Oksana Berezovska

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid β-peptide

The pathogenic event common to all forms of Alzheimers disease is the abnormal accumulation of the amyloid β-peptide (Aβ). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Aβ. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Aβ production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Aβ through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Aβ generation, indicating their potential for use in the treatment of Alzheimers disease.

Notch1 inhibits neurite outgrowth in postmitotic primary neurons

Notch plays an important role in cell fate decisions in uncommitted proliferative cells, including neurogenesis, but is believed to not have a role in postmitotic cells. We have shown previously that Notch1 is highly expressed in embryonal mouse and human brain, but surprisingly it continues to be expressed at low levels in the adult brain. The function of Notch1 in postmitotic neurons in mammals is unknown. To better understand the potential role of Notch1 in mature central nervous system neurons we studied the effect of Notch1 transfection on neurite outgrowth in primary neocortex hippocampal neurons. Transfection at two days in vitro with full length Notch1 inhibited neurite outgrowth. Transfection at five to six days in vitro, after neurite outgrowth was established, led to apparent regression of neurites. These effects were enhanced when truncated constitutively active forms of Notch1 were introduced. Co-transfection with Numb, a physiological inhibitor of Notch, blocked Notchs effect on neurite outgrowth. We also examined whether Notch1 could activate C-promoter binding factor (CBF1) transcription factor using C-promoter binding factor-luciferase constructs, and demonstrated that this signal transduction pathway is present and can be activated in postmitotic neurons. Our results show that in postmitotic neurons Notch1 influences neurite morphology, and can activate its native signal transduction pathway. These data strongly suggest that Notch1 may play a physiologically important role in the central nervous system beyond neurogenesis.

Nonsteroidal anti-inflammatory drugs lower Aβ42 and change presenilin 1 conformation

Recent reports suggest that some commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) unexpectedly shift the cleavage products of amyloid precursor protein (APP) to shorter, less fibrillogenic forms, although the underlying mechanism remains unknown. We now demonstrate, using a fluorescence resonance energy transfer method, that Aβ42-lowering NSAIDs specifically affect the proximity between APP and presenilin 1 and alter presenilin 1 conformation both in vitro and in vivo, suggesting a novel allosteric mechanism of action.

γ-secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer’s Disease

Tactical Target Intramembrane proteolysis by the γ-secretase complex is important during development and in the pathology of Alzheimers disease. γ-Secretase has usually been considered as a homogeneous activity. Serneels et al. (p. 639, published online 19 March; see the Perspective by Golde and Kukar) now show that the Aph1B component of the γ-secretase complex is responsible for the generation of long β-amyloid species involved in Alzheimers disease. In a mouse model of Alzheimers disease, full knockout of Aph1B improved disease phenotypes, without the sort of toxicity previously observed when targeting γ-secretase more generally. Targeted knockout of only part of the γ-secretase complex lessens toxicity and still improves disease phenotypes. The γ-secretase complex plays a role in Alzheimer’s disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the γ-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different γ-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B γ-secretase in a mouse Alzheimer’s disease model led to improvements of Alzheimer’s disease–relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total γ-secretase activity in the human brain, and thus specific targeting of Aph1B-containing γ-secretase complexes may help generate less toxic therapies for Alzheimer’s disease.

γ-Secretase/presenilin inhibitors for Alzheimer’s disease phenocopy Notch mutations in Drosophila

Signaling from the Notch (N) receptor is essential for proper cell‐fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)‐dependent transmembrane‐cleaving activity that is closely related or identical to the γ‐secretase proteolysis of the amyloid‐β precursor protein (APP) involved in Alzheimers disease pathogenesis. Here, we show that N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐(S)‐phenylglycine t‐butyl ester, a potent γ‐secretase inhibitor reported to reduce amyloid‐β levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N‐dependent signaling molecules established its biochemical mechanism of action in vivo. Other γ‐secretase inhibitors caused similar effects. Thus, the three‐dimensional structure of the drug‐binding site(s) in Drosophila γ‐secretase is remarkably conserved vis‐à‐vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.

Notch is expressed in adult brain, is coexpressed with presenilin-1, and is altered in Alzheimer disease

In C. elegans, the Notch family member lin-12 has been shown to have a genetic interaction with sel-12, the homologue of the Alzheimer disease-associated presenilin (PS) genes in humans. Mutations in PS genes cause autosomal dominant Alzheimer disease, with age of onset frequently in the 40s. Notch is known as a developmental protein that plays an important role in lateral Inhibition and specifying cell fate decisions in proliferating immature cells, and is not known to be present in adult neurons. We reasoned that, if Notch 1/PS-1 interaction is relevant in Alzheimer disease, Notchl would also need to be expressed in neurons in adult brain and colocalized with PS-1. We found that Notchl, Notch2, and a Notch ligand. Jagged 1, are expressed in adult brain in mouse and in human, with strongest expression in the hippocampal formation and Purkinje cells of the cerebellum. Double immunofluorescent staining demonstrates neuronal colocalization of Notchl with PS-1. Moreover, Notchl expression in sporadic Alzheimer disease hippocampus is elevated more than 2-fold in comparison to that in control human hippocampus by both immunohistochemistry and Western blot analysis (p<0.007). These results support the hypothesis that Notchl continues to play a role in terminally differentiated neurons, and that Notchl/PS-1 interactions may occur in adult mammalian brain. The alteration in Notchl expression in sporadic Alzheimer disease raises the possibility that disruption of Notch 1/PS-l functional interactions may occur in Alzheimer disease.

