Isolation and characterization of patient-derived, toxic, high mass amyloid beta-protein (Abeta) assembly from Alzheimer disease brains.

Amyloid beta-protein (Abeta) 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, Abeta 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 Abeta 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, Abeta-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 Abeta assemblies.

The biochemical study on the etiology of Alzheimer's disease.

Alzheimer's disease has been characterized by senile plaque and neurofibrillary tangle in the brain. However, their relation to etiology of this disease has been left unclear. Recently it has been clarified that neurofibrillary tangle consists of highly phosphorylated tau protein. Then we have started to identify the enzyme(s) responsible for this phosphorylation and obtained tau protein kinase I and II. Tau protein kinase I phosphorylated not only tau protein but also pyruvate dehydrogenase, phosphorylation of which caused inactivation of this enzyme and finally led the cell to death. Then we have proved that TPKI is upregulated in AD brain but not in control brain. Upregulation of TPKI was induced by treating the neuronal cells with Abeta protein. Finally we have identified oligomeric aggregation of Abeta protein named Amylospheroid is highly potent to degenerate neuronal cells both in vitro and in vivo systems.

Thiol Protease Inhibitor, E-64-d, Prevents Spindle Formation during Mouse Oocyte Maturation1 : (meiosis/maturation-promoting factor/spindle formation/Ca2+ -activated neutral protease/chromosome condensation).

E-64-d, a membrane permeant derivative of E-64, the thiol protease inhibitor, was found to prevent meiotic maturation of mouse oocytes in a dose dependent manner. When immature mouse oocytes were incubated with E-64-d for up to 14 hr, first polar body emission was blocked to 36% at 200 µg/ml and 6% at 400 µg/ml, but germinal vesicle breakdown occurred normally. Cytological analysis revealed that meiotic spindles were not formed, while chromosome condensation occurred. Thus, E-64-d prevents oocytes from progressing to the first meiotic metaphase. When exposed to E-64-d after 8 hr of incubation without E-64-d, one-fourth of oocytes completed the first meiotic division but never progressed to the second metaphase. In three-fourth of the oocytes inhibited to emit the first polar body, spindles disappeared after incubation with E-64-d. The results suggest that E-64-d promotes disassembly of meiotic spindles resulting in inhibition of meiotic maturation. We propose that thiol protease is involved in spindle formation in mouse meiotic maturation.