Mark S. Shearman

Involvement of Caspases in Proteolytic Cleavage of Alzheimer’s Amyloid-β Precursor Protein and Amyloidogenic Aβ Peptide Formation

Abstract The amyloid-β precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-β (Aβ) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimers disease brains and colocalization of its APP cleavage product with Aβ in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for Aβ peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimers disease.

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Selective activation of the γ-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites

The γ‐subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. γ‐PKC from bovine cerebellum, but not the α‐or βI/βII‐subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)‐11‐cis‐isomer) was a potent, selective activator of the γ‐subspecies, and also, to a lesser extent, 12(S)‐hydroxy‐5,8,10,14‐eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the γ‐subspecies of PKC.

Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target γ-Secretase at a Novel Site EVIDENCE FOR AN ALLOSTERIC MECHANISM

γ-Secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-β (Aβ) peptides from processing intermediates of the β-amyloid precursor protein. Because Aβ peptides are thought to be causative for Alzheimers disease, inhibiting γ-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of γ-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Aβ(1–42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Aβ(1–42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective γ-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the γ-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of γ (42)-cleavage over alternative cleavages in the β-amyloid precursor protein and, more importantly, alternative substrates.

Presenilin-dependent γ-secretase activity modulates thymocyte development

In neuronal cells, presenilin-dependent γ-secretase activity cleaves amyloid precursor proteins to release Aβ peptides, and also catalyzes the release of the intracellular domain of the transmembrane receptor Notch. Accumulation of aberrant Aβ peptides appears to be causally related to Alzheimers disease. Inhibition of Aβ peptide production is therefore a potential target for therapeutic intervention. Notch proteins play an important role in cell fate determination in many different organisms and at different stages of development, for example in mammalian T cell development. We therefore addressed whether structurally diverse γ-secretase inhibitors impair Notch function by studying thymocyte development in murine fetal thymic organ cultures. Here we show that high concentrations of the most potent inhibitors blocked thymocyte development at the most immature stage. In contrast, lower concentrations or less potent inhibitors impaired differentiation at a later stage, most notably suppressing the development of CD8 single-positive T cells. These phenotypes are consistent with an impairment of Notch signaling by γ-secretase inhibitors and define a strict Notch dose dependence of consecutive stages during thymocyte development.

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

The mechanism by which aggregates of the β‐amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42‐amino‐acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron‐binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild‐type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron‐binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co‐expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation.

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.

Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of β-amyloid in rat PC12 cells

Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimers disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

The common structure and activities of four subspecies of rat brain protein kinase C family

Elucidation of the complete sequences of four cDNA clones (α, βI, βII, and γ) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C1‐C4) and five variable (V1‐V5) regions. Although these sequences are highly homologous and closely related to one another V3‐, V4‐, and V5‐regions of γ‐subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C1, contains a tandem repeat of cysteine‐rich sequence (6, total 12 cysteine residues). The third constant region, C3, has an ATP‐binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of α‐, βI, βII, and γ‐subspecies are roughly 16, 8, 55, and 21%, respectively. γ‐Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

Sulfonated dyes attenuate the toxic effects of β-amyloid in a structure-specific fashion

We recently reported that several sulfate-containing glycosaminoglycans, a class of compounds associated with the beta-amyloid plaques of Alzheimers disease, attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. The amyloid-binding sulfonated dye Congo Red was shown to have a similar effect. Using two clonal cell lines, we now demonstrate that several sulfonated dyes attenuate beta-amyloid toxicity and that the protective effect appears specific for compounds whose sulfonate groups can interact with the beta-pleated structure of aggregated amyloid. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.