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Sporadic Alzheimer's disease is a polygenic disease and the relation between many genetic risk factors and the development of Alzheimer's disease has been controversial. Accumulation of amyloid β-protein (Aβ) in the brain is the neuropathological hallmark and thought to be a key event in the upstream stage of pathological cascade of the disease. Although increased production of Aβ is established in the pathogenesis of familial Alzheimer's disease due to mutations in presenilin 1, 2, and amyloid β-protein precursor genes, there is no evidence of up regulated synthesis of Aβ in the brains of patients with sporadic Alzheimer's disease. In addition, aging is the most major risk factor for the disease. These findings suggest the possibility that reduction of the catabolic system of Aβ due to aging causes the formation of senile plaques in sporadic disease. Therefore, proteolytic enzymes of Aβ might be related to the development of sporadic Alzheimer's disease.
One of the enzymes responsible for the degradation of Aβ is neprilysin (NEP).1 This is a membrane bound metallopeptidase which is widely expressed in many tissues including the CNS. It cleaves Aβ 1–42 between amino acids 9 and 10 and between amino acids 37 and 38.2 Reduced mRNA and protein concentrations of NEP in the brain from patients with Alzheimer's disease were reported, suggesting that low concentrations of NEP contributed to the accumulation of Aβ.3 Recent investigation showed that NEP inhibitor infusion into the brain resulted in increased deposition of Aβ, indicating that NEP regulates proteolytic catabolism of Aβ in vivo.2 There is a dinucleotide repeat polymorphism in the 5′ region of the NEP gene. A lower molecular weight allele of NEP gene polymorphism is associated with low amplitude of P300 and increased risk of psychiatric disorders such as alcoholism, conduct disorders of children, and depression.4 As there is a positive correlation between serum concentration of NEP and psychiatric problems, increased number of dinucleotide repeats of the NEP gene is presumed to be related to lower serum concentrations of NEP.4 Therefore, decreased NEP correlated with dinucleotide repeat polymorphism in the 5′ region and might be related to the accelerated accumulation of Aβ through reduced catabolism of Aβ. To verify a role of NEP in the pathogenesis of Alzheimer's disease, we examined the relation of NEP gene polymorphism with the development of Alzheimer's disease and Alzheimer's type neuropathological changes.
Subjects comprised 75 postmortem confirmed patients who had had sporadic Alzheimer's disease (age at death range 62–104 years; mean 85.8 (SD) 7.8 years) and 89 non-demented people (age at death range 65–101 years; mean 85.3 (SD 7.8) years) from a postmortem series at Yokufukai Geriatric Hospital in Tokyo. There was no significant difference of ages at death between two groups. Non-demented patients were without any neurodegenerative disorders. The clinical and postmortem diagnosis was performed according to criteria of the NINCDS-ADRDA and DSM III-R and the Consortium to Establish a Registry for Alzheimer's disease (CERAD), respectively.5
We evaluated Alzheimer's disease-type neuropathological changes quantitatively as previously described.5 Briefly, we counted the densities of the senile plaques, senile plaques with dystrophic neurites and neurofibrillary tangles in the hippocampus and superior temporal gyrus from the brains of all patients. Ten×100 microscopical fields (field size 2.56 mm2) for senile plaques and dystrophic neurites, and 10×200 microscopical fields (field size 0.64 mm2) for neurofibrillary tangles were examined using specimens treated with methenamine-Bodian stain.
