Some effects of aluminium on rat brain protein synthesis

0306492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd

Camp.B&hem. Physiol.Vol. 103C,No. 3, pp. 58S591, 1992 Printed in Great Britain

SOME EFFECTS OF ALUMINIUM ON RAT BRAIN PROTEIN SYNTHESIS SERBANSAN-MARINA and D. MCEWEN NICHOLLS* Department of Biology, York University, North York (Toronto), Ontario, Canada M3J lP3. Tel. 416-736-2100; 416-736-5698 (Received 30 March 1992; accepted for publication 1 May 1992) Abstract-l. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, ICIor 20 mg Al/kg body weight) every third day from one to four weeks of age. 2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [14C] leucine, [I%] phenylalanine, or [“S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits. 3. The incorporation of [“Slmethionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction. 4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.

INTRODUCI’ION The toxicity of aluminium (Al) in biological systems is of increasing importance because of the tendency towards acidification in the environment and because

of the increased use of Al in industry (Sigel and Sigel, 1988; Lewis, 1989). In particular, the effect of Al on the nervous system and its possible link to Alzheimer’s disease has received considerable attention (Still and Kelly, 1980; Martyn et al., 1989; Rifat et al., 1990; Crapper McLachlan et al., 1991), especially since non-genetic factors appear to play an important role in the development of Alzheimer’s disease (Katzman and Saitoh, 1991). The interaction of genetic and other factors in certain individuals who exhibit susceptibility to Al toxicity is poorly understood (Yano et al., 1989; Crapper McLachlan et al., 1989, 1991). In species, such as rabbits, that are susceptible to Al neurotoxicity, there is an increased binding of Al to nuclear chromatin, an accumulation of intraneuronal 10 nm filaments and progressive encephalopathy. Species, such as rodents and primates, that are resistant to Al exhibit greater cytoplasmic accumulation of Al and little encephalopathy (Crapper et al., 1980). In our laboratories we have studied infant rabbits following S.C.chronic Al exposure as a model system for studying protein synthesis during mammalian Al neurotoxicity (Nicholls et al., 1990, 1991). The mRNA fraction obtained from brain polysomes following high doses of aluminium maltolate or lactate exhibited decreased activity in a protein-synthesizing system. Such a decrease resembled that following acute or chronic exposure to lead, another neurotoxic metal (Kennedy ef al., 1983). The exposure of rats and other species to heavy metals, such as cadmium or mercury, is well known *For reprint segments.

to increase liver and kidney metallothionein levels which may provide protection from toxic effects (for a review, see Kagi and Kojima, 1987). In addition, these metals stimulate the translation of metallothionein mRNA and certain other mRNAs (Kuliszewski and Nicholls, 1982; Samji et al., 1985; Harrison and Nicholls, 1986; Angelow and Nicholls, 1991) The iron (Fe) binding protein, ferritin, like metallothionein, binds a number of metals in the cytoplasm and may thus protect rats against toxic effects (Price and Joshi, 1983; Fleming and Joshi, 1987). Since Al exhibits little neurotoxicity in rats and remains in the cytoplasmic fraction, we have designed experiments to see whether Al induces the synthesis of ferritin and other proteins which might protect this species. METHODS Animals and chemicals Litters of 10 newborn rats and their dam (Wistar) were obtained from Charles River Canada, St Constant, Quebec. Litters of six newborn New Zealand White rabbits and their doe were from Maple Lane Rabbits, Clifford, Ontario. At one week of age the infants were injected i.p. with 5, 10 or 20 mg Al/kg as AlC1, and on every third day thereafter to a total of six injections. Control animals received an equivalent volume of distilled water (4ml/kg body weight). The animals were cared for according to the principles of the Canadian Council on Animal Care. Animals were sacrificed by decapitation two days after the last injection. Aluminium chloride (AlCl, .6H, 0) was from J. T. Baker Chemical Co., Phillipsdurg, hJ. Affinity isolated anti-horse spleen ferritin (developed in rabbits), rabbit serum, aprotinin protease inhibitor, protein A-Sepharose, and Nonidet P-40 were from the Sigma Chemical Co., St Louis, MO. Polyuridylic acid was from Boehringer Mannheim, Laval, Quebec. Tachisorb-R immunoadsorbant for rabbit antiserum was from Calbiochem-Behring, La Jolla, CA. Rabbit reticulocyte lysate, L-[U-‘4C] leucine (340 mCi/mmol) L-[U‘“Cl phenylalanine (5 13 mCi/mmol), and L-[~%]methionine 585

586

SERBAN

( > 800 Ci/mmol), were obtained Ltd., Oakville, Ontario.

