A beta 1-40, a major component of Alzheimer's disease cerebral amyloid, is present in the cerebrospinal fluid and remains relatively soluble at high concentrations (less than or equal to 3.7 mM). Thus, physiological factors which induce A beta amyloid formation could provide clues to the pathogenesis of the disease. It has been shown that human A beta specifically and saturably binds zinc. Here, concentrations of zinc above 300 nM rapidly destabilized human A beta 1-40 solutions, inducing tinctorial amyloid formation. However, rat A beta 1-40 binds zinc less avidly and is immune to these effects, perhaps explaining the scarcity with which these animals form cerebral A beta amyloid. These data suggest a role for cerebral zinc metabolism in the neuropathogenesis of Alzheimer's disease.

A candidate gene for the chromosome 1 Alzheimer's disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.

BACE1 activity is significantly increased in the brains of Alzheimer's disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Na(v)1) beta2-subunit (beta2), a type I membrane protein that covalently binds to Na(v)1 alpha-subunits, is a substrate for BACE1 and gamma-secretase. Here, we find that BACE1-gamma-secretase cleavages release the intracellular domain of beta2, which increases mRNA and protein levels of the pore-forming Na(v)1.1 alpha-subunit in neuroblastoma cells. Similarly, endogenous beta2 processing and Na(v)1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimer's disease patients with high BACE1 levels. However, Na(v)1.1 is retained inside the cells and cell surface expression of the Na(v)1 alpha-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving beta2, thus regulates Na(v)1 alpha-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimer's disease patients with elevated BACE1 activity.

Most cases of early-onset familial Alzheimer's disease (FAD) are caused by mutations in the genes encoding the presenilin 1 (PS1) and PS2 proteins, both of which undergo regulated endoproteolytic processing. During apoptosis, PS1 and PS2 were shown to be cleaved at sites distal to their normal cleavage sites by a caspase-3 family protease. In cells expressing PS2 containing the asparagine-141 FAD mutant, the ratio of alternative to normal PS2 cleavage fragments was increased relative to wild-type PS2-expressing cells, suggesting a potential role for apoptosis-associated cleavage of presenilins in the pathogenesis of Alzheimer's disease.

Synthesis of the iron-storage protein ferritin is thought to be regulated at the translational level by the cytosolic content of chelatable iron. This response to iron is regulated by the iron-modulated binding to ferritin mRNAs of a repressor protein, the iron regulatory element-binding protein. From measurements made in a cell-free system, regulation of the iron regulatory element-binding protein has been recently suggested to involve direct interaction with hemin. The following observations on the synthesis of ferritin and of heme oxygenase (HO), the heme-degrading enzyme, in rat fibroblasts or hepatoma cells lead us to conclude that chelatable iron is a direct physiological regulator of ferritin synthesis in intact cells: (i) the inhibitor of heme degradation, tin mesoporphyrin IX, reduces the ability of exogenous hemin to induce ferritin synthesis but enhances HO synthesis; (ii) the iron chelator desferal suppresses the ability of hemin to induce synthesis of ferritin but not of HO; (iii) the heme synthesis inhibitor succinylacetone does not block iron induction of ferritin synthesis; (iv) there is no apparent relationship between the ability of various metalloporphyrins to inactivate the iron regulatory element-binding protein in cell-free extracts and their capacity to induce ferritin synthesis in intact cells; (v) administered inorganic iron significantly induces the synthesis of ferritin but not of HO; (vi) addition of delta-aminolevulinic acid to stimulate heme synthesis represses the ability of inorganic iron to induce ferritin synthesis while activating HO synthesis. Taken together, our results demonstrate that (i) release of iron by HO plays an essential role in the induction of ferritin synthesis by heme and (ii) chelatable iron can regulate ferritin synthesis independently of heme formation.

Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an ϳ53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an ϳ20-kDa C-terminal fragment and an ϳ34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.A significant portion of Alzheimer's disease (AD) 1 is attributed to specific gene defects leading to familial Alzheimer's disease (FAD) (1-5). Two homologous genes, presenilin 1 (PS1) and presenilin 2 (PS2), are responsible for at least 50% of early onset (Ͼ60 years old) FAD (2, 3). PS1 and PS2 are serpentine proteins consisting of six to nine predicted transmembrane domains interspersed with one large and multiple smaller hydrophilic loops (4, 5). At the amino acid level, the two proteins are 67% identical and exhibit significant homology to two Caenorhabditis elegans gene products, sel-12 (approximately 50% identity) which has been predicted to facilitate Notch receptor function (6), and spe-4 (approximately 26% identity) which is involved in cytoplasmic trafficking of proteins during spermatogenesis (7). PS1 and PS2 are ubiquitously expressed (4, 5) and in brain are expressed primarily in neurons, with similar regional distributions (8 -10). The presenilins are localized to the endoplasmic reticulum (ER) and the Golgi apparatus but not the plasma membrane suggesting a potential role in protein processing (8,11,43). To date, the PS1 and PS2 genes have been shown to contain 35 different mutations which are inherited in an autosomal dominant fashion in over 60 kindreds with early onset FAD (4, 5, 12; for summary, see Ref.3). Recent studies suggest that the presenilins may directly or indirectly affect the processing of APP leading to increased production of A␤42 (13-16). These results help to explain the relatively high degree of amyloid burden in the brains of FAD patients carrying PS1 and PS2 mutations. The pathogenic mechanism by which presenilin mutations lead to increased ␤-amyloid deposition and other neuropathological features of AD remains unclear. To begin understanding the role(s) of PS2 in normal cellular metabolism and AD pathogenesis, we investigated the processing and degradation pathways of PS2. EXPERIMENTAL PROCEDURESMat...

The voltage-gated sodium channel ␤2-subunit (␤2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that ␤2 undergoes ectodomain shedding followed by presenilin (PS)-dependent ␥-secretase-mediated cleavage. 12-OTetradecanoylphorbol-13-acetate treatment or expression of an ␣-secretase enzyme, ADAM10, resulted in ectodomain cleavage of ␤2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa Cterminal fragment (␤2-CTF) was independently inhibited by three specific ␥-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. ␥-Secretase inhibitor treatment also increased endogenous ␤2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free ␥-secretase assay, we detected ␥-secretase activity-dependent generation of a 12 kDa ␤2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of ␤2 processing by ␥-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific ␥-secretase inhibitor, would alter ␤2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing ␤2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased ␤2-CTF levels, we also tested whether ␤2-CTFs or ␤2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of ␤2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that ␤2 is a PS/␥-secretase substrate and ␥-secretase mediated cleavage of ␤2-CTF is required for cell-cell adhesion and migration of ␤2-expressing cells.A major pathologic hallmark of Alzheimer disease is the deposition of amyloid ␤-peptide (A␤) 1 into senile plaques. A␤ is produced from ␤-amyloid precursor protein (APP) by sequential ␤-and ␥-secretase-mediated cleavages. An alternative to the ␤-secretase pathway is a cleavage mediated by ␣-secretase, which cuts in the middle of the A␤ region of APP (1). APP has been shown to serially undergo either ␤-␥ and ␣-␥ cleavages. Both A␤ and the ␤-secretase-mediated C-terminal cleavage product of APP, C99 or CTF␤, have been shown to contribute to neurodegeneration in Alzheimer disease (2, 3). ␥-Secretase is a multisubunit aspartyl protease complex, harboring presenilin 1 (PS1) or 2 (PS2), nicastrin, aph-1, pen2, and likely additional factors (4). The presenilins are polytopic membrane proteins that constitute either catalytic subunits or necessary cofactors of the ␥-secretase complex (5, 6). Besides APP, PS/␥-secretase is required for the intramembraneous cleavages of at least 15 additional proteins, including Notch, nectin-1␣, and recently NRADD (4, 7-11). All the known substrates...

No abstract