl buffer, pH 7±6, containing protease inhibitors PMSF (1 m) and leupeptin (1 µg ml−"). The cells were lysed by freeze-thawing 4–5 times in a dry ice}ethanol bath. The cell lysate was then spun at 15 000 g in a microfuge at 4°C for 10 min and the soluble supernatant was collected and stored at ®20°C for subsequent experiments and referred to as cell extract. Protein concentration of the cell lysate was determined (Bradford, 1976) using a Bio-Rad protein assay kit (Bio-Rad laboratories, Hercules, CA, U.S.A.). To determine the activities in the media, 5 % FBS containing media from the culture was collected after 72 hours, spun at 15 000 g for 15 minutes, and the supernatant was collected and stored at ®20°C. All analyses were performed in duplicates within the linear range of activities in 3–5 cultures from the same donor, and the reported values represent the mean³standard error of mean (...) of these results. β-Glucuronidase GUSB enzyme activity was measured fluorimetrically using the conditions previously optimized by Ray et al. (1997) with the synthetic

LYSOSOMAL ENZYMES IN AGING HUMAN RPE

233

T I Eye donor information Donor agea (year)

Sex

Source

Cause of death

Race

Intervalb (hours)

26 27 33 39 46 (1) 46 (2) 46 (3) 55 56 64 (1) 64 (2) 67

Female Male Female Female Female Female Female Male Male Female Male Male

NDRI Rochester Eye Bank NDRI NDRI Cleveland Eye Bank Rochester Eye Bank NDRI NDRI NDRI NDRI NDRI NDRI

Black Black White White White White White White White White White White

36 36 24 36 48 36 48 24 36 24 46 36

72 82 85 (1)

Male Male Male

White White White

24 36 48

85 (2)

Female

NDRI NDRI Central Florida Lions Eye Bank NDRI

Sepsis Stab wound to heart Chest trauma Subarachnoid hemorrage Cardiac arrest Cardiac arrhythmia Acute cardiac events Cardiac arrest Emphysema pulmonar Acute cardiac events Cerebrovascular accident Respiratory failure secondary to renal failure Coronary artery disease Bronchial pneumonia Ischemic cardiopathy Acute cardiac crisis

White

36

a Donor number is indicated within parenthesis where more than one donor is available in the same age group ; b Interval between time of death of the donor and time of harvesting the RPE cells for culture ; NDRI, National Disease Research Interchange.

substrate 4-methyl-umbellyferyl β--glucuronide (4-MUG) (Sigma, St. Louis, MO, U.S.A.). The crude cell extract or media was incubated with 2 m 4-MUG in 100 m Na-acetate buffer, pH 4±5 at 60°C for 1 hour. The reaction was terminated with 0±4  ethylenediamine (EDA). The amount of 4-methyl umbelliferone (4-MU) released by enzymatic digestion was measured at 360 nm excitation and 450 nm emission with an Hitachi F1200 fluorimeter (Hitachi, Stoughton, MA, U.S.A.). GUSB activity was quantitated using 4-MU as standard. Enzyme activities were expressed as nmole substrate cleaved}hour incubation}mg protein for the cell layer, and as nmole substrate cleaved}hour incubation}10& cells for the media. To determine Km for GUSB, the enzyme activity was measured at different substrate concentrations (ranging from 0±1–3±2 m) using the cell extract from the donor of different ages. Lineweaver-Burk plot using reciprocal values of substrate concentration and velocity of reaction yielded Km. Thermal stability of GUSB was measured in samples of different ages as described before (Ray et al., 1997). The aliquots of RPE cell extracts were pre-incubated at 69°C for different time periods (0, 0±5, 1, 1±5, 2, 2±5 hours). The preincubated samples were then analysed for GUSB activity as described above. The residual GUSB activity was then plotted against preincubation time. The sample without preincubation at 69°C was used as control (100 % activity). Arylsulfatase B ASB activity was measured fluorimetrically (Chang, Rosa and Davidson, 1981) using the substrate, 4-methyl-umbelliferyl-sulfate (Sigma,