SAN-MARINA and D. MCEWEN NICHOLLS

from Amersham

Canada

Preparation of brain pol.vsomes Brain polysomes were prepared as described by Nicholls et al. (1991). Brains of 3-6 animals were homogenized in 2 vols of 0.25 M sucrose in ice cold buffer containing 50 mM Tris-HCI (pH 7.7 at 22”C), 80 mM potassium acetate, 6 mM magnesium acetate, and 10 mM 2-mercaptoethanol (TKM buffer) to which had been added 1.5 ml of a liver postmicrosomal supernatant preparation containing ribonuclease inhibitor (Bishay and Nicholls, 1973). The microsomal fractions and postmicrosomal supernatant (S105) fractions were obtained and processed as described previously (Kuliszewski and Nicholls, 1983). The polysomal fractions were prepared by treatment with 2% (w/v) deoxycholate and the polysomal pellet was rinsed with buffer, resuspended in 0.25 ml of 0.25 M sucrose in TKM buffer and made 10% (v/v) with glycerol prior to storage at -70°C. The preparation of polysomal RNA was as described by Kuliszewski and Nicholls (1982). In some experiments the mRNA of the post-ribosomal supernatant fraction (i.e. cytoplasmic ribonucleoprotein (RNP) particles) was prepared as described by Zahringer et al. (1976) and the RNA extracted as before. Since brain contains considerable mRNA in the poly(A)RNA fraction, no further fractionation was done (Sutcliffe, 1988). Preparation fraction

qf liter postmicrosomal

supernalant

(S 105)

Rat liver was homogenized at 0-4°C in 4 vols of 0.25 M sucrose in TKM buffer in a glass homogenizer with 8 strokes of a Teflon pestle and the postmicrosomal supernatant fraction was prepared as described above and stored at -70°C with 10% (v/v) glycerol. This preparation served both as a source of ribonuclease inhibitor for the preparation of brain polysomes and as a source of aminoacyl tRNA synthetases, tRNA, and protein synthesizing enzymes for the translation of brain mRNA bound to polysomes. Incorporation of labelled amino acids into proteins Polysomes were incubated for 20 min at 37°C in 250 pl of TKM buffer containing 4 mM ATP, 0.4 mM GTP, 0.25 PCi [‘*Cl leucine or [“‘Cl phenylalanine and an amount of SlO5 fraction that did not limit the reaction. Preliminary experiments showed that 20 min was the optimal incubation time. The reaction was stopped and the proteins precipitated with 10% (w/v) trichloroacetic acid and unlabelled carrier leucine or phenylalanine (0.2% w/v) and carrier protein (1 mg bovine serum albumen). The protein was heated, washed and solubilized for measurement of the radioactivity as described previously (Kennedy et al., 1983). The results were corrected for blank tubes that did not contain polysomes. Polysomal or postribosomal supernatant mRNA was translated using rabbit reticulocyte lysate treated with micrococcal nuclease so that translation depended upon exogenous mRNA. Lysate (18 ~1) (containing 200 PM hemin which blocks repressor protein, Lin et al., 1990). 45 PCi of ‘5[S] methionine, and an mRNA fraction (or in some cases polysomes) in a final volume of 25 ~1 were incubated as described by Amersham. The reaction was stopped after 60 min at 30°C by the addition of unlabelled methionine and chilling on ice. Triplicate aliquots (3 ~1) of the translation mixture were applied to squares (1 cm*) of Whatmann 3 MM filter paper and the proteins were precipitated, heated and washed as described by Chin-See and Nicholls (1982). After drying, the filters were placed in scintillation fluid, counted and corrected for blank tubes (no mRNA).

