The SAMP8 mouse a model of Alzheimer disease

Biogerontology3:57–60,2002.©2002Kluwer Academic Publishers.Printed in the Netherlands.57 The SAMP8mouse:a model of Alzheimer disease?∗John E.MorleyDivision of Geriatric Medicine,Saint Louis University,St.Louis,Missouri,USA and GRECC,St.Louis Veterans Affairs Medical Center,St.Louis,MO63104,USA(e-mail:morley@;fax:+1-314-771-8575)Key words:β-amyloid,amyloid precursor protein,antisense,cognition, 9desaturaseAbstractThe SAMP8mice develops early abnormalities in learning and memory.These are related to abnormalities in septo-hippocampal function with a decrease in serotonin leading to an increase in GABA and a decrease in acetylcholine. The cognitive defects in these animals are due to overproduction ofβ-amyloid and can be reversed by antibodies to β-amyloid or specific antisense oligonucleotides.The major defect produced byβ-amyloid in these mice appears to be reduction in 9desaturase activity leading to altered membrane phospholipid content.The SAMP8mouse appears to be an excellent model to examine the pathophysiology of early defects seen in Alzheimer disease.Professor Takeda developed the SAM(Senescence Accelerated Mouse)models at Kyoto University (Takeda et al.1991).The SAM mice were derived from an AKR/J breeding colony–a number of these mice showed characteristics of rapid aging.Selective breeding led to the development of a number of sublines.The SAMP8was found to have age-related learning and memory deficits without displaying other signs of premature aging.In our laboratory these animals have been continuously bred for over a decade and have a median lifespan of18.9months and the controls(SAMR1)have a median lifespan of21 months of age(Flood and Morley1992).Animals in our colony are free of viral and bacterial infections and their behavioral characteristics have bred true for the last decade.In1986,Miyamoto et al.(1986)demonstrated an impairment in learning on the one-way footshock avoidance task run in a two chamber box in SAMP8 mice at12months of age.In our original study we reported that SAMP8mice had a significant failure of acquisition by9months of age,compared to SAMR1 mice that did not demonstrate an acquisition deficit until20months of age(Flood and Morley1992). To demonstrate this we utilized an aversive T-maze ∗Presented at the1st ASCMG meeting,Melbourne,Australia, March19–22,2001.paradigm,a task that is hippocampally dependent (Farr et al.2000).The SAMP8mice also learn lever press acquisition for milk reward poorly(Flood and Morley1992)as well as a complex black-white,left-right ten unit maze(Flood et al.1995a).SAMP8mice have a retention deficit for passive avoidance training in some(Miyamoto et al.1986)but not all studies(Flood and Morley1992).In the T-maze footshock avoidance task,SAMP8mice had retention deficits from8to12months of age(Flood and Morley 1993).R1mice showed similar defects at21to22 months of age and C57BL/6Nnia mice at24months (Flood and Morley1990).Female SAMP8mice also have an acquisition defect on the T-maze footshock avoidance(Flood et al.1995a).However,they failed to show a retention deficit.In males we did backcrosses between SAMP8 male mice and CD-1females(Flood et al.1995b). Groups of SAMP8mice with88%P8genes showed no acquisition or retention defects but those with94%, 97%,or100%P8genes showed poor acquisition and retention by12months of age.Lipofuscin content as a marker of aging did not differ across the lifespan in any of the backcross substrains.These studies establish the SAMP8as a good model of premature acquisition and memory deficits.58Neurotransmitters and memoryUtilizing a series of neuropharmacological studies we have established that a number of neurotransmitters are involved in the regulation of memory in SAMP8 mice(Flood and Morley1994;Flood et al.1993, 1998).The predominant defect appears to be in the septo-hippocampal pathway.A decline in serotonin input from the median raphe to the septum results in a disinhibition of GABA inhibition in the septum. This increase in GABA results in a decrease in acetyl-choline in the hippocampus.Beta-amyloid and memoryBeta-amyloid injected intracerebroventricularly or directly into the hippocampus,amygdala or septum results in a decrease in memory retention(Flood et al.1991,1994a).Utilizingβ-amyloid fragments we found thatβ-amyloid18–22was the key component for producing amnesia(Flood et al.1994b).DFFVG, an inert series of amino acids when administered by itself,blocked the amnestic effect ofβ-amyloid.This compound enhanced retention in older SAMP8mice (J.E.Morley and E.Roberts,unpublished data).Western blot analysis of hippocampal tissue from SAMP8mice showed a three-fold increase in amyloid precursor protein(APP)from4to12months(Morley et al.2000).APP did not increase significantly in R1mice over the same time period.APP mRNA was measured using an RNAase protection assay.The SAMP8mice showed an increase in APP mRNA from 4to12months with no increase inβ-actin mRNA over the same period.The increase in mRNA was less than that observed for its protein,APP.Beta-amyloid levels were significantly increased in8month SAMP8mice compared to4month mice,and were further increased at12months of age(Kumar et al.2000a).Kumar et al.