Promising Targets to Combat Alzheimer's Disease

5 November/3 December, 1999

Promising Targets to Combat Alzheimer's Disease

Approximately, 20 million people suffer from dementia caused by Alzheimer's disease (AD) world-wide. An inherited form (familial) of AD cases constitute a small portion of AD patients but its study sheds light on the mechanism of occurrence for major sporadic AD cases. Familial AD patients carry autosomal dominant mutations in the genes encoding presenilin proteins 1 and 2 (PS1 and PS2), and beta-amyloid precursor protein (APP). All presenilin PS1 and PS2 mutations in familial AD are missense mutations, which cause single amino acid substitutions in the primary structure of the presenilins. These mutations result in the increase of 42-amino acid amyloid_beta (A_b42, 4K) that is the primary component of insoluble amyloid plaques and vascular deposits in AD brains (Haass C and De Strooper B, 1999: uid=10542139 for general discussion).
APP is a type-1 transmembrane protein that resembles glycosylated membrane receptor, and in species as diverse as Drosophila and humans, APP or closely related proteins are expressed in multiple tissues and cell types. Different APP isoforms are derived from a single gene by alternative splicing. The most prevalent forms of APP are 695- to 770-amino acid glycoproteins with a large , a single hydrophobic transmembrane domain, and a short cytoplasmic portion. Three enzymes, alpha-, beta- and gamm-secretases, are thought to cleave APP into A_b fragments of different sizes. APP is first cleaved by alpha- or beta-secretase in its ectodomain . beta-Secretase cleaves APP at the N-terminus to release APPs_beta (a 100-kD soluble N-terminal fragment and C99, a 12-kD C-terminal fragment which remains membrane bound). Cleavage by alpha-secretase produces APPs_alpha (a large soluble N-terminal fragment and C83, a 10-kD membrane-bound C-terminal fragment). Both C-terminal fragments, C99 and C83, then become the substrates for one or more gamma-secretases which cleave the fragments within their transmembrane domains, leading to the release and secretion of A_b and the nonpathogenic p3 peptide, respectively.

Intense efforts have been directed toward the identification of alpha-, beta-, and gamma-secretases. In the case of beta-secretase, the enzyme is expressed in most cell cultures examined. The cleavage site is specific (between residues 671 and 672 of APP), and in human embryonic cells, the majority of A_b starts at Asp1, but a minority of peptides start at Val-3 and Glu11. Human embryonic kidney 293 cells express beta- and gamma-secretases involved in A_b production, and also overexpress APP containing pathogenic Swedish mutation (Met to Leu at P1 position, or K670M671 to N670L671 at the beta_secretase cleavage site) that somehow dramatically enhances beta-secretase cleavage. A cDNA expression library from this cell line was constructed and transfected 293 cells, and A_b was assayed by ELISA . One clone was positive in the primary screening and encoded a novel protein, which was termed BACE (beta-site APP cleaving enzyme; Vassar R et al, 1999: uid=10531052). BACE cDNA (2526 base pairs, 501 amino acid open reading frame) shared significant sequence similarity with members of the pepsin subfamily of aspartic proteases. When BACE cDNA transfected 293 cells overexpressing APP, beta-secretase activity increased 2-fold as assayed by A_b production, and BACE induced cleavage only at the known beta-secretase positions, Asp1 and Glu11. Together with other observations such as BACE distribution in the brain, BACE matches all known properties of beta-secretase.

More recently (namely a few weeks later in this intense race), Sinha S et al (1999: uid=10591214) also reported a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and found it to be the predominant beta-cleavage activity in human brain. The researchers cloned the gene and purified the enzyme to homogeneity from human brain using a substrate analog inhibitor. The purified enzyme has all the properties predicted for beta-secretase and is a new membrane-bound aspartic proteinase. Another research group (Yan et al,1999: uid=10591213) simultaneously identified a membrane-bound aspartyl protease (termed Asp2) with beta-secretase activity. Asp2 is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduced amyloid_b production. Solubilized Asp2 protein cleaved a synthetic APP peptide substrate at the beta-secretase site, and remarkably the rate of cleavage was increased tenfold by the Swedish mutation associated with early-onset Alzheimer's disease.