Immunoprecipitation of ferritin Samples containing translation products were mixed with 300 ~1 of immunoprecipitation buffer (0.5% w/v aprotinin, 0.25% w/v methionine, 150 mM NaCI, 5 mM EDTA. 1% v/v Nonidet P-40, and 50 mM Tris-HCI, pH 7.4), and incubated at room temperature with 4~1 of control rabbit serum for 10min and then with 30~1 of protein ASepharose (binding capacity 3.1 fig immunoglobulin/~l) for 30 min essentially as described by Davis et al. (1986). The resulting non-specific antigen-antibody complexes were precipitated by centrifugation in a benchtop microfuge at 2000g for 10 min. The ferritin-containing supernatant was incubated overnight at 5°C with an optimal amount (5 pg) of antiferritin antibody. The following day, all samples were incubated with 60 ~1 of protein A-Sepharose for 1 h at room temperature and then centrifuged for 10 min at 2000g. The resulting pellet was washed three times with 300 11 of fresh buffer. sedimented by centrifugation at 2OOOg for IOmin, and boiled with 40 ~1 of SDS sample buffer for 10 min in

order to solubilize the protein. Protein A-Sepharose “beads” were removed by centrifugation at 2000g for 10 min and the ferritin-containing supernatant was applied to cylindrical 15% (w/v) polyacylamide gels containing SDS. Following fixation in methanol/acetic acid/water (50: 10:40). the gels were sliced, solubilized and counted for radioactivity at 90% efficiency using a Searle Model 6892 liquid scintillation counter. Tubes without mRNA served as blanks for correction of the samples. A small aliquot of supernatant (5-10 ~1) was also counted for radioactivity. In other experiments Tachisorb was used. first for the precipitation of non-specific antigen-antibody complexes (sedimented by centrifugation at 12,OOOg for 5 min). and then for the precipitation of antiferritin-ferritin complexes. The supernatant from the non-specific step was heated at 75-80°C for 15 min to denature lysate proteins except ferritin which is heat stable. Denatured proteins were precipitated at 0°C for 30min and then sedimented by centrifugation at 20,OOOg for 20 min. The clear supernatant was incubated overnight at 5’C with an optimal amount of antiferritin antibodies. The following day, all samples were incubated with 250~1 Tachisorb for 1h at room temperature and then centrifuged for 10 min at 2000 g. The pellet containing antigen-antibody complexes was freed of nonspecific contaminants by centrifugation at 12,OOOg through 250 ~1 of a 1 M solution of sucrose in immunoprecipitation buffer. The resulting pellet was washed twice with 400 pl of immunoprecipitation buffer, re-sedimented by centrifugation at 2000g for IOmin, solubilized and counted for radioactivity. Miscellaneous Protein concentration was determined according to the procedure of Lowry et al. (1951) using bovine serum albumen as a standard. The RNA concentration was determined by the absorbance at 260 nm using an absorbance of 1.0 = 50pg/ml. The Al analyses were carried out by D. R. C. McLachlan and B. Krishnan using an automated atomic absorption spectrometer (PE Zeeman/300) as described previously (Krishnan et al., 1988). Values in the figures and tables are means + S.E.M. Means for Al exposed animals compared to control animals that had P < 0.05 by Student’s r-test are indicated with an asterisk.

RESULTS Protein

synthesis

using the polysomal

fraction

The mRNA bound to the brain polysomal fraction from rats (Fig. 1) and rabbits (Fig. 2) was translated in a mixture containing [‘“Cl leucine and an excess of protein-synthesizing factors. The incorporation of

Rat brain mRNA after AI

[‘“Cl leucine into protein in the rat brain preparations from animals exposed to IO or 20 mg Al/kg (Fig. I), was increased statistically significantly above the control preparations except for the lowest concentration of polysomes at the lower dose of AI. However, for rats exposed to oniy 5 mg Al/kg, the synthesis of labelled proteins by brain preparations was not statistically significantly different from that of control preparations (not shown). At 20 mg Al/kg there was about 50% mortality so that all of the subsequent experiments used 1Omg Al/kg over 3 weeks (i.e. a total of 60mg Al/kg). Fig. 2 shows the results for brain preparations from rabbits that received 5 or 10 mg Al/kg over 3 weeks. No statistically signi~cant difference in the synthesis of proteins was detected in this species exposed to AK?, by the i.p. route. At the highest dose, 20 mg Al/kg, no rabbits survived.