St. Louis, MO, U.S.A.). The cell extracts from donors of different ages were incubated with 5 m substrate in 0±05M Na-acetate buffer (pH 5±6), 3 m lead acetate, 0±3 m silver nitrate, 10 µg BSA at 37°C for 15 min. The reaction was terminated by adding 0±4  EDA. The 4-MU released by enzymatic digestion was measured and the activity expressed as described for the GUSB assay. α-Mannosidase α-Mann activity was measured fluorimetrically (Lee, Little and Yoshida, 1982) using the substrate, 4-methyl-umbelliferyl α--mannopyranoside (Sigma, St. Louis, MO, U.S.A.). The cell extracts or media from donors of different ages were incubated with 5 m substrate in 0±2  phosphate}0±12  citrate buffer (pH 4±5) at 45°C for 1 hour. The reaction was terminated by adding 0±4  EDA. The 4-MU released by enzymatic digestion was measured and the activity expressed as described for the GUSB assay. Cathepsin D Cat D activity was measured using the method of Burke and Twining (1988). Cell extracts and media were incubated with 2 % bovine hemoglobin (Sigma, St. Louis, MO, U.S.A.) as substrate in 0±25  formate buffer (pH 3±3) for 60 minutes at 37°C, both in the presence and absence of 10 µ pepstatin (Sigma, St. Louis, MO, U.S.A.), a cathepsin D inhibitor. The reaction was terminated by adding 3 % trichloroacetic acid (TCA). The TCA soluble proteolytic products were separated by centrifugation at 14 000 g in a microfuge at 4°C for 15 minutes and then measured by the method of Lowry et al. (1951) using tyrosine (Sigma, St. Louis, MO, U.S.A.) as a standard.

234

Cathepsin D activity was expressed as ng tyrosine} hour incubation}mg protein. Western Analysis Fifty micrograms of protein from 46, 56 and 67 year donors were electrophoresed in a 12±5 % SDS-polyacrylamide gel (Laemmli, 1970) alongside prestained protein molecular weight size markers (Life Technologies, Gaithersburg, MD, U.S.A.) and then electrotransferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH, U.S.A.) (Towbin, Staehelin and Gordon, 1979). Western analysis was performed using polyclonal goat anti-human GUSB immunoglobulin (kindly provided by William S. Sly, St. Louis University Medical Center, St. Louis, MO, U.S.A.) as primary antibody and rabbit anti-goat immunoglobulin as secondary antibody and "#&I-protein A (Amersham Corp., Arlington Heights, IL, U.S.A.) for detection of immune complexes by autoradiography (Ray et al., 1990). Glycosaminoglycans (GAGs) Analysis Since glycosaminoglycans (GAGs) are the natural substrates of the GUSB, we examined the synthesis and turnover of GAGs in the RPE cell layer and media. Confluent cell cultures were metabolically labeled in 35 mm dishes with Na $&SO (100 µCi ml−") in low # % sulfate media (made by mixing regular DMEM media with sulfate free DMEM media in a 1 : 1 ratio) for 72 hours. Total radiolabeled GAGs were isolated from the cultured cell layer and media as described before (Ray et al., 1997). Briefly, the proteins from the cell layer and media were digested with pronase (5 mg ml−" ; Sigma, St. Louis, MO, U.S.A.) after adding standard carrier GAGs (a mixture of HS, DS and CS, 0±08 mg per ml each of cell extract or media), and the GAGs were precipitated by adding 2 % cetylpyridinium chloride and 15 % ethanol. The GAGs were then purified and solubilized in water. The isolated GAGs were analysed by cellulose-acetate electrophoresis in barium acetate buffer, pH 8±1 using known GAG standards (Stramm, 1987). The GAGs were visualized by staining with Alcian blue. The radioactive GAGs were first identified by fluorography, and then quantified using the Fuji Mac Bas 1000 Phosphor Imager. The specific GAG classes were ascertained by electrophoresis following enzymatic digestion as described before (Stramm, 1987 ; Stramm et al., 1990). 3. Results Lysosomal Enzyme Activities Effect of cell passage For this study it was important to culture the RPE cells for generating sufficient amount of cells to be used in relevant experiments. Also studies made on primary cells might have

M. E. V E R D U G O A N D J. R A Y

T II Lysosomal enzyme activities in human RPE : effect of cell passage Donor agea (years) 64 (1)

80

85 (1)

Passage

GUSB (units)b

Cat D (units)c

P ! P " P # P ! P " P # P ! P " P #

2300³20 430³50 180³85±5 974³290 905³30 767³432±5 ND 1117³217±5 550³130

3520³300 2776³37±5 2776³185 9207³607 7176³981 5822³600 9343³185 8462³110 6432³169

a Numbers in parenthesis indicate the donor number among the donors of same age group ; b units ¯ nmole substrate cleaved}hr incubation}mg protein ; c units ¯ ng tyrosine}hr incubation}mg protein ; ND, not determined ; Values represent means³... of replicate analyses from three or more cultures.

