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Three novel alternatively spliced transcripts of the beta-site amyloid precursor protein cleaving enzyme (BACE) were cloned from human brain. Alternative splicing of the RNA occurs at an internal donor in exon 3 and/or an internal acceptor in exon 4. The splicing events lead to a deletion of 25 (BACE-I-476), 44 (BACE-I-457) and 69 (BACE-I-432) amino acids and the latter two caused the loss of two of four N-linked glycosylation sites. Although the mature form of BACE-501 was resistant to endoglycosidase H treatment, glycosylated forms of BACE-I-457 and BACE-I-476 were sensitive. This result suggests that BACE-I-457 and BACE-I-476 underwent different post-translational modifications. Moreover, the beta-secretase activity of BACE-I-457 and BACE-I-476 was significantly weaker than that of BACE-501. Thus, these isoforms may contribute to a physiological function of BACE.

We report the cDNA sequence of human Fe65L2. The human Fe65L2 encoded 486 amino acids; the deduced amino acid sequence was shorter by 18 amino acids than the rat protein and had 86% identity to the rat protein Three protein-protein interaction domains, a WW and two PID/PTB elements, were conserved among the Fe65 protein family. Human Fe65L2 mRNA was expressed in various tissues; a transcript of about 2.2 kb was mainly expressed in the brain. A splicing variant lacking two amino acids in the first PID/PTB element was detected. We also confirmed that the carboxyl-terminal region of PID/PTB of the Fe65L2 interacted with the intracellular domain of the Alzheimer's beta-amyloid precursor protein (APP) and APP-like proteins.

Proteolytic cleavage of amyloid beta-peptide (Abeta) from amyloid precursor protein (APP) is a key event in the pathogenesis of Alzheimer's disease. Beta-site amyloid precursor protein cleaving enzyme (BACE) cleaves the APP at the N-terminus of Abeta. We investigated whether particular stress conditions modify the expression and activity of BACE, and found that treatment of human neuroblastoma cells with protein synthesis inhibitors induced expression of a novel splice variant of BACE. This unusual transcript, I-127, is produced by usage of an internal splicing donor site in exon 3. The splicing event leads to a premature termination codon, as well as elimination of one of two conserved aspartic protease active sites, a transmembrane domain, and a C-terminal cytoplasmic tail from BACE. Low levels of this mRNA were found in the human brain. When expressed in cells, I-127 had no effect on Abeta secretion and was retained in the endoplasmic reticulum without propeptide removal. It was also unstable with a turnover t(1/2) of approximately 2h; normal BACE had a turnover t(1/2) of approximately 8h. Finally, I-127 was degraded in a proteasome-dependent manner. Thus, I-127 is regulated by both nonsense-mediated mRNA decay (NMD) and proteasome-dependent degradation.

The mutations of presenilins (PSs) gene and their clinicopathological correlations to Alzheimer's disease (AD) have lately attracted considerable attention. In this report we analyzed fifteen Japanese familial Alzheimer's disease (FAD) including 12 early-onset FAD and 13 sporadic AD patients for the mutation of PS-1 gene by direct sequence analysis. We found the mutations, G384A, E280A in two FAD and H163R in one sporadic AD patient, and no N141I or M239V mutation in PS-2 gene, and no mutation in exons 16 and 17 in amyloid precursor protein (APP) gene. Families in which we failed to find the mutation by this screening may have mutations elsewhere in PSs or in APP gene, or yet unidentified other AD loci may exist. This is the first report to find a sporadic AD patient having PS-1 mutation.

Apolipoprotein E (apo E) genotypes in 350 leprosy patients were examined and compared with those of 870 age-matched controls. The allelic frequencies of the apo E gene did not differ between demented patients with leprosy and controls. However, the frequency of apo E epsilon 4 allele was significantly higher in non-demented leprosy patients than in controls (P0.001). Of special interest is that the prevalence of E3/4 genotype in non-demented leprosy patients increased significantly with age, being 14.1%, 24.4%, and 28.3% in the 60s, 70s, and 80s, respectively (P0.05). These data suggest that apo E epsilon 4 is not a risk factor for senile dementia in elderly leprosy patients, and there exist factors to overcome the risk of apo E4 in leprosy patients.

We investigated the secretion of amyloid beta-protein (beta AP) in a mouse neuronal cell line SN49. SN49 cells stably transfected with mouse beta-amyloid precursor protein (APP) 695 cDNA released approximately three times greater amounts of a 4 kDa protein immunoreactive with anti-beta AP antibodies than untransfected and mock-transfected cells. This 4 kDa protein was further identified as mouse beta AP by direct amino acid sequence analysis. These results strongly suggest that the beta AP secretion occurs in mouse neuronal cells as in human cells.

To clarify the effects of the proteolytic cleavage of presenilin 1 (PS1) and presenilin 2 (PS2) proteins on their functions, we established stable cell lines which expressed the physiologically cleaved N-terminal fragment (NTF) with or without mutations of familial Alzheimer's disease (FAD). We found that exogenous expression of the PS1-NTF or PS2-NTF harboring FAD mutations was insufficient for increased production of amyloidogenic A beta X-42 peptide and that the overexpressed NTFs had no effect on the accumulation of endogenous presenilin fragments.

We examined expression and production of interleukin-3 (IL-3) mRNA and IL-3 protein in mouse primary cultured neurons and glia by the reverse transcription and polymerase chain reaction and a bioassay using an IL-3-dependent cell line. IL-3 mRNA was demonstrated mainly in hippocampal neurons but not in glia, while a small but definite production of bioactive IL-3 was detected in septal and hippocampal neuronal cultures. Thus, endogenous IL-3 might be produced by certain neurons in situ.

Whereas loss of CA3 neurons in the hippocampus of monkeys which died of stress ulcers suggests that some structural changes may occur, there is no direct evidence that shows stress-induced irreversible changes of neurons. When rats were orchidectomized (castrated) and stressed by restraint and immersion in water for 15 min/day for 30 days, significant loss of hippocampal CA3 and CA4 neurons was observed. Furthermore, primary cultured hippocampal neurons survived shorter when treated with corticosterone. This neuronal loss was prevented by simultaneous administration of testosterone in vivo and in vitro. These findings indicate that stress can contribute to neuronal degeneration associated with hypogonadal conditions such as aging.