587

4.0 3.0 2.0

1.0

& I_

OLl

50

‘0

m-

X

4.0

100

150

100

150

r

3.0 2.0

a 1.0

a

J

;,I’

OLU 50

$ -

’

1.0 0 t

?P ‘0

r

5W

150 b

Fig. 2. Incorporation of [“C] leucine into protein using polysomes prepared from the brain of control or Al-exposed rabbits. The experiments were carried out as described for Fig. I. Mean f S.E.M. of 2 polysomal preparations, assayed in triplicate, each using 2 or 3 brains. O-0, control, A-A, 5 mg Al/kg, W--m, 10 mg Al/kg.

Protein synthesis due fo mRNA rather than ribosomes

I’9

RNA

Fig. 1. Incorporation of [‘%I] leucine into protein using polysomes prepared from the brain of control or Al-exposed rats. Polysomes were isolated from the brain of control and aluminium injected rats and varying amounts (SO-150 ng RNA) were incubated in a cell-free system which consisted of the following in a total incllbation volume of 0.25 ml: 0.25 M sucrose, SOmM Tris-HCI (pH 7.7), 80mM potassium acetate, 6mM magnesium acetate, 10 mM 2mercaptoethanoi, 5mM ATP, 0.5 mM GTP, 0.25 PCi of [“C] leucine, together with an excess (245 pg) of postmicrosomai supernatant fraction (S 10.5) obtained from control liver. The incorporation per tube into newly synthesized proteins was determined as described in the Methods section. Mean + S.E.M. of 4 or more polysomal preparations, assayed in duplicate, each using 2-5 brains. O-0, control, l -e t0 mg AI/kg, m--B, 20 mg Al/kg. a. b, P < 0.05.

Because of the possibility that the results shown in Fig. 1 could arise if the exposure to Al produced an increase in the activity of the ribosomes themselves, rather than in the mRNA bound to the ribosomes, the experiment shown in Table I was carried out. In this experiment [‘“Cl phenylalanine was used to measure protein synthesis, firstly using the mRNA bound to the brain polysomes and, secondly using the synthetic mRNA, polyuridylic acid (poly (U)). In order to use poly(U) as mRNA, the brain polysomes were first preincubated for 60 min in a complete protein-synthesizing system that had no labelled amino acids added, in order to free the polysomes of translatable mRNA. Then poly(U) and [‘“Cl phenylalanine were added and protein synthesis carried out for 20min. Table la shows that control brain polysomes incubated with [‘“Cl phenylalanine for 60min exhibited new protein synthesis that was comparable to that found using [‘“Cl leucine (Fig. 1). Similarly, brain polysomes from Al-exposed rats exhibited a statistically significant increase in protein synthesis (approx. 60%) over that of the controls. Furthermore, when the addition of [‘“Cl phenylalanine was delayed until after the first 40min of incubation there was little or no further synthesis of Iabelled protein, showing the depletion of mRNA activity by incubation for 40min.

588

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SAN-MARINAand D. MCEWENNICHOLLS

Table 1. Effect of exposure to Al on brain ribosomes studied by measuring the incorporation of [14q phenylalanine into protein using endogenous or exogenous mRNA source of Ribosomes

Incubation time (mitt)

Time [‘+Z] phe added (mm)

(a) Translation of endogenous mRNA 60 Control Aluminium 60 Control Aluminium (b) Translation of exogenous mRNA Control 20 Aluminium

0 40

0

Incorporation (cpmitube) 17lOi 2750 f 95 f 68k

238 114’ 25 14

1380 f 173 t510& I72

(a) Polysomes (90 pg+dx) were incubated with excess post-microsomal supernatant fraction as described under Methods. ]‘*C] phenylalanine (0.25 pCi) was added at 0 or 40 mitt in order to test the translational activity during a further 20 min incubation period. The reaction was stopped and the protein precipitated, washed and counted as described under Methods. Mean k S.E.M. of 3-6 samples. *P < 0.05. (b) Polysomes that had been incubated 60 min as in (a) without the addition of [“C] phenylalanine were isolated by centrifugation through a sucrose “cushion” as described under Methods and then reincubated as before with the addition of [‘%I phenylalanine and 6Opg of poly(U) per tube. Mean + S.E.M. of 36 samples.