variability due to different amount of in vivo phagocytic load and the local tissue environment which might still be present in the primary culture (Wyszynski et al., 1989). Since we have previously observed that GUSB activity in the RPE of several species decreases with passages (Ray et al., 1997), we first determined the effect of subculturing on the RPE lysosomal enzymes selected for this study. Towards this goal we cultured RPE from donors of different ages (Table I), and measured lysosomal enzyme activities at different passages (P , P , and P ). GUSB enzyme ! " # activities decreased with cell passage irrespective of the age of the donor. However, the decrease in activity did not follow any consistent pattern with the age of the donor (Table II). In contrast, Cat D activity did not change remarkably with passaging (Table II). In order to obtain maximum number of cells for biochemical study, yet not adversely decrease the enzyme activities that occurs with passaging, we elected to use first passage culture (P ) throughout this study. " Distribution of the enzymes between cell layer and cultured media To determine the distribution of enzymatic activity between the cell layer and media, GUSB, Cat D and α-Mann activities were measured in donors of different ages (Table III). All three enzymes were detected in cell layer, but in media only GUSB and α-Mann activity were detected in relatively much lower amount and no activity for Cat D was detectable. Effect of donor age To determine the influence of donor-age on lysosomal enzymes, activities for GUSB, Cat D, ASB and α-Mann were measured in the RPE cell lysates from donors between 26–85 years of age. Among all the donors, those three age groups (i.e. 46, 64, and 85 years) in which multiple donors were obtained (Table I), results are presented as the mean

LYSOSOMAL ENZYMES IN AGING HUMAN RPE

235

T III Lysosomal enzyme activities in human RPE—distribution in cell layer and media Donor agea (years)

Cell layer}media

GUSB (units)b

α-Mann (units)b

Cat D (units)c

Cell layer Media Cell layer Media Cell layer Media

120³22 0±90³0±025 430³50 3±3³0±33 1126³28 5±1³0±35

56³4±55 4±3³0±36 36³1±25 1±4³0±05 62³1±2 4±6³0±10

1219³298 0±0 2843³37±5 0±0 8666³68 0±0

27 64 (1) 85 (1)

a Numbers in parenthesis indicate the donor number among the donors of same age group ; b units ¯ nmole substrate cleaved}hr incubation}mg protein (for cell layer) or 10& cells (for media) ; c units ¯ ng tyrosine}hr incubation}mg protein (for cell layer) or 10& cells (for media) ; values represent means³... of replicate values from more than three cultures.

(B)

(A) 9000 Cat D (ng tyrosine/hr/mg protein)

GUSB (nmole/hr/mg protein)

1200 1000 800 600 400 200

7000

5000

3000

1000 0

0 (C)

(D) 250 ASB (nmole/hr/mg protein)

α-Mann (nmole/hr/mg protein)

140 120 100 80 60 40

200

150

100

50

20 0

20

30

40

50 60 Age (years)

70

80

0

20

30

40

50 60 Age (years)

70

80

F. 1. Age dependent changes in the activity of lysosomal enzymes. Activities of GUSB (A), Cat D (B), α-Mann (C), and ASB (D) were measured in RPE cultures (P ) from donor of different ages (26–85 years). Values represent mean³... of replicate " analysis from three or more cultures. Air bars represent mean³... from multiple donors (see Table I).

value and the standard error of mean is taken into account [Fig. 1, (A)–(C)]. ASB activities were measured from single donors of different age [Fig. 1(D)]. The increase in GUSB activity with age appears to take place in two distinct phases. The first phase consisted of a slower increase in the enzyme activity observed

between age 27 to 72 years followed by a sharp increase beyond 72 years [Fig. 1(A)]. Similarly increase in Cat D activity was moderate between 46 and 67 year donors and an abrupt increase was observed in 72 years and older donors [Fig. 1(B)]. In contrast, α-Mann [Fig. 1(C)] and ASB [Fig. 1(D)]

236

M. E. V E R D U G O A N D J. R A Y

T IV

Age (year)

46

56

67

Km and Vmax determined for β-glucuronidase in donors of different age Donor age (year)

Km

Vmax

78 kDa

27 46 55 57 64 72 80 82 85

0±385 0±476 0±30 0±32 0±666 0±555 0±432 0±23 0±341

48 62 89±3 118 53 69 200 48 365

60 kDa

Km and Vmax represent the average from two independent plots from the same donor.