(2001)cloned the APP cDNA from the hippocampus of an8month old SAMP8mouse. The nucleotide clones was99.7%homologous with that of other mice and89.2%with human homologs. The SAMP8mice displayed only one nucleotide substitution that was unique to the SAMP8mice viz.a substitution of alanine for valine at position300. We then expressed the cDNA from SAMP8mice in HeLa cells.This resulted in production of glycosylated mature APP.Utilizing an antisense oligonucleotide of APP(613–626),we demonstrated that in these cells it decreased the expression of APP in a close and time dependent manner.Smaller antisense oligo-nucleotides and two random oligonucleotides had no effect on expression of APP.Phosphorothionation of the active antisense oligonucleotide resulted in lower doses being effective,as did encapsulation of the oligonucleotide by lipofectamine.Synthesis of mRNA was unaffected by these treatments.By16months of age SAMP8mice demonstrate Beta amyloid plaques in the hippocampus.This would suggest that plaque formation is an epiphenomenona, unrelated to the effects of beta-amyloid on memory. The effect of decreasing beta-amyloid on behavior in SAMP8miceWe next examined the effect of Beta-amyloid anti-body,normal rabbit serum(a rich source of antibodies) and anti-IgG on acquisition in12months old P8mice (Morley et al.2000).Antibodies to Beta-amyloid produced a return of the acquisition testing to the level seen in a4-month-old SAMP8mouse.There were no effects of normal rabbit serum or Anti-IgG.In ongoing studies we have found that the Beta-amyloid antibody is effective for approximately14days(J.E.Morley, unpublished).In addition,antibodies to beta-amyloid resulted in a normalization of the pharmacological patterns of dose responses to a variety of neurotrans-mitters in12months SAMP8mice to those seen in 4month old SAMP8mice.Utilizing microdialysis techniques we have shown that Beta-amyloid antibody into the septum resulted in an increase in acetylcholine release from the hippocampus(S.A.Farr and J.E. Morley,unpublished observations).We next examined the effect of antisense oligo-nucleotides on cognition.For these experiments we utilized phosphorothiolated antisense oligonucleotides (Kumar et al.2000a).Two or more injections of the APP antisense oligonucleotide resulted in reversal of the acquisition and retention deficits seen in12month old SAMP8mice.A random antisense had no effect on cognition.The APP antisense oligonucleotide reduced the C-terminal but not the N-terminal level of APP in the hippocampus.It had no effect on mRNA for APP.In a series of studies we demonstrated that the anti-sense oligonucleotide crossed the blood-brain barrier (Kumar et al.2001).We then used this data to calculate the peripheral dose necessary to improve ing this dose we demonstrated that the antisense oligonucleotide when given peripherally to59SAMP8mice at12months of age could reduce the acquisition deficit.Overall these studies support the contention that memory deficits due to Beta-amyloid are not due to irreversible neurotoxic reactions but rather represent reversible neurotoxic reactions.Whether Beta-amyloid is the direct effector of these effects or it creates a cascade of events such as generation of free radical compounds(e.g.,H2O2or CO)remains to be determined.Sex hormonesNumerous studies have suggested,but failed to prove,a protective effect of estrogen on Alzheimer disease.In addition other steroid hormones have been suggested to enhance retention in mice(Flood et al.1992).In preliminary studies we have found an improvement in acquisition in female SAMP8mice given estradiol(S.A.Farr and J.E.Morley,unpub-lished results).Male SAMP8mice have a rapid decline in circu-lating testosterone levels from4to12months of age(Flood et al.1995c).This led us to examine the effect of testosterone replacement on retention in these mice.Testosterone decreased the memory deficit.This improvement in memory was associated with a decline in amyloid precursor peptide in limbic system structures.Fishing for memory associated genes:Micro arraysIn an attempt to demonstrate specific age-dependent gene expression in the SAMP8mouse,we compared the changes in gene expression in the hippocampus between SAMP8mice aged between4and12monhts and C57BL/6mice of similar ages.We utilized a microarray that contained stress protein and toxi-cology genes(Kumar et al.2000b).We found significant changes in the chaperone family which is involved in protein folding.These proteins demon-strate a significant decrease with age and may result in improper protein folding leading to desposition of amyloid into plaques.There was also a decrease in cytochrome P450IIB9-testosterone16∝-hydroxylase. Stress response regulars such as the JNK stress acti-vated protein kinase and p38MAP kinase were also decreased with age.The increase inubiquitin-like Figure1.Pathophysiology of the memory deficit in the SAMP8 mouse.protein NEDD8suggests an increase in free radical activity.In a separate study we found a decrease in the calcium-binding protein,calbindin and protein kinase Cγwhich correlated with a decline in retention testing in SAMP8mice(Armbrecht et al.1999). Downstream effects of beta-amyloid:Altered membrane mobilitySAMP8mice have a marked decrease in delta-9-desa-turase by10months of age923).This is accompanied by a decrease in the mRNA for delta-9-desaturase.The levels of unsaturated fatty acids are lowered to the same levels in brains of older SAMP8mice.Thesefindings suggest that beta-amyloid may be altering membrane mobility.This would result in different binding and/or effect of neurotransmitters in relationship to the neuronal membranes.These changes have been demonstrated in SAMP8mice and would explain the early deterioration in acquisition and retention in these mice.60ConclusionThe SAMP8mouse develops deficits in learning and memory early in its lifespan.This appears to be due to overproduction of beta-amyloid.By decreasing 9desaturase activity,beta-amyloid modulates membranefluidity and decreases the effectiveness of neurotransmitters involved in the upregulation of memory(Figure1).The data presented here suggest that the SAMP8mouse may be an excellent spontan-eous model for early Alzheimer Disease. 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