Familial Alzheimer's Disease Presenilin 1 Mutations Cause Alterations in the Conformation of Presenilin and Interactions with Amyloid Precursor Protein

Presenilin 1 (PS1) is a critical component of the γ-secretase complex, an enzymatic activity that cleaves amyloid β (Aβ) from the amyloid precursor protein (APP). More than 100 mutations spread throughout the PS1 molecule are linked to autosomal dominant familial Alzheimers disease (FAD). All of these mutations lead to a similar phenotype: an increased ratio of Aβ42 to Aβ40, increased plaque deposition, and early age of onset. We use a recently developed microscopy approach, fluorescence lifetime imaging microscopy, to monitor the relative molecular distance between PS1 N and C termini in intact cells. We show that FAD-linked missense mutations located near the N and C termini, in the mid-region of PS1, and the exon 9 deletion mutation all change the spatial relationship between PS1 N and C termini in a similar way, increasing proximity of the two epitopes. This effect is opposite of that observed by treatment with Aβ42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) (Lleo et al., 2004b). Accordingly, treatment of M146L PS1-overexpressing neurons with high-dose NSAIDs somewhat offsets the conformational change associated with the mutation. Moreover, by monitoring the relative distance between a PS1 loop epitope and the APP C terminus, we demonstrate that the FAD PS1 mutations are also associated with a consistent change in the configuration of the PS1-APP complex. The nonpathogenic E318G PS1 polymorphism had no effect on PS1 N terminus-C terminus proximity or PS1-APP interactions. We propose that the conformational change we observed may therefore provide a shared molecular mechanism for FAD pathogenesis caused by a wide range of PS1 mutations.

The γ Secretase-generated Carboxyl-terminal Domain of the Amyloid Precursor Protein Induces Apoptosis via Tip60 in H4 Cells

The amyloid precursor protein (APP), a large glycoprotein highly expressed in neurons, is cleaved in its intramembranous domain by γ secretase to generate amyloid-β and a free carboxyl-terminal intracellular fragment (APP-CT), which has previously been suggested to interact with the adapter protein Fe65 and the histone acetyltransferase Tip60. An identical γ secretase activity mediates cleavage of Notch, releasing an intracellular signaling domain that translocates to the nucleus. We examined the effect of an ectopically expressed 58-amino acid APP-CT fragment (APP-C58) on human H4 neuroglioma cells. We demonstrate by confocal microscopy and fluorescence resonance energy transfer analysis that APP-C58 translocates to the nucleus and forms a complex in the nucleus with the Tip60, independent of interactions with Fe65. APP-C58 transfected H4 cells undergo apoptosis within 48–72 h, marked by nuclear blebbing, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, and blockade by a caspase inhibitor. When nuclear access of APP-C58 is prevented by fusing with a strong membrane-targeting farnesylation domain, apoptosis is blocked. APP-C58-induced apoptosis was markedly enhanced by co-transfection with wild type Tip60 and decreased by mutant Tip60 lacking histone acetyltransferase activity, suggesting that Tip60 mediates APP-CT-induced cell death. Thus, γ secretase cleavage of APP may contribute to Alzheimers disease-related neurodegeneration in two ways: release of amyloid-β and liberation of a bioactive carboxyl-terminal domain from membrane-bound APP.

Aspartate Mutations in Presenilin and γ‐Secretase Inhibitors Both Impair Notch1 Proteolysis and Nuclear Translocation with Relative Preservation of Notch1 Signaling

It has been hypothesized that a presenilin 1 (PS1)‐related enzymatic activity is responsible for proteolytic cleavage of the C‐terminal intracellular protein of Notch1, in addition to its role in β‐amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand‐induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full‐length Notch1‐enhanced green fluorescent protein‐transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C‐promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notchs physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ‐secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ‐secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ‐secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ‐secretase inhibitors. Our results support the hypotheses that (a) PS1‐dependent APP γ‐secretase‐like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimers disease by targeting γ‐secretase processing of APP to reduce Aβ production.

CHIP Targets Toxic α-Synuclein Oligomers for Degradation

α-Synuclein (αSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric αSyn intermediates may represent the most toxic αSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on αSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced αSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic αSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of αSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric αSyn may contribute to the successful development of drug therapies that target oligomeric αSyn by mimicking or enhancing the powerful effects of CHIP.