Elevation of cerebral proteases after systemic administration of aluminum

The levels of three proteases in the cerebral cortex of rats following a three week exposure to aluminum\ were measured[ The activity of apopain "CPP21#\ an interleukin 0 b converting enzyme "ICE#!like cysteine protease speci_cally associated with apoptosis\ was increased following dosing with aluminum[ The activity of calcium!activated neutral protease\ calpain\ was also increased[ However\ the enzyme activity of trypsin!like serine protease\ known to be elevated by oxidative events\ was unchanged[ Since aluminum is suspected as a possible factor in the pathogenesis of Alzheimer|s disease and other neurological diseases\ it is speculated that changed levels in proteolytic enzymes may relate to the neurotoxicity of aluminum[ (cid:11) 0887 Published by Elsevier Science Ltd[ All


0[ Introduction
Aluminum is a neurotoxin which has been proposed to be associated with neurodegenerative diseases such as Alzheimer|s disease "AD# "Crapper et al[\ 0862^Perl and Brody\ 0879^Trapp et al[\ 0867# and amyotrophic lateral sclerosis "ALS# "Garruto et al[\ 0875^Perl et al[\ 0871#\ but such a relationship has hitherto not been clearly established[ AD is pathologically characterized by the appearance of senile plaques\ neuro_brillary tangles "NFT#\ and loss of neuronal cells[ Accumulation of pro! teins or peptides\ speci_cally b!amyloid and tau\ within these lesions are hallmarks of AD and may be\ in part\ due to disregulation of protease enzymes[ Certain pro! teases may play important roles in AD[ Modi_cations of chymotrypsin!like proteases and trypsin!like serine pro! teases have been shown to be involved in accumulation of b!amyloid and tau protein in AD "Smith et al [ Iron is a very potent pro!oxidant and aluminum is known to potentiate iron!related reactive oxygen species formation in isolated systems "Otieza\ 0883^Bondy and Kirstein\ 0885#[ For this reason\ the current study also used groups of rats treated with iron\ both alone and combined with aluminum\ in order to determine whether aluminum modulates iron!induced ROS production in living animals[

2[0[ Apopain protease activities
Using Ac!asp!glut!val!asp!aminomethylcoumarin as a speci_c apopain "capsase 2# substrate\ aluminum treated animals had signi_cantly elevated apopain activities in the brain\ while iron!treated rats showed no signi_cant change " Fig[ 0#[ Following combined treatment with aluminum and iron\ there was a signi_cant elevation of apopain activity that mirrored the changes found fol! lowing exposure to aluminum alone[ To test the possibility that elevated apopain activity was associated with apoptosis\ we measured cellular DNA fragmentation by assaying~uorescently labeled enzymatically incorporated dideoxynucleotide at 2?!OH ends of fragmented DNA[ However\ no signi_cant chan! ges between control animals and aluminum treated animals were found "data not shown#[

2[1[ Calpain proteases
Using tert!butoxycarbonyl!leu!met amide 6!amino!3! chloromethylcoumarin as a model calpain substrate\ total calpain activity in brains of rats treated with aluminum for three weeks was signi_cantly elevated in comparison to control values " Fig[ 1#[ No signi_cant di}erences were found in rats treated with iron alone\ while combined treatment with aluminum and iron signi_cantly increased total calpain activity relative to controls in a manner parallel to changes found using aluminum alone[ When calpain was assayed in the absence of EDTA and in the presence of 49 mM Ca 1¦ \ activity was too low to allow accurate determination "data not shown#[ Thus the cal! pain subclass that was studied here was almost solely calpain II[

2[2[ Trypsin!like serine proteases
The activities of trypsin!like serine proteases in rat brain were slightly elevated in the aluminum\ iron and alumi! num with iron treated animals relative to the control group as judged by rates of hydrolysis of rhodamine 009\ bis!"benzyloxycarbonyl!ile!pro!arg amide#[ However\ these elevations were not statistically signi_cant " Fig[ 2#[