F. 3. Donor age dependent GUSB synthesis. Equal amount of protein (50 µg) from donors of ages 46–67 years were separated in a SDS-polyacrylamide gel and analysed by western blotting using human GUSB antibody. The size of the proteins were determined by comparing known molecular size marker run on a parallel lane (not shown). The two GUSB bands (78 kDa and 60 kDa) are shown by arrows.

100

samples, no correlation between the donor age and the kinetic parameter for GUSB was observed. Thermal stability of GUSB widely varies among different species and is pH dependent (Brot, Bell and Sly, 1978 ; Ray et al., 1997). To determine the effect of donor-age on thermal stability of GUSB, thermal inactivation of GUSB was measured at 69°C in RPE samples from 46 to 85 years old donors. The decay of GUSB activity was observed to be 25–35 % in 2±5 hours of preincubation in all the samples tested with similar rate of decay (Fig. 2). This suggests that the thermal stability of GUSB is not influenced by the age of the donors.

GUSB activity (%)

80

60

40

20

0

0.5

1 1.5 2 Time of preincubation (hour)

2.5

F. 2. Effect of donor age on GUSB thermal stability. Cell lysates from cultured RPE (P ) of donors of different ages " [46 (*), 64 (U), 72 (_), 80 (E), 85 (+) years] were preincubated at 69°C up to 2±5 hours. The residual GUSB activity after preincubation was measured fluorimetrically (see Materials and Methods) and expressed as percent activity of the sample without preincubation at 69°C (control). Values represent the average of duplicate analysis from two cultures of the same donor.

activities did not alter with aging. We found a fairly steady level of activity with some variability without any distinct pattern. Kinetic studies of GUSB To determine whether age related increase in GUSB activity is due to changes in the kinetic properties of the enzyme, we measured the Km and thermal stability of the enzyme. The Km and Vmax were determined from the Lineweaver-Burk plot using RPE cell extracts from donors of different ages (Table IV). Although differences were observed between the determinations made in different donor

Western blot analysis of GUSB To substantiate further that the age-related increase of GUSB activity is not due to post-translational modification, equivalent amount of the RPE cell extracts from donors (46, 56 and 67 years) were resolved in a denaturing gel and immunoreactive GUSB bands were detected (Fig. 3). Two expected size bands (78 and 60 kDa) were detected in all donors examined irrespective of age. It has been reported that the 60 kDa polypeptide is derived by nicking 78-kDa GUSB subunit at Val"&*Gly"'! (Tanaka et al., 1992). The intensity of bands increased with the age of the donors which suggests that the rate of biosynthesis of GUSB is influenced by aging. Effect of Aging on Glycosaminoglycan Profile Glycosaminoglycans (GAGs) are the natural substrates for GUSB, ASB and other lysosomal hydrolases present in the RPE. Three individual GAG classes heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (CS), were identified in the RPE cell layer and media (Fig. 4). Among these three GAGs, HS is the predominant one in the cell layer of all donors regardless of their age, and DS constitutes the minor component of the DS}CS doublet (Fig. 4, left panels). With increase of age of the donors, a change in the

LYSOSOMAL ENZYMES IN AGING HUMAN RPE (A)

237

Cell

Media

HS DS CS

Age (yr)

27

46

64

85

27

46

64

85

(B) 400.0

HS

CS

300.0

200.0

100.0

0

CS DS

5.0 Migration (mm)

10.0

46 yr GAG level (PSL)

GAG level (PSL)

46 yr 400.0

HS

200.0 DS

0

5.0 Migration (mm)

10.0

F. 4. Donor age dependent glycosaminoglycan level in RPE. The fluorogram (A) shows an age dependent variation in the level of heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (CS) separated by cellulose-acetate electrophoresis from metabolically labeled cell layer (A, left panel) and media (A, right panel). Densitometric scanning (B) of the fluorogram for 46 year old donor shows relative amount of HS, DS, and CS in the cell layer (B, left panel) and media (B, right panel). PSL represents an arbitrary unit expressing the intensity of the GAG bands in the fluorogram.

