Luisa Onstead

Aβ42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimers disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.

Aβ40 Inhibits Amyloid Deposition In Vivo

Numerous studies have established a pivotal role for Aβ42 in Alzheimers disease (AD) pathogenesis. In contrast, although Aβ40 is the predominant form of amyloid β (Aβ) produced and accumulates to a variable degree in the human AD brain, its role in AD pathogenesis has not been established. It has generally been assumed that an increase in Aβ40 would accelerate amyloid plaque formation in vivo. We have crossed BRI-Aβ40 mice that selectively express high levels of Aβ40 with both Tg2576 (APPswe, K670N+M671L) mice and BRI-Aβ42A mice expressing Aβ42 selectively and analyzed parenchymal and cerebrovascular Aβ deposition in the bitransgenic mice compared with their singly transgenic littermates. In the bitransgenic mice, the increased steady-state levels of Aβ40 decreased Aβ deposition by 60–90%. These results demonstrate that Aβ42 and Aβ40 have opposing effects on amyloid deposition: Aβ42 promotes amyloid deposition but Aβ40 inhibits it. In addition, increasing Aβ40 levels protected BRI-Aβ40/Tg2576 mice from the premature-death phenotype observed in Tg2576 mice. The protective properties of Aβ40 with respect to amyloid deposition suggest that strategies that preferentially target Aβ40 may actually worsen the disease course and that selective increases in Aβ40 levels may actually reduce the risk for development of AD.

A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

Alzheimers disease is characterized by the deposits of the 4-kDa amyloid β peptide (Aβ). The Aβ protein precursor (APP) is cleaved by β-secretase to generate a C-terminal fragment, CTFβ, which in turn is cleaved by γ-secretase to generate Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 generates the C-terminal fragment, CTFα. In addition to Aβ, endoproteolytic cleavage of CTFα and CTFβ by γ-secretase should yield a C-terminal fragment of 57–59 residues (CTFγ). However, CTFγ has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected thein vitro generation of Aβ as well as an ∼6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTFγ, and we describe an in vitro assay for γ-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTFγ fragment. Three compounds previously identified as γ-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTFγ, providing additional evidence that the fragment arises from γ-secretase cleavage. Consistent with reports that presenilins are the elusive γ-secretases, subcellular fractionation studies showed that presenilin-1, CTFα, and CTFβ are enriched in the CTFγ-generating fractions. The in vitroγ-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for γ-secretase inhibitors.

A novel gamma secretase assay based on detection of the putative C-terminal fragment-gamma of APP

Alzheimers disease is characterized by the deposits of the 4-kDa amyloid β peptide (Aβ). The Aβ protein precursor (APP) is cleaved by β-secretase to generate a C-terminal fragment, CTFβ, which in turn is cleaved by γ-secretase to generate Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 generates the C-terminal fragment, CTFα. In addition to Aβ, endoproteolytic cleavage of CTFα and CTFβ by γ-secretase should yield a C-terminal fragment of 57–59 residues (CTFγ). However, CTFγ has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected thein vitro generation of Aβ as well as an ∼6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTFγ, and we describe an in vitro assay for γ-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTFγ fragment. Three compounds previously identified as γ-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2,2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTFγ, providing additional evidence that the fragment arises from γ-secretase cleavage. Consistent with reports that presenilins are the elusive γ-secretases, subcellular fractionation studies showed that presenilin-1, CTFα, and CTFβ are enriched in the CTFγ-generating fractions. The in vitroγ-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for γ-secretase inhibitors.

Mutations in LRRK2 increase phosphorylation of peroxiredoxin 3 exacerbating oxidative stress‐induced neuronal death

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are responsible for autosomal dominant and sporadic Parkinson disease (PD), possibly exerting their effects via a toxic gain of function. A common p.G2019S mutation (rs34637584:A>G) is responsible for up to 30–40% of PD cases in some ethnic populations. Here, we show that LRRK2 interacts with human peroxiredoxin 3 (PRDX3), a mitochondrial member of the antioxidant family of thioredoxin (Trx) peroxidases. Importantly, mutations in the LRRK2 kinase domain significantly increased phosphorylation of PRDX3 compared to wild‐type. The increase in PRDX3 phosphorylation was associated with decreased peroxidase activity and increased death in LRRK2‐expressing but not in LRRK2‐depleted or vector‐transfected neuronal cells. LRRK2 mutants stimulated mitochondrial factors involved in apoptosis and induced production of reactive oxygen species (ROS) and oxidative modification of macromolecules. Furthermore, immunoblot and immunohistochemical analysis of postmortem human PD patients carrying the p.G2019S mutation showed a marked increase in phosphorylated PRDX3 (p‐PRDX3) relative to normal brain. We showed that LRRK2 mutations increase the inhibition of an endogenous peroxidase by phosphorylation promoting dysregulation of mitochondrial function and oxidative damage. Our findings provide a mechanistic link between the enhanced kinase activity of PD‐linked LRRK2 and neuronal cell death. 32:1390–1397, 2011. ©2011 Wiley Periodicals, Inc.