When the prein~ubation procedure was carried out and fresh synthetic mRNA and [““Cl phenylaianine were provided for a second incubation, maximal synthesis of Iabelled polyphenylalanine occurred at 20 min (Table lb). In this reincubation, however, the results for brain polysomes incubated with exogenous mRNA were the same for preparations from Alexposed rats as they were for preparations from control rats. In other words, the activity of the endogenous mRNA bound to brain polysomes from Al-exposed animals, and not the activity of the ribosomes per se, accounts for the difference in protein synthesis in the two preparations.

and Tachisorb as described in Methods. There was a statistically significant increase in the iabelling of the immunoprecipitate synthesized by preparations from Al exposed rats (4600 f 862 cpm/tube, n = 5) compared to that synthesized by those from control rats (759 & 211 cpm/tube, n = 5). Ferritin mRNA in the RNP fraction and in the polysomal fraction It is now well known that, in the liver, ferritin mRNA is stored in cytoplasmic ribonucleoprotein particles which occur in the postribosomal supernatant fraction, and that this mRNA is bound to a protein that inhibits its translation (Leibold and Munro, 1988; Walden et al., 1989). Upon an increase in cytoplasmic iron (Fe) levels this mRNA is no longer inhibited and is released for translation by the polysomes. Therefore an experiment was carried out to compare the mRNA of the brain polysomal fraction with that of the cytoplasmic ribonucleoprotein particle fraction (i.e. postribosomal fraction) (Table 2). Following translation of this mRNA in a reticulocyte lysate, aliquots were obtained to measure total protein synthesis and then the immunoprecipitation of ferritin was carried out using protein A-Sepharose and antiferritin antibodies. The immunoprecipitate was subjected to SDS tube gel electrophoresis and the ferritin containing slices were eluted and counted for radioactivity. The proportion of ferritin synthesized relative to total proteins synthesized using polysomal mRNA was almost 2 times greater in preparations from Al-exposed rats than in those from control rats. Using mRNA that was obtained from the ribonucleoprotein particles the opposite result was observed, i.e., the proportion of ferritin synthesized relative to total proteins synthesized in the preparations from AI exposed animals

Protein synthesis using rat mRNA in a reticu~ocyte &sate Since the Al-induced increase in the incorporation of labelled amino acids into protein depended upon the endogenous mRNA bound to the ribosome, the polysomal mRNA was tested in a reticulocyte lysate protein synthesizing system that had been preincubated to remove endogenous mRNA. Figure 3 shows that the incorporation of [35S]methionine into protein depended upon the amount of polysomes containing mRNA that was added to the incubation mixture both for controls and for preparations from the brain of the Al-exposed rats. However, the incorporation was increased statistically significantly above the control values in the latter Al-derived preparations. Synthesis of brain ferritin in vitro Since it was possible that part of the increased protein synthesis was due to an increased synthesis of ferritin, the labelled translation products were treated with ferritin antibodies. RNA was extracted from Al-exposed or control brain polysomes and was translated in a reticulocyte lysate as described for Fig. 3. The [3sS] methionine-labelled proteins synthesized from equivalent amounts of control or test mRNA were precipitated with antiferritin antibodies

L

0

I 5

I IO

I 15

I 20

mRNP.

Fig. 3. Incorporation of [“S] methionine into proteins using brain polysomes from control or Al-exposed rats. Polysomes (5-1.5 pug RNA) were incubated in a reticulocyte lysate with 45 PCi [35S]methionine in a final volume of 25 ~1 for 60min as described in Methods. The reaction was stopped and the proteins were heated, washed and counted as described under Methods section. Mean f S.E.M. of 3 samples. O---0, control, O--0. Al. *P -c 0.05.