2[3[ Addition of aluminum to cerebral preparations in vitro
Aluminum sulfate was added directly to isolated cerebral S1 fractions and protease activity was determined[ As the concentrations of aluminum tested "0Ð49 mM#\ there was no detectable e}ect on the levels of any of the three proteases assayed "data not shown#[ Thus the enzymatic changes found in dosed animals re~ected a metabolic [ This protease has been shown to activate PARP "poly "ADP!ribose#!polymerase#\ which in turn recognizes DNA strand breaks "such as those that occur during the controlled degradation of DNA when cells undergo apoptosis#[ Apopain has been implicated as the capsase which best _ts the evidence for a central role in apoptosis "Mukasa et al[\ 0886#[ It may be relevant that both of the proteases found to be elevated in this study\ "calpain and apopain# are associated with apoptic cells and appear to operate in conjunction "Nath et al[\ 0885#[ Aluminum treatment signi_cantly increased apopain activity relative to controls while iron treatment had no e}ect on activity levels of the enzyme[ The com! bination of aluminum and iron also had a signi_cant e}ect on activity elevation which closely paralleled the increase due to aluminum exposure alone[ Thus alumi! num\ administered systemically\ may modulate cortical apoptotic pathways[

3[1[ Calpain
Since the level of calpain assayed in the presence of a low calcium concentration "49 mM# was very low\ the calpain subclass assayed in this study was predominantly calpain II which is known to be activated under pathological conditions[ The role of calpain protease in the patho! physiology of neurodegenerative disease has been the subject of several studies "Ostwald et al [\ 0882^Saito et al[\ 0883#[ In the current study\ calpain activity increased following aluminum but not iron treatment[ The com! bination of aluminum and iron also resulted in an increase that most likely solely re~ected the contribution of aluminum alone since the aluminum and iron level was roughly equal to aluminum alone[ Subsequent to intracellular Ca 1¦ elevation\ calpain undergoes autolysis and becomes activated "Shea et al[\ 0885#[ Protein kinase C "PKC# is a substrate for calpain and becomes constitutively activated as the catalytic domain is cleaved from the regulatory domain of the PKC[ This activation has been shown to result in hyper! phosphorylation of the cytoskeletal protein tau in human neuroblastoma cells\ which renders tau a less e.cient substrate for proteolysis by calpain with subsequent abnormal accumulation of tau "Shea et al[\ 0884#[ Alumi! num has previously been found to increase the resistance of a variety of calpain substrates to hydrolysis "Nixon et al[\ 0883#[ The presence of aluminum may exacerbate these events by inducing high intracellular Ca 1¦ levels "Julka and Gill\ 0885#\ as well as directly interacting with hyperphosphorylated tau "Guy et al[\ 0880^Savory et al[\ 0884#\ thereby making tau even more resistant to proteolysis by calpain[ However\ since saturating con! centrations of calcium were used in our assay\ the increase in calpain activity described here cannot re~ect alteration of intracellular Ca 1¦ levels in the brain[ Aluminum did not directly stimulate calpain in vitro\ and so the e}ect of this metal in vivo\ involved more complex mechanisms than direct interactions with proteins[ In the absence of added calcium\ calpain II has previously been found to be inhibited by millimolar concentrations of aluminum chloride added in vitro "Zhang and Johnson\ 0881# but such concentrations are unlikely to be encountered in intact tissues[

3[2[ Trypsin!like serine proteases
The trypsin!like serine protease group is one of the most abundant protease classes found in the brain[ It has approximately 4!fold higher activity in cortical tissue than in rat liver "unpublished results#[ However\ treat! ment of rats with aluminum\ iron\ or the two metals together had no signi_cant e}ect on the activity of this protease in rat brain[ This enzyme is known to be upre! gulated following oxidative damage to proteins "Dean\ 0876^Davies\ 0882#[ Despite reports suggesting that pro! oxidant status may be enhanced following treatment with aluminum salts "