relative distribution of the cell layer GAGs was observed. There was a modest increase of HS, with concomitant decreases of CS and DS [Table V and Fig. 4(A) left panel]. Interestingly, the decrease of DS was most remarkable with a 8-fold difference between donors of ages 27 to 85 years (Table V). In contrast, in the media no consistent age dependent change in the GAG profiles was observed (Fig. 4, right panels). 4. Discussion Our study with the cultured retinal pigment epithelium (RPE) clearly demonstrates age related increase of at least two lysosomal acid hydrolases cathepsin D and β-glucuronidase. Cat D, the major protease of RPE, increased between 26–85 years. Between 27–46 years, cat D increased 2–3 fold ; the increased level maintained up to 67 years then again another sharp increase was noted between 67–85 years. On the other hand the activity of GUSB, the important enzyme responsible for ECM remodeling, increased 3–5 fold between 27–46 years then increased gradually from 64 to 85 years. No such age dependent effect was observed while we measured

ASB and α-mann. The age-related changes of enzyme activities are well documented in animal tissues (Rothstein, 1975 ; Finch, 1972 ; Wilson, 1973 ; Lombardo et al., 1981). Boulton et al. (1994) have demonstrated an age-related increase of cat D and acid phosphatase in the freshly isolated RPE. The increased level of lysosomal enzymes with age may be due to an association of lysosomes with pigment granules (melanosomes and lipofuscin granules) as hypothesized by Boulton et al. (1994). An age-dependent decrease of α-mannosidase and few other glycosidases (e.g., β-galactosidase, N-acetyl glucosaminidase and N-acetyl-β-galactosaminidase) have been reported in freshly isolated RPE (Cingle et al., 1996). A similar decrease of α-mann in primary cultured RPE has also been reported but the activity of several other glycosidases, including GUSB, did not alter (Wyszynski et al., 1989). In the present study, however, we did not observe any age-related changes of α-mann activity in cultured, passaged (P ) RPE. The reported decrease of " α-mann activity might be dependent upon in vivo phagocytic load and the local tissue environment which might still be present in the primary culture (P ) ! (Wyszynski et al., 1989) but abolished completely in

238

M. E. V E R D U G O A N D J. R A Y

T V Donor age dependent distribution of GAGs in human RPE % GAGs at different donor agea GAG

27

46 (1)

64 (1)

85 (1)

HS DS CS

52 24 24

64 15 21

74 12 14

83 3 14

a Numbers in parenthesis indicate the donor number among the donors of same age group ; relative amounts of individual GAGs (HS, DS and CS) in RPE cell layer from donors of different ages (27, 46, 64 and 85 years) are expressed as percent (%) of total cell layer GAGs.

the passaged culture (present study). Region specific expression of lysosomal enzyme ( β-glucuronidase and acid phosphatase) activities in canine RPE were lost when cells were grown in culture (Cabral et al., 1990). We observed in dog RPE, GUSB activity in P culture ! varies remarkably depending on the time elapsed between enucleation and initiation of RPE harvest. GUSB activity in P culture generated from freshly ! enucleated dog eye is 2–3 fold higher than eye harvested 24 hours after enucleation. This difference at P culture was not maintained in passaged (P , P ) ! " # culture (Ray et al., 1997). To avoid the effect of factors that might be contributed from in vivo and to determine the lysosomal enzyme activity in aging RPE we chose P " culture throughout this study. Our study demonstrate a remarkable age-related increase of GUSB activity even at P culture, a 8–10 fold increase of GUSB " activity between donors of ages 27–85 years were found. GUSB kinetic studies in donors of different ages, i.e., determination of Km and thermal stability showed no age-dependent effect, suggesting increase of GUSB activity in aging donor is not due to the posttranslational modification(s) of the enzyme. This was confirmed by western blot analysis using GUSBantibody which detected 2 bands (78 and 60 kDa) in all donors tested with increase in band intensity in older donors. This suggests increased level of GUSB synthesis with aging. During visual transduction RPE plays an essential role in photoreceptor renewal (Young and Bok, 1969 ; 1985 ; Berman, 1991) by degrading ingested outer segment through a highly developed phagolysosomal system (Feeney, 1973). The ingested components in the phagolysosomes are thought to be degraded by RPE lysosomal enzymes such as α-mannosidase (Wyszynski et al., 1989), cathepsin D (Regan et al., 1980) and phospholipase A (Zimmerman et al., 1983) either singly or in concert. The major end products of lysosomal enzyme action in the phagolysosomal system of RPE cells (Feeney, 1978 and Wing et al., 1978) are lipofuscin granules, the autofluorescent particles that accumulate in an age-related manner in