Reduction of Aβ accumulation in the Tg2576 animal model of Alzheimer’s disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin 1

The abnormal accumulation of the amyloid β protein (Aβ) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimers disease (AD). Compounds that reduce Aβ accumulation may therefore be useful therapeutically. In cell‐based screens we detected a significant reduction in Aβ concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Aβ accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non‐overlapping 40%–50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Aβ in the brain of treated animals indicates that both Aβ40 and the longer, more amyloidogenic form of the peptide, Aβ42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Aβ concentration in vitro can reduce Aβ accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Aβ accumulation.

Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer’s Amyloid β-Protein

The Alzheimer’s amyloid protein (Aβ) is released from the larger amyloid β-protein precursor (APP) by unidentified enzymes referred to as β- and γ-secretase. β-Secretase cleaves APP on the amino side of Aβ producing a large secreted derivative (sAPPβ) and an Aβ-bearing C-terminal derivative that is subsequently cleaved by γ-secretase to release Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 releases the secreted derivative sAPPα. In yeast, α-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Aβ40 and Aβ42 in the medium but had no effect on sAPPα. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Aβ40, Aβ42, and sAPPβ than its parental line (GPI+). When this parental line was treated with PI-PLC, Aβ40, Aβ42, and sAPPβ decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in β-secretase activity and Aβ secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Aβ biogenesis may be excellent therapeutic target(s) in Alzheimer’s disease.

Expression of BRI–amyloid β peptide fusion proteins: a novel method for specific high-level expression of amyloid β peptides

In order to develop transgenic animal models that selectively overexpress various Abeta peptides, we have developed a novel expression system that selectively expresses Abeta40 or Abeta42 in the secretory pathway. This system utilizes fusion constructs in which the sequence encoding the 23-amino-acid ABri peptide at the carboxyl terminus of the 266-amino-acid type 2 transmembrane protein BRI is replaced with a sequence encoding either Abeta40 or Abeta42. Constitutive processing of the resultant BRI-Abeta fusion proteins in transfected cells results in high-level expression and secretion of the encoded Abeta peptide. Significantly, expression of Abeta42 from the BRI-Abeta42 construct resulted in no increase in secreted Abeta40, suggesting that the majority of Abeta42 is not trimmed by carboxypeptidase to Abeta40 in the secretory pathway.

Convertases other than furin cleave β-secretase to its mature form

An aspartyl protease, Beta‐Site APP cleaving enzyme (BACE), was identified as the β‐secretase responsible for generating the Amyloid β protein that is believed to cause Alzheimers disease. BACE has a short propeptide domain that is absent in the mature enzyme because of proteolytic cleavage after the sequence RLPR. This sequence is a predicted substrate for proprotein convertases such as furin. To determine the role of furin and other proprotein convertases, we expressed proBACE in a furin‐deficient mutant Chinese hamster ovary (CHO‐K1) line, RPE.40. ProBACE signal was higher in RPE.40 than in the CHO‐K1 parent, which confirmed that furin plays a role in propeptide removal. However, two independent approaches showed that proBACE is cleaved to mature BACE in RPE.40: proBACE was rapidly turned over in RPE.40 although total BACE was stable, and decanoyl‐RVKR‐chloromethylketone, an inhibitor of the proprotein convertase family, substantially increased proBACE levels in both RPE40 and CHO‐K1. Transient transfection shows that furin, PACE4, PC5/6, and PC7 mediate BACE cleavage in vivo, at least when overexpressed. RPE.40 is proficient in BACE activity despite its furin deficiency. Therefore, our finding that proBACE is cleaved in this mutant leaves open the possibility that maturation is an important regulatory step and a therapeutic target.

Apolipoprotein E4 and tau allele frequencies among Choctaw Indians

Apolipoprotein genotyping and tau haplotyping were carried out on a series of cases with dementia and controls from the Choctaw Nation of Oklahoma. Both the Apolipoprotein E4 allele frequency and the tau H2 haplotype frequency were low in the Choctaw compared with Caucasians and there was the possibility that the association between dementia and the E4 allele was weaker than in Caucasians.