Rat brain

mRNA

Table 2. Effect of Al exposure on the incorporation of [“S] methionine into immunoprecipitable ferritin relative to total protein using brain mRNA of the polysomal fraction compared to brain mRNA of the postribosomal fraction Ferritin Total protein (%)

Source of brain mRNA Polysomal fraction Postribosomal fraction

Control Aluminium Control Aluminium

0.12 0.22 0.28 0.11

f 0.03 * 0.03’ i_ 0.02 +0.01*

Brain mRNA was prepared from the polysomal (3 brains pooled) and postribosomal (5 brains pooled) fractions obtained from control or Al-exposed rats. Equal amounts of RNA from control or Al preparations were incubated in reticulocyte lysate with [“‘S] methionine and the labelled translation products were counted and then immunoprecipitated using antiferritin and protein A-Sepharose as described in Methods. The immunoprecipitate was subjected to SDS gel electrophoresis on 15% (w/v) polyacrylamide cylindrical gels. The ferritin bands were determined by the simultaneous migration of horse spleen ferritin and these bands were sliced (2 mm) and counted for radioactivity. Mean + S.E.M. of 4 samples obtained in 2 separate experiments. ‘P < 0.05.

was only about one-third of that in the control preparation. In other words, in rats exposed to Al, the ferritin mRNA in the RNP particles is decreased while that bound to polysomes is increased relative to control preparations. When the brain of these rats was analyzed for total Al content after 1Omg Al/kg for 3 weeks, a statistically significant increase was found in the Al exposed rats (controls, 1.38 f 0.18 pg Al/g dry weight, n = 8; Al exposed, 3.23 + 0.30 pg Al/g dry weight n = 8). No change in brain weight, either on a wet weight or dry weight basis, could be detected, however. DISCUSSION

The results show that there is a marked increase in the synthesis of proteins in oitro using polysomes from the brain of young rats exposed to Al (lOmg/kg) for three weeks by i.p. administration. One of these proteins was ferritin which is known to be abundant in brain. The mRNA fraction was responsible for the increase and not the polysomes per se. When young rabbits were similarly exposed through the injection of AlCI,, no such effect was detected at levels of administered Al comparable to the levels responsible for an effect in rats. Intracerebra1 injection of the chloride, however, induces neurofibrillary degeneration (Wisniewski et al., 1980). In rabbits, using Al lactate (16 mg Al/kg body wt) or Al maltolate (3 mg Al/kg body wt) a decrease in mRNA activity was observed (Nicholls et al., 1990, 1991). Both Al lactate and Al maltolate are able to cross the blood-brain barrier more readily than the chloride, and especially the maltolate which is a ligand that is soluble both in water and in lipophilic solvents (Bertholf et al., 1989; Kruck and McLachIan, 1989). In these rabbits the level of brain Al was significantly increased from 0.5-l .Og Al/g dry weight to 2.5-3.5 pg Al/g dry weight (Nicholls et al., 1990), i.e. about the same level as that found in the brain of the rats studied here. Thus, similar accumulations of Al in the two species result in quite different overall mRNA responses. The increased synthesis of rat proteins but not rabbit proteins following elevated