the cytoplasm of RPE cells. The RPE cells are for the most part non-mitotic cells, continuous phagocytic load leads to a striking age-related accumulation of lipofuscin from about age 40 (Feeney, 1973 ; Marshall, 1987). This accumulation is striking in the macular region. Other age-related structural changes in Bruch’s membrane as well as drusen formation, the extracellular excrescences of RPE, have been studied extensively. Drusen formation is not a threat to vision but changes in Bruch’s membrane initiated by abnormal substances secreted by aging RPE cells, might lead to change in structure and elasticity which will affect photoreceptor function (Marshall, 1987). GAGs are present in extracellular matrix and on the cell surface as proteoglycans. These proteoglycans regulate the thickness of collagen fibrils in matrices, stabilize and maintain the association of adjacent collagen fibers (Van Kuppevelt et al., 1985) and play a role in cell adhesion, motility and differentiation (Ruoslahti, 1990 ; Hardingham and Fosang, 1992). Changes in the proteoglycan structure during aging has been reported. In our study measurement of GAGs clearly shows a progressive decrease of DS in RPE with aging. Determination of the relative distribution of GAGs (e.g. HS, DS, CS) present in the RPE of different aged donors demonstrated a remarkable decrease of DS (8 fold) and a modest decrease of CS with an increase of HS level. DS is a substrate for both GUSB and ASB. In the DS degradative pathway ASB acts prior to GUSB. Since ASB activity remained unaltered, the decrease of DS suggests higher level of catabolism is due to increased GUSB activity in aged donors. The GAGs metabolism is also altered by their structural modification, e.g., lack of sulfation, altered sulfation (Caparas, Cintron and Hernandez-Neufeld, 1991) and}or shorter chain synthesis (Sawaguchi et al., 1993). In any of these events distribution of GAG classes will be different. Lack of sulfated GAG might make the cells less resilient since negatively charged GAG attracts water and helps to maintain ocular pressure. Increase of total GAG in general and HS in particular has been implicated in age-related maculopathy (Kliffen et al., 1996). It appears that there is an association between ARMD and the aging RPE together with its basal extracellular matrices. The primary failure in RPE degradative functions causes a progressive accumulation of basal laminar deposit between BM and RPE causing photoreceptor dysfunction leading to blindness. The most compelling evidence for an association between an abnormality in RPE extracellular matrix metabolism and ARMD is the detection of a mutation in the tissue inhibitor of metalloproteinase-3 (TIMP3), which is known to play a pivotal role in extracellular matrix remodeling, in patients with Sorsby’s fundus dystrophy (SFD) (Weber et al., 1994). Similarities between SFD and ARMD suggest that mutations affecting genes that determine the structure or

LYSOSOMAL ENZYMES IN AGING HUMAN RPE

maintenance of Bruch’s membrane might be involved in ARMD (Carrero-Valenzuela et al., 1996). The biochemical basis of the age-related changes is largely unknown. These age related changes may reflect alterations in the regulation of synthesis and secretion pathways or may be related to extracellular modifications of the biosynthetic products generated during the course of normal aging. In either case these could lead to adverse consequences for the cell physiology. Clinical pathology might be the advanced manifestation of the above effects. The studies presented here demonstrates age related changes in the activity of specific lysosomal enzymes in the RPE. Further studies to understand the molecular basis of such changes with aging and its relevance to the ARMD (if any) is underway.

Acknowledgements This work is supported by the American Federation for Aging Research (AFAR)}The Starr Foundation Grant, NEI}NIH Grant EY 11142, Consolidated Research Grant, College of Veterinary Medicine, Cornell University, Donnelley Development Award. Authors like to thank Dr Gustavo Aguirre and Dr Kunal Ray for reviewing the manuscript ; appreciate Sue Pearce-Kelling for editorial assistance and Virginia Scarpino for technical assistance ; Linda Spear, Rochester Eye and Tissue Part Bank and NDRI for helping in getting the eyes.

