␤-Amyloid (A␤) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of A␤ aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized A␤1-40 peptide. We found that slow rotation of A␤1-40 solution reproducibly gave self-aggregated A␤1-40 containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other A␤1-40 aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10-to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when A␤1-42 formed ASPD by slow rotation. However, A␤1-42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed Ϸ100-fold-higher toxicity than A␤1-40-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break ␤-sheet interactions, A␤ may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I͞gly-cogen synthase kinase-3␤ in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimer's disease.A 40-to 42-residue peptide named ␤-amyloid (A␤) is a major constituent of senile plaques in Alzheimer's disease (AD) (1). Although multiple pathways have been suggested to lead to AD, recent advances indicate a causal link between A␤ and AD (2), and this idea is supported further by findings that vaccination against A␤ ameliorates behavioral deficits in transgenic mice (3-5). Among various in vivo A␤ species, A␤ 1-42 generally is considered as the primary vehicle of toxicity, whereas A␤ 1-40 , a major species under physiological conditions, is considered less harmful and more resistant to the formation of oligomers than A␤ 1-42 (6). However, it remains controversial which A␤ species contributes predominantly to AD pathogenesis, because both in vitro and in vivo studies have confirmed toxicity of A␤ 1-40 aggregates (7,8).It has been widely accepted that toxicity of A␤ requires aggregation of native A␤ monomers (9-11). Besides fibrils, several types of nonfibrillar aggregates have been reported: A␤ 1-40 oligomers from dimers-hexamers (6, 12-14); a mixture of A␤ 1-42 oligomers named A␤-derived diffusible ligands (ADDLs), ranging from trimers-hexamers up to 24-mers (15); and fibril intermediates named protofibrils (PFs) (16,17). All of these aggregates are mixtures of A␤ oligomers with a variety in oligomer size, and precise morphological analysis of each A␤ a...

Amyloid ␤-protein (A␤) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, A␤ assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10 -15-nm spherical A␤ assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (>100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, A␤-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other A␤ assemblies.Neurodegenerative diseases, such as Alzheimer disease (AD), 2 Parkinson disease, prion diseases, and the polyglutamine diseases, arise from abnormal protein interactions in the central nervous system (1). In these diseases, complex multistep processes of protein conformational change and accretion produce various nonfibrillar assemblies, leading finally to fibrils (1-5). Recent studies have suggested that the early assemblies in this process might be the most toxic, possibly through the exposure of buried moieties and the formation of surface tertiary structures not present in physiologic monomers (6). These surface tertiary structures could mediate abnormal interactions with other cellular components (1).In AD, extensive studies have suggested that accumulation of amyloid ␤-protein (A␤), a physiologic derivative of amyloid precursor protein (APP), plays a primary pathogenic role (7-9). Various forms of assemblies ranging in mass from dimers up to multimers of ϳ1 MDa have been reported as neurotoxins (10 -13) as follows: protofibrils (14); dimers/trimers (natural low-n oligomers) (15); 3-24-mer A␤-(1-42) assemblies termed A␤-derived diffusible ligands (ADDLs) (16); 12-mers termed globulomers (17) or A␤*56 (18); 15-20-mer A␤ assemblies te...

Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease) is an autosomal-recessive disorder caused by a deficiency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS; E.C.3.1.6.4). GALNS is required to degrade glycosaminoglycans, keratan sulfate (KS), and chondroitin-6-sulfate. Accumulation of undegraded substrates in lysosomes of the affected tissues leads to a systemic bone dysplasia. We summarize information on 148 unique mutations determined to date in the GALNS gene, including 26 novel mutations (19 missense, four small deletions, one splice-site, and two insertions). This heterogeneity in GALNS gene mutations accounts for an extensive clinical variability within MPS IVA. Seven polymorphisms that cause an amino acid change, and nine silent variants in the coding region are also described. Of the analyzed mutant alleles, missense mutations accounted for 78.4%; small deletions, 9.2%; nonsense mutation, 5.0%; large deletion, 2.4%; and insertions, 1.6%. Transitional mutations at CpG dinucleotides accounted for 26.4% of all the described mutations. The importance of the relationship between methylation status and distribution of transitional mutations at CpG sites at the GALNS gene locus was elucidated. The three most frequent mutations (over 5% of all mutations) were represented by missense mutations (p.R386C, p.G301C, and p.I113F). A genotype/phenotype correlation was defined in some mutations. Missense mutations associated with a certain phenotype were studied for their effects on enzyme activity and stability, the levels of blood and urine KS, the location of mutations with regard to the tertiary structure, and the loci of the altered amino acid residues among sulfatase proteins.

Development of the airways, alveoli, and the pulmonary vasculature in the fetus is a process that is precisely controlled. One of the growth factors involved, vascular endothelial growth factor (VEGF), is so critical for embryonic development that in the mouse, elimination of just a single allele is lethal. In the early stages of lung development, the mouse VEGF gene expresses three isoforms (120, 164, and 188) in a distinct temporo-spatial pattern, suggesting a specific function for each. We engineered mice that express only VEGF 120, to study the role of VEGF isoforms in lung development. Lung vessel development in these mice was studied by scanning electron microscopy of Mercox casts of lung vasculature. Airway and air-blood barrier development was analyzed by light microscopy, transmission electron microscopy, immunohistochemistry, and morphometry. In all VEGF120/120 fetuses and pups, lung vascular casts were smaller and less dense compared with 120/+ and wild-type littermates. Although the generation count of pre-acinar vessels was similar in all three genotypes, the most peripheral vessels were dilated and were more widely separated in 120/120 fetuses of all ages, compared with 120/+ and wild-type littermates. In addition, 120/120 animals had fewer air-blood barriers and a decreased airspace-parenchyma ratio compared with 120/+ and wild-type littermates. We concluded that the absence of VEGF 164 and 188 isoforms impairs lung microvascular development and delays airspace maturation, indicating an essential role for heparin-binding VEGF isoforms in normal lung development.