after

Al

589

brain Al concentrations suggests that in the rat a number of these proteins, in addition to ferritin, might contribute a protective effect. When ferritin, which occurs in brain at about one-third of the level in liver (Fleming and Joshi, 1987; Dexter et al., 1990; Hill, 1990), was immunoprecipitated from the newly synthesized proteins, a significant increase in labelling was detected in the Al-exposed rats. Thus, it appears that Al may induce the synthesis of brain ferritin much as Fe induces the synthesis of liver ferritin. In this regard, administration by the oral route of low doses of AlCl, over a period of one year resulted in a significant increase in ferritin concentration in the liver of aged rats, though not in their brain (Fleming and Joshi, 1987). Since the brain continues to grow during the first 2-3 weeks of life in the rat (Robertson et al., 1985) and the blood-brain barrier remains incomplete and permeable to metals until after about 2 weeks of age it is not surprising that we can detect increased ferritin synthesis in brain preparations from infant rats. Furthermore, at two weeks of age, the brain of rats exhibits resistance to Fe overload (Taylor et al., 1991) suggesting ferritin involvement. It is well known that during this period the infant rat brain is particularly susceptible to changes induced by another metal, lead (Michaelson, 1973; Kennedy et al., 1983), while the present study shows that it is also susceptible to changes induced by Al. Thus, if the situation in vivo resembles that in vitro, there is likely an increased synthesis of brain ferritin based on mRNA translation (and perhaps transcription). If the degradation rate is not much changed, then increased levels of ferritin may result. In the liver considerable ferritin mRNA is bound to an inhibitory protein and is located in cytoplasmic mRNP particles. Increased levels of iron release this mRNA from the repressor protein and make it available for translation by the ribosomes (Leibold and Munro, 1988; Walden et al., 1989). In brain the situation may be similar following Al administration in view of our results which suggest a translocation of mRNA from the RNP fraction to the polysome fraction in preparations from the brain of Al-exposed animals. The mechanism of the Al effect on brain ferritin synthesis could be directly on the ferritin repressor protein, similar to the Fe effect on liver ferritin synthesis. Alternatively, the effect of Al could be an indirect effect. For example, Al might displace some of the Fe bound to ferritin and make Fe available for binding to the ferritin repressor protein. Studies of free and protein-bound metal may help to resolve this. While brain ferritin in mammals has not been studied extensively it may resemble other ferritins, composed of 24 protein subunits of the L and H type. There is a high amino acid sequence homology between L subunits of different species and about a 50% sequence homology between L and H subunits (Theil, 1987). In our experiments the immunoprecipitated ferritin is mainly the L subunit since the antibodies were raised to horse spleen ferritin which is composed of > 90% L subunits and since the heat treatment used has been reported to favour the isolation of a ferritin rich in L subunits (Frenkel et al.,

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1984). In liver ferritin there is evidence that after iron loading there is an increase in the L to H subunit ratio which is believed to lead to an increase in iron storage content per molecule (Dickey et al., 1987; White and Munro, 1988). Thus an increased synthesis of L subunits in brain, resulting either directly from Al induction or indirectly from induction by Fe that is mobilized by Al, may provide increased storage for Al. relieving some of the neurotoxic effect of this metal in young rats. The cytoplasmic localization of brain Al in an Al-resistant species, such as rats (Crapper et al., 1980), further suggests ferritin as a major protein capable of binding Al, since ferritin is one of the more abundant proteins in the cytosol. Acknowledgements-The authors are grateful to Professors D. R. Crapper McLachlan and T. P. A. Kruck for their encouragement and support throughout this work and to B. Krishnan for analysis of brain Al. The investigation was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. Mrs Ilse Fulton provided expert assistance in preparing the manuscript. REFERENCES Angelow R. W. and Nicholls D. M. (1991) The effects of mercury exposure on liver protein synthesis in rainbow trout. Comp. Biochem. Physiol. lOOC, 439-444. Bertholf R. L., Herman M. M., Savory J., Carpenter R. M., Sturgill B. C., Katsetos C. D., VandenBerg S. R. and Wills M. R. (1989) A long-term intravenous model of aluminium maltol toxicity in rabbits: Tissue distribution, hepatic, renal, and neuronal cytoskeletal changes associated with systemic exposure. Toxic. appl. Pharmac. 98, 58-74. Bishay E. S. and Nicholls D. M. (1973) Purification and characterization of alkaline ribonuclease inhibitor from normal and nephrotic rat kidney. Archs. Biochem. Biophys. 158, 1855194. Crapper D. R., Quittkat S., Krishnan S. S., Dalton A. J. and DeBoni U. (1980) Intranuclear aluminium content in Alzheimer’s disease, dialysis encephalopathy, and experimental aluminium encephalopathy. Acra neuropath. (Berl.) 50, 19-24. Crapper McLachlan D. R., Lukiw W. J. and Kruck T. P. A. (1989) New evidence for an active role of aluminium in Alzheimer’s disease. Can. J. neural. Sci. 16, 490-497. Crapper McLachlan D. R., Kruck T. P., Lukiw W. J. and Krishnan S. S. (1991) Would decreased aluminium ingestion reduce the incidence of Alzheimer’s disease? Can. med. Ass. J. 145, 793-804. Davis L. G., Dibner M. D. and Battey J. F. (1986). Basic Methods in Molecular Biology, pp. 3022305. Elsevier, New

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