References Aguirre, G. and Stramm, L. (1991). The RPE : A model system for disease expression and disease correction. Prog. Retinal Res. 11, 153–91. Berman, E. R. (1994). Retinal pigment epithelium : lysosomal enzymes and aging. Br. J. Ophthalmol. 78, 82–3. Berman, E. R. (1991). Biochemistry of the eye. Pp. 380–406. Plenum Press : New York. Bok, D. (1979). Phagocytic properties of the retinal pigment epithelium. In The retinal pigment epithelium (Eds Zinn, K. M. and Marmour, M. F.) Pp. 148–74. Harvard University Press : Cambridge. Bok, D. (1985). Retinal photoreceptor-pigment epithelium interactions. Invest. Ophthalmol. Vis. Sci. 26, 1659–94. Boulton, M., Moriarty, P., Jarvis-Evans, J. and Marcyniuk, B. (1994). Regional variation and age-related changes of lysosomal enzymes in the human retinal pigment epithelium. Br. J. Ophthalmol. 78, 125–9. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–54. Bressler, N. M., Bressler, S. B. and Fine, S. L. (1988). Age related macular degeneration. Surv. Ophthalmol. 32, 375–413. Brot, F. E., Bell, C. F., Jr. and Sly, W. S. (1978). Purification and properties of β-glucuronidase from human placenta. Biochemistry 17, 385–91. Burke, J. M. and Twining, S. S. (1988). Regional comparisons of cathepsin D activity in bovine retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 29, 1789–93.

239

Cabral, L., Unger, W., Boulton, M., Lightfoot, R., McKechnie, N. and Grierson, I. (1990). Regional distribution of lysosomal enzymes in the canine retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 31, 670–6. Caparas, V. L., Cintron, C. and Hernandez-Neufeld, M. R. (1991). Immunohistochemistry of proteoglycans in human lamina cribrosa. Am. J. Ophthalmol. 112, 489–95. Carrero-Valenzuela, R. D., Klein, M. L., Weleber, R. G., Murphey, W. H. and Litt, M. (1996). Sorsby fundus dystrophy. A family with the Ser181Cys mutation of the tissue inhibitor of metalloproteinases 3. Arch. Ophthalmol. 114, 737–8. Chang, P. L., Rosa, N. E. and Davidson, R. G. (1981). Differential assay of arylsulfatase A and B activities : A sensitive method for cultured human cells. Anal. Biochem. 117, 382–9. Cingle, K. A., Kalski, R. S., Bruner, W. E., O’Brien, C. M., Erhard, P. and Wyszynski, R. E. (1996). Age-related changes of glycosidases in human retinal pigment epithelium. Curr. Eye Res. 15, 433–8. Feeney, L. (1973). The phagolysosomal system of the pigment epithelium. A key to retinal disease. Invest. Ophthalmol. Vis. Sci. 12, 635–8. Feeney, L. (1978). Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical and ultrastructural studies. Invest. Ophthalmol. Vis. Sci. 17, 538–600. Feeney-Burns, L. and Ellersieck, M. (1985). Age related changes in the ultrastructure of Bruch’s membrane. Am. J. Ophthalmol. 100, 686–97. Finch, C. E. (1972). Enzyme activities, gene function and aging mammals (review). Exp. Gerontol. 7, 53–67. Fukuda, M. N., Papermaster, D. S. and Hargrave, P. A. (1979). Rhodopsin carbohydrate : Structure of small oligosaccharides attached at sites near the NH ter# minus. J. Biol. Chem. 254, 8201–7. Hardingham, T. E. and Fosang, A. J. (1992). Proteoglycans : many forms and many functions. FASEB J. 6, 861–70. Haskins, M. E., Aguirre, G. D., Jezyk, P. and Patterson, D. F. (1980). The pathology of the feline model of mucopolysaccharidosis VI. Am. J. Pathol. 101, 657–73. Hayasaka, S. (1974). Distribution of lysosomal enzymes in the bovine eye. Jpn. J. Ophthalmol. 18, 223–39. Hogan, M. F. (1972). Role of the retinal pigment epithelium in macular disease. Trans. Am. Acad. Ophth. Otolaryng. 76, 64–80. Ishikawa, T. and Yamada, E. (1970). The degradation of the photoreceptor outer segment within the retinal pigment epithelial cell of rat retina. J. Electron. Micros. 19, 85–99. Klagsbrun, M. and D’Amore, P. A. (1991). Regulators of angiogenesis. Ann. Rev. Physiol. 53, 217–39. Kliffen, M., Mooy, C. M., Luider, T. M., Huijmans, J. G. M., Kerkvliet, S. and de Jong, P. T. V. M. (1996). Identification of glycosaminoglycans in age-related macular deposits. Arch. Ophthalmol. 114, 1009–14. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (Lond.) 227, 680–5. Lazarus, H. S., Sly, W. S., Kyle, J. W. and Hageman, G. S. (1993). Photoreceptor degeneration and altered distribution of interphotoreceptor matrix proteoglycans in the mucopolysaccharidosis VII mouse. Exp. Eye Res. 56, 531–41. Lee, J. E., Little, T. E. and Yoshida, A. (1982). Purification and chemical characterization of human acidic alpha-mannosidase. Enzyme 28, 33–40. Lombardo, A., Goi, G. C., Marchesini, S., Caimi, L., Moro, M.

