P.E. Fraser

The cellular prion protein binds copper in vivo

The normal cellular form of prion protein (PrPC) is a precursor to the pathogenic protease-resistant forms (PrPSc) believed to cause scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeldt–Jakob disease. Its amino terminus contains the octapeptide PHGGGWGQ, which is repeated four times and is among the best-preserved regions of mammalian PrPC. Here we show that the amino-terminal domain of PrPCexhibits five to six sites that bind copper (Cu(II)) presented as a glycine chelate. At neutral pH, binding occurs with positive cooperativity, with binding affinity compatible with estimates for extracellular, labile copper. Two lines of independently derived PrPCgene-ablated (Prnp0/0) mice exhibit severe reductions in the copper content of membrane-enriched brain extracts and similar reductions in synaptosomal and endosome-enriched subcellular fractions. Prnp0/0mice also have altered cellular phenotypes, including a reduction in the activity of copper/zinc superoxide dismutase and altered electrophysiological responses in the presence of excess copper. These findings indicate that PrPCcan exist in a Cu-metalloprotein form in vivo.

Therapeutically effective antibodies against amyloid-β peptide target amyloid-β residues 4−10 and inhibit cytotoxicity and fibrillogenesis

Immunization of transgenic mouse models of Alzheimer disease using amyloid-β peptide (Aβ) reduces both the Alzheimer disease–like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Aβ42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4–10 of Aβ42, and that these antibodies inhibit both Aβ fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.

The Presenilin 1 Protein Is a Component of a High Molecular Weight Intracellular Complex That Contains β-Catenin

The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. We have used linear glycerol velocity and discontinuous sucrose gradient analysis to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a ∼250-kDa complex. This complex also contains β-catenin but not β-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a ∼180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (β-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF.

Two novel (M233T and R278T) presenilin-1 mutations in early-onset Alzheimer's disease pedigrees and preliminary evidence for association of presenilin-1 mutations with a novel phenotype

Eleven early-onset dementia families, all with affected individuals who have either presented clinical symptoms of early onset familial Alzheimers disease (EOFAD) or have been confirmed to have EOFAD by autopsy, and two early onset cases with biopsy-confirmed AD pathology, were screened for missense mutations in the entire coding region of presenilin-1 (PS-1) and -2 (PS-2) genes. Missense mutations were detected by direct sequence analysis of PCR products amplified from genomic DNA templates of affected individuals. Three pedigrees were attributable to known mutations in the PS-1 gene: P264L, E280A and the splice acceptor site (G to T) mutation, which results in the deletion of residues 290–319 of PS-1(PS-1 Δ290–319). In a fourth pedigree, a novel PS-1 mutation was identified in exon 7 (M233T), which is homologous to a pathogenic PS-2 mutation (M239V), and is characterized by a very early average age of onset (before the age of 35). In one early onset case, another novel PS-1 mutation was identified in exon 8 (R278T). Of the five remaining families and the other early onset case, none have missense mutations in the PS-1 or PS-2 genes, or in exon 16 and 17 of the APP gene. Moreover, two of the PS-1 mutations, PS-1 Δ290–319 and ρ278T, are associated with the co-presentation of familial spastic paraparesis (FSP) in some of the affected family members. Our data raise the possibility that the phenotypic spectrum associated with PS-1 mutations may extend beyond typical FAD to include FSP, a disease heretofore unsuspected to bear any relationship to FAD. In addition, our data suggest that other novel EOFAD loci, in addition to APP and the presenilin genes, are involved in the aetiology of up to 50% of EOFAD cases.

Presenilins Interact with Armadillo Proteins Including Neural‐Specific Plakophilin‐Related Protein and β‐Catenin

Abstract : Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early‐onset familial Alzheimers disease. We have used yeast‐two‐hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including β‐catenin, p0071, and a novel neuronal‐specific armadillo protein—neural plakophilin‐related armadillo protein (NPRAP). The PS1 : NPRAP interaction occurs between the arm repeats of NPRAP and residues 372‐399 at the C‐terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm‐like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.

