Excessive cerebral accumulation of the 42-residue amyloid ␤-protein (A␤) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of A␤ from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how A␤ is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted A␤ under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic A␤ at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its A␤-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade A␤, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic A␤ at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of A␤, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted A␤ extracellularly is IDE.Converging lines of evidence support the hypothesis that progressive cerebral accumulation of the 40 -42-residue amyloid ␤-proteins (A␤s) 1 is an early, invariant, and necessary step in the pathogenesis of Alzheimer's disease (AD). As a result, there is growing interest in decreasing cerebral A␤ levels as a therapeutic and preventative approach to the disease. A␤ is generated by endoproteolysis of the ␤-amyloid precursor protein (APP) and secreted constitutively by most mammalian cells throughout life. Whereas many studies have examined the proteolytic processing of APP and the mechanisms of A␤ production, almost nothing is known about how A␤ peptides are normally degraded and cleared following their secretion. We recently screened the conditioned media of several different cell lines for A␤-degrading activity and found that the principal such activity was conferred by a nonmatrix metalloprotease that was released by microglial cells and other cells and efficiently degraded both endogenous and synthetic A␤ (1). The release of the protease from microglial cells was augmented by activating the cells with lipopolysa...

Progressive cerebral accumulation of amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease. Little is known about how Abeta, after being secreted, is degraded and cleared from the extracellular space of the brain. Defective Abeta degradation could be a risk factor for the development of Alzheimer's disease in some subjects. We reported previously that microglial cells release substantial amounts of an Abeta-degrading protease that, after purification, is indistinguishable from insulin-degrading enzyme (IDE). Here we searched for and characterized a role for IDE in Abeta degradation by neurons, the principal cell type that produces Abeta. Whole cultures of differentiated pheochromocytoma (PC12) cells and primary rat cortical neurons actively degraded endogenously secreted Abeta via IDE. However, unlike that in microglia, IDE in differentiated neurons was not released but localized to the cell surface, as demonstrated by biotinylation. Undifferentiated PC12 cells released IDE into their medium, whereas after differentiation, IDE was cell associated but still degraded Abeta in the medium. Overexpression of IDE in mammalian cells markedly reduced the steady-state levels of extracellular Abeta(40) and Abeta(42), and the catalytic site mutation (E111Q) abolished this effect. We observed a novel membrane-associated form of IDE that is approximately 5 kDa larger than the known cytosolic form in a variety of cells, including differentiated PC12 cells. Our results support a principal role for membrane-associated and secreted IDE isoforms in the degradation and clearance of naturally secreted Abeta by neurons and microglia.

Recent studies suggest that insulin-degrading enzyme (IDE) in neurons and microglia degrades Abeta, the principal component of beta-amyloid and one of the neuropathological hallmarks of Alzheimer's disease (AD). We performed parametric and nonparametric linkage analyses of seven genetic markers on chromosome 10q, six of which map near the IDE gene, in 435 multiplex AD families. These analyses revealed significant evidence of linkage for adjacent markers (D10S1671, D10S583, D10S1710, and D10S566), which was most pronounced in late-onset families. Furthermore, we found evidence for allele-specific association between the putative disease locus and marker D10S583, which has recently been located within 195 kilobases of the IDE gene.

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibitioninduced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria. The proteasome is a multicatalytic complex that is involved in the degradation of many cellular proteins, including those that are rapidly turning over, oxidized, or misfolded. As a general rule, in order to be targeted to the proteasome, proteins need to be ubiquitinated through a series of enzymatic steps that involve the attachment of chains of a small molecule, ubiquitin, to lysine residues (Ciechanover 1998). In addition, some proteins that have a natively unfolded structure may not need ubiquitination to be targeted for degradation at the proteasome (Tofaris et al. 2001). Proteasomal dysfunction has been linked increasingly to neurodegeneration, and in particular to Parkinson's disease (PD) and related Lewy body (LB) diseases (Chung et al. 2001;McNaught et al. 2001;Lang-Rollin et al. 2003). For example, expression of mutant forms of a-synuclein that lead to PD in certain families is associated with proteasomal inhibition (Tanaka et al. 2001;Stefanis et al. 2001;Petrucelli et al. 2002).The availability of selective pharmacological proteasomal inhibitors has led to a number of studies examining the effects of proteasomal inhibition on cultured neuronal cells (Sadoul et al. 1996;Lopes et al. 1997;Lin et al. 1998;Canu et al. 2000;Pasquini et al. 2000;Qiu et al. 2000;Rideout et al. 2001a;McNaught et al. 2002;Petrucelli et al. 2002;Rideout and Stefanis 2002

Amyloid beta-protein (Abeta) has been implicated as an early and essential factor in the pathogenesis of Alzheimer's disease. Although its cellular production has been studied extensively, little is known about Abeta clearance. Recently, insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, was found to degrade both endogenously secreted and synthetic Abeta peptides. Surprisingly, IDE-mediated proteolysis of [(125)I]Abeta(1-40) in microglial cell-culture media was accompanied by the formation of (125)I-labelled peptides with higher apparent molecular masses, raising the possibility that the degradation products act as 'seeds' for Abeta oligomerization. To directly address the role of IDE in Abeta degradation and oligomerization, we investigated the action of purified recombinant wild-type and catalytically inactive IDEs. Our data demonstrate that (i) IDE alone is sufficient to cleave purified Abeta that is either unlabelled, iodinated or (35)S-labelled; (ii) the initial cleavage sites are His(14)-Gln(15), Phe(19)-Phe(20) and Phe(20)-Ala(21); and (iii) incubation of IDE with [(125)I]Abeta, but not with [(35)S]-Abeta, leads to the formation of slower migrating species on gels. Since iodination labels N-terminal fragments of Abeta, and (35)S labels C-terminal products, we analysed unlabelled synthetic fragments of Abeta and determined that only the N-terminal fragments migrate with anomalously high molecular mass. These results indicate that IDE alone is sufficient to degrade Abeta at specific sites, and that its degradation products do not promote oligomerization of the intact Abeta peptide.

Background: Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. Methods: Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. Results: Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated αsyn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking β-sheet content after subjecting them to proteinase K digestion. Conclusions: Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.

The Bcl-2 and Bcl-x proteins suppress programmed cell death, whereas Bax promotes apoptosis. We investigated the pattern of expression of Bcl-2, Bax and Bcl-x during neuronal differentiation and development. All three proteins were widely expressed in neonatal rats but, in the adult, Bax levels were 20- to 140-fold lower in the cerebral cortex, cerebellum and heart muscle, whereas Bcl-x was not downregulated in any of the tissues examined. In the cerebral cortex and cerebellum, the decrease in Bax levels occurred after the period of developmental cell death. Further, microinjection of a Bax expression vector into cultured sympathetic neurons, which depend on nerve growth factor for survival, induced apoptosis in the presence of survival factor and increased the rate of cell death after nerve growth factor withdrawal. This effect could be blocked by co-injection of an expression vector for Bcl-xL or for the baculovirus p35 protein, an inhibitor of caspases (ICE-like proteases). These results suggest that, during development, the sensitivity of neurons to signals that induce apoptosis may be regulated by modulating Bax levels and that Bax-induced death requires caspase activity.

The worm appears to be robust to this form of (pulsed) radiation, at least under the exposure conditions used.