240

and Tettamanti, G. (1981). Influence of age and sex on five human plasma lysosomal enzymes assayed by automated procedures. Clin. Chim. Acta. 113, 141–52. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–75. Marshall, J. (1987). The ageing retina : physiology or pathology. Eye 1, 282–95. Planter, J. J. and Kean, E. L. (1976). Carbohydrate composition of bovine rhodopsin. J. Biol. Chem. 251, 1548–52. Ray, J., Okamura, H., Kelly, P. A., Liebhaber, S. A. and Cooke, N. E. (1990). Alteration in the receptor binding specificity of human growth hormone by genomic exon exchange. Mol. Endocrinol. 4, 101–7. Ray, J., Wu, Y. and Aguirre, G. D. (1997). Characterization of β-glucuronidase in the retinal pigment epithelium. Curr. Eye Res. 16, 131–43. Regan, C. M., deGrip, W. J., Daemen, F. J. M. and Bonting, S. L. (1980). Degradation of rhodopsin by a lysosomal fraction of retinal pigment epithelium. Biochemical aspects of the visual process. Exp. Eye Res. 30, 183–91. Rothstein, M. (1975). Aging and the alteration of enzymes : a review. Mech. Age Dev. 4, 325–38. Ruoslahti, E. (1990). Proteoglycans in cell regulation. J. Biol. Chem. 264, 13369–72. Sawaguchi, S., Yue, B. Y. J. T., Fukuchi, T., Iwata, K. and Kaiya, T. (1993). Age-related changes of sulfated proteoglycans in the human lamina cribrosa. Curr. Eye Res. 12, 685–92. Steinberg, R. H. and Miller, S. S. (1979). Transport and membrane properties of the retinal pigment epithelium. In The retinal pigment epithelium (Eds Zinn, K. and Marmor, M.) Pp. 205–225. Harvard University Press : U.S.A. Stramm, L. (1987). Synthesis and secretion of glycosaminoglycans in cultured retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 28, 618–27. Stramm, L., Haskins, M., McGovern, M. and Aguirre, G. (1983). Tissue culture of cat retinal pigment epithelium. Exp. Eye Res. 36, 91–101. Stramm, L., Wolfe, J., Schuchman, E., Haskins, M., Patterson, D. F. and Aguirre, G. (1990). β-

M. E. V E R D U G O A N D J. R A Y

glucuronidase mediated pathway essential for retinal expression and in vitro disease correction using retroviral mediated cDNA transfer. Exp. Eye Res. 50, 521–32. Tanaka, J., Gasa, S., Sakurada, K., Miyazaki, T., Kasai, M. and Makita, A. (1992). Characterization of the subunits and sugar moiety of human placental and leukemic beta-glucuronidase. Biol. Chem. Hoppe-Seyler. 373, 57–62. Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some application. Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4. VanKuppevelt, T. H. M. S. M., Cremers, F. P. M., Domen, J. G. W., Van Beuuingen, H. M., Van den Brule, A. J. C. and Kuyper, C. M. A. (1985). Ultrastructural localization and characterization of proteoglycans in human lung alveoli. Eur. J. Cell Biol. 36, 74–80. Verdugo, M. E., Aguirre, G. D. and Ray, J. (1996). Studies of lysosomal enzymes in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 37, S379. Weber, B. H. F., Vogt, G., Pruett, R. C., Stohr, H. and Felbor, U. (1994). Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby’s fundus dystrophy. Nat. Genet. 8, 352–6. Wilson, P. D. (1973). Enzyme changes in aging mammals. Gerontologia 19, 79–125. Wing, G. L., Blanchard, G. C. and Weiter, J. J. (1978). The topography and age-relationship of lipofuscin concentration in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 17, 601–7. Wyszynski, R. E., Bruner, W. E., Cano, D. B., Morgan, K. M., Davis, C. B. and Sternberg, P. (1989). A donor-agedependent change in the activity of alpha-mannosidase in human cultured RPE cells. Invest. Ophthalmol. Vis. Sci. 30, 2341–7. Young, R. W. and Bok, D. (1969). Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. Cell Biol. 42, 392–403. Zimmerman, W. F., Godchaux, W. III and Belkin, M. (1983). The relative proportions of lysosomal enzyme activities in bovine retinal pigment epithelium. Exp. Eye Res. 36, 151–8.