Aβ1–40 but not Aβ1–42 levels in cortex correlate with apolipoprotein E ϵ4 allele dosage in sporadic Alzheimer's disease

Apolipoprotein E (ApoE) epsilon4 allele is established to be a risk factor for the development of late-onset Alzheimers disease (AD) which is associated with increased frequency of senile plaques and extent of amyloid angiopathy. In a recent report, we demonstrated that ApoE epsilon4 dosage correlates with an increase in A beta1-40 but not A beta1-42/43-immunoreactive plaques. In the present study, we sought to confirm this relationship at a biochemical level by using a sensitive ELISA to measure the amounts of A beta1-42/43 and A beta1-40 in cerebral cortex in 36 cases of sporadic AD. We found that dosage of ApoE epsilon4 allele correlated significantly with cortical A beta1-40 levels, while levels of A beta1-42 showed no significant association with genotype. These results parallel our immunohistochemical findings and suggest that A beta1-40 may play a key role in the pathogenesis of late-onset sporadic AD.

Spatial learning in transgenic mice expressing human presenilin 1 (PS1) transgenes

Dominant mutations in the Presenilin 1 gene are linked to an aggressive, early-onset form of familial Alzheimers Disease (FAD). Spatial memory of transgenic (Tg) mice expressing either mutant (lines Tg(M146L)1, Tg(M146L)76, Tg(L286V)198) or wild type (line Tg(PS1wt)195) human PS1 transgenes was investigated in the Morris water maze (WM) test at 6 and 9 months of age. The results showed that the mutated Tg mice had increased swim speed when compared to non-Tg littermates or Tg PS1 wild type mice. The swim speed difference did not, however, significantly affect the spatial learning in the WM test and all groups showed comparable search paths during training and similar spatial bias during probe trials. When re-tested at 9 months, all mice showed significantly improved learning acquisition of spatial information. The lack of progressive spatial learning impairment in mice expressing the mutated human PS1 transgene in the WM does not preclude impairments in other cognitive tasks but suggests that full phenotypic expression of mutant PS1 alleles may require co-expression of human versions of other AD-associated genes.

Presenilin 1 is Actively Degraded by the 26S Proteasome

The metabolic pathways governing the turnover of presenilin 1 (PS1) have been incompletely worked out. The PS1 holoprotein has low abundance in many cells and appears to undergo endoproteolytic cleavage near residue 298. We provide evidence that one mechanism by which the PS1 holoprotein is degraded is through the action of the 26S proteasome. We also show that the proteasome does not participate in the endoproteolytic cleavage.

Analysis of the functional effects of a mutation in SOD1 associated with familial amyotrophic lateral sclerosis

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene have been reported in some pedigrees with Familial Amyotrophic Lateral Sclerosis (FALS). We have investigated the functional and structural effects of a Gly-->Ser mutation at codon 41 of SOD1 in a pedigree with FALS and the topography of SOD1 expression in the mammalian CNS. These analyses show that the 41Gly-->Ser mutation causes a 27% reduction in Cu, Zn SOD activity. SOD1 is transcribed at high levels in rat motoneurons and four other types of neurons homologous to upper motoneurons that degenerate in human ALS. However, SOD1 is transcribed at lower levels in other types of neurons, such as cerebellar Purkinje cells, which are not usually involved significantly in human ALS. On the other hand, immunocytochemical studies indicate that most types of rat neurons contain similar levels of Cu, Zn SOD immunoreactive protein. Nevertheless, these results suggest that the essential feature causing this subtype of ALS is either a reduction in Cu, Zn SOD activity in cell types that presumably critically require Cu, Zn SOD for protection against oxidative damage or the fact that the mutation in SOD1 associated with FALS results in a novel gain of function that is particularly deleterious to those cell types expressing SOD1 at high levels.

Proteolytic processing of presenilin-1 (PS-1) is not associated with Alzheimer's disease with or without PS-1 mutations.

Cerebral presenilin‐1 protein (PS‐1) is normally composed of the amino‐terminal fragment (NTF) with M r 28 kDa and the carboxy‐terminal fragment (CTF) with 18 kDa. We analyzed human PS‐1 in brains with early‐onset familial Alzheimers disease (FAD) with and without PS‐1 mutations to study whether mutated PS‐1 was abnormally metabolized. Cerebral PS‐1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS‐1 mutation in human brains. A small portion of PS‐1 was recently found to suffer another processing by caspase‐3, an apoptosis‐related cysteine protease. In contrast to the recent finding that the Volga‐German mutation on presenilin‐2 (PS‐2) affects the increasing caspase‐3 PS‐2 fragment, the PS‐1 mutation did not cause a significant change in PS‐1 fragmentation. We conclude that PS‐1 fragmentation and other (probably caspase‐3‐mediated) digestion following apoptosis occur independently of PS‐1 mutations.