Thus, beta_secretase (membrane-bound aspartic protease) is a new protein target for drugs to be designed to block the production of amyloid_b peptide. Such drugs should block the consequent formation of amyloid plaque in Alzheimer's disease. The ultimate validation may require further analysis of the effect of the beta_secretase deficiency in vivo, for instance made by gene targeting in the model system, and of behavior of beta_secretase alleles for the risk of Alzheimer's disease.

In addition to beta-secretase involvement in the risk of Alzheimer's disease, gamma-secretase also draws much attention. Recent findings indicate that APP processing is affected through the effects of PS proteins on gamma-secretase, resulting in the increased production of A_b peptide fragments. PS proteins are integral membrane proteins with a proposed structure implying eight transmembrane domains, and in neurons have been localized to the endoplasmic reticulum (ER) and the Golgi compartments. This localization overlaps to some extent on the site of amyloidgenic A_b42 generation. PS proteins somehow control the proteolysis of the integral membrane domains of APP, and their proteolytic activity is relatively selective. When PS1 is absent, cleavage by gamma-secretase of the transmembrane domain of APP was prevented, causing carboxylterminal fragments of APP to accumulate and thus resulting in a fivefold drop in the production of A_b peptide (De Strooper B et al,1998: uid=9450754). Cleavage by alpha- and beta-secretase of the extracellular domain of APP was not affected by the absence of PS1. PS1 specifically controls the proteolytic activity of gamma-secretase. In fact, PS proteins not only control proteolysis of other proteins, but also are themselves proteolytically processed. In vivo, only very small amounts of the holoprotein (full-length PS) can be detected, while rather high levels of 30K amino-terminal fragment and 20K carboxyl-terminal fragment of PS proteins are observed in all tissues and cell lines so far examined. These fragments are assembled into active complexes together with other proteins such as beta-catenin (Capell A et al, 1998: uid=9452432; Kang DE et al, 1999: uid=10341227).

It is still a long way from treating AD patients effectively. However, beta-secretase, PS proteins and gamma-secretase may present valuable molecular targets for the development of drugs to lower the production of highly amyloidgenic A_b and to combat AD. There is no guarantee that lowering the A_b in the brain indeed reduces neuronal degeneration. One of the encouraging findings in this line is the report by (Schenk, D et al, 1999: uid=10408445) showing that immunization with A_b attenuates Alzheimer-disease-like pathology in the PDAPP mouse. The PDAPP transgenic mouse overexpresses mutant human APP (Val717Phe) and progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. The mice were immunized with A_b42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when A_b deposition and several of the subsequent neuropathological changes were well established. Immunization of the young mice prevented the development of A_b plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older mice also markedly reduced the extent and progression of these AD-like neuropathologies. Thus immunization with A_b may be possibly effective in preventing and treating Alzheimer's disease.

How does neuronal degeneration occur in AD brains. Xu X et al (1999: uid=10377452) recently assessed the potential effects of APP on neuronal death and survival. When APP-deficient rat neuroblastoma cells (B103) were transfected with DNA constructs encoding wild-type or familial AD-mutant human APP (V717I, found at least 16 different AD families), wild-type, but not AD-mutant, APP effectively protected cells against apoptosis induced by ultraviolet irradiation, staurosporine, or p53. Wild-type APP also strongly inhibited p53 DNA-binding activity and p53-mediated gene transactivation, whereas AD-mutant APP did not. APP appears to protect neuronal cells against apoptosis by controlling p53 activation at the post-translational level. Whatever the mechanism, it is most likely that apoptotic process is involved in the neuronal degeneration and that APP mutation affects that process.

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