We extracted genomic DNA from the brain with phenol/chloroform. Genomic DNA was amplified by polymerase chain reaction (PCR) using the primer pairs described by Comings et al.4 Counting GT repeats in the 5′ region of the NEP gene was performed with an ALF DNA sequencer II (Pharmacia Biotech). Direct sequence analysis of PCR products of some samples with representative genotypes using ABI PRISM model 310 verified the number of GT repeats (Perkin-Elmer). The apolipoprotein E (ApoE) genotype was also determined.5
The distributions of NEP gene polymorphism in patients with Alzheimer's disease and non-demented subjected were examined by χ2 test. The same analysis was performed in the subgroups divided by ApoE ε4 status. The correlations between NEP gene polymorphism and the densities of the senile plaques, dystrophic neurites, and neurofibrillary tangles in the hippocampus and superior temporal gyrus in the brains from patients with Alzheimer's disease and non-demented patients, and ages at onset and durations of illness in Alzheimer's disease were examined by Kruskal-Wallis test. We used five sets of data, NEP genotype, NEP allele, longer allele, shorter allele, and the sum of GT repeats of two alleles as NEP gene polymorphism to classify our samples. Statistical significance was defined as two tailed probabilities of <0.01. All analyses were performed using the computer software StatView J-4.5 (Abacus Concepts).
The genotypic and allelic frequencies of the NEP gene in Alzheimer's disease and non-demented patients are shown in table 1. Five types of alleles, which represent 19–23 GT repeats and nine genotypes, were found in our samples. There were no significant differences in the frequency of NEP genotypes between Alzheimer's disease and non-demented in the total patients (table 1), ApoE ε4 carriers or non-ApoE ε4 carriers (data not shown). The densities of the senile plaques, dystrophic neurites, and neurofibrillary tangles in the hippocampus and superior temporal gyrus in the total cases, and ages at onset and durations of illness in Alzheimer's disease divided by NEP genotype are shown in table 2. The NEP genotype was not associated with the densities of the senile plaques, dystrophic neurites, or neurofibrillary tangles in the hippocampus or superior temporal gyrus. The NEP genotype also did not influence Alzheimer's disease-type neuropathological changes even when we divided the total cases into the subgroups with or without Alzheimer's disease. There was no correlation between NEP genotype and ages at onset or durations of illness. The results remained non-significant when we performed similar analysis using the other types of NEP polymorphism such as longer allele, shorter allele, and the number of sum of GT repeats of two alleles to classify our samples. The genotype, longer allele, shorter allele, or the number of sum of GT repeats of two alleles were not associated with the genotype or allele of the ApoE ε4 (data not shown). The ApoE ε4 allele was significantly associated with Alzheimer's disease (p=0.0001).
To our knowledge, this is the first study to examine the genetic relation between a catabolic enzyme of Aβ and sporadic Alzheimer's disease. Although the present study does not demonstrate association of NEP with the development of Alzheimer's disease or Alzheimer's-type neuropathological changes, we suppose that the investigation of the catabolic system of Aβ is important for four reasons. Firstly, it links to elucidation of the mechanism of accumulation of Aβ. As NEP is thought to be a main peptidase which accounts for the degradation of Aβ in the brain,1 it is necessary to examine the influence of the NEP gene on the severity of the senile plaques and dystrophic neurites to search for a role of clearance of Aβ in the deposition of Aβ. Secondly, this research contributes in clarifying a role for senile plaques and dystrophic neurites in the development of Alzheimer's disease. Thirdly, the detection of key molecules in the degradation of Aβ might directly lead to the treatment of Alzheimer's disease. Fourthly, recent analyses disclosed that families with late onset Alzheimer's disease are linked to genetic markers near the insulin degrading enzyme gene, which is thought to be one of the catabolic enzyms of Aβ.6 Genes of the degradating enzymes of Aβ such as the NEP gene still remain potential risk factors for sporadic Alzheimer's disease. The examination of other polymorphisms in the NEP gene or multivariate analysis taking in the related gene except ApoE which modifies the processing of Aβ might detect potential correlation of the NEP gene with Alzheimer's disease.
The study was supported in part by a Health Science Research Grant (to MY) from the Ministry of Health and Welfare, Japan and grants in aid for scientific research (to HM and MY) from the Ministry of Education, Science, Sports, and Culture, and (to NS) from the Japan Society for the Promotion of Science, Japan.