ELISA analysis of β-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways

Limiting beta amyloid (Aβ) production could become an important therapeutic target in Alzheimer's disease (AD). Aβ is derived by the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Aβ40 and Aβ42 production. Aβ production and, therefore, β-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of β-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the β-cleaved product, APPswβ. Using this ELISA, we provide new information regarding the relative amount of β-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the β-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no β-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswβ and Aβ secretion, suggesting that protein interactions with this region normally impede β-secretase cleavage. These results suggest that APPsw is cleaved by β-secretase late in the secretory pathway.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66393/1/j.0022-3042.2002.00764.x.pd

Implicating Calpain in Tau-Mediated Toxicity In Vivo

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo

TOR-Mediated Cell-Cycle Activation Causes Neurodegeneration in a Drosophila Tauopathy Model

SummaryBackgroundPrevious studies have demonstrated reexpression of cell-cycle markers within postmitotic neurons in neurodegenerative tauopathies, including Alzheimer's disease (AD). However, the critical questions of whether cell-cycle activation is causal or epiphenomenal to tau-induced neurodegeneration and which signaling pathways mediate cell-cycle activation in tauopathy remain unresolved.ResultsCell-cycle activation accompanies wild-type and mutant tau-induced neurodegeneration in Drosophila, and genetically interfering with cell-cycle progression substantially reduces neurodegeneration. Our data support a role for cell-cycle activation downstream of tau phosphorylation, directly preceding apoptosis. We accordingly show that ectopic cell-cycle activation leads to apoptosis of postmitotic neurons in vivo. As in AD, TOR (target of rapamycin kinase) activity is increased in our model and is required for neurodegeneration. TOR activation enhances tau-induced neurodegeneration in a cell cycle-dependent manner and, when ectopically activated, drives cell-cycle activation and apoptosis in postmitotic neurons.ConclusionsTOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model, identifying TOR and the cell cycle as potential therapeutic targets in tauopathies and AD

Calpain mutants suppress tau-induced toxicity in the fly eye.

<p>As shown using scanning electron microscopy, compared to the normal external appearance of control flies (<i>w¹¹¹⁸</i> in A; <i>GMR-GAL4/+</i> in B), flies expressing wild-type human tau (tau^WT; C) displayed the moderately rough eye phenotype characterisitc of tau toxicity. Genetic disruption of calpain A (D–F) or calpain B (G–I) using either deficiency lines (D and G) or P-element insertions (E–F and H–I) effectively suppressed tau toxicity.</p

Colocalization of tau and calpain in <i>Drosophila</i> neurons.

<p>Neuronal cells cultured from late 3^rd instar larvae expressing tau^WT were stained for tau in red (A and D), calpain A and calpain B in green (B and E, respectively), with colocalization shown in yellow (C and F). The expression pattern of calpain A and B was similar to that of tau, with the primary site of colocalization in the neuronal cell body. Scale bar  = 5 µM.</p

Calpain protein expression level and enzyme activity are decreased in calpain mutants.

<p>A. Using antibodies specific to calpain A or calpain B, western blot analysis showed that compared to tau^WT-expressing flies, all of the calpain mutant lines have lower levels of calpain expression (<i>top</i>). Tau expression levels for all of the calpain mutants are equivalent to the expression level of tau in tau^WT-expressing flies using both Tau 5 and Tau-1 antibodies (<i>bottom two panels</i>). B. When normalized to the level of calpain in tau^WT-expressing flies, densitometric analysis revealed reduced calpain expression in all calpain mutant lines tested (<i>top</i>). The values shown represent the mean of three or more independent experiments. When normalized to calpain activity in tau^WT-expressing flies, all calpA and calpB mutants have reduced calpain activity (<i>bottom</i>). The values shown represent the mean of enzyme activity measured in triplicate. Error bars for both graphs represent the standard error of the mean and data points indicated with asterisks are significant. *P<0.05, **P<0.01 (unpaired student t-test).</p

Cleavage of tau by calpain creates the 17 kD fragment.

<p>A. Schematic of 0N4R tau with the nine putative calpain cleave sites indicated. Proteolysis at lysine 44 (K44) and arginine 230 (R230) yields a 17 kD fragment of tau. Abbreviations: methionine (M), lysine (K), arginine (R), and tyrosine (Y). B. The K44Q/R230Q mutation is predicted to disrupt the recognition motif for calpain at K44 and R230 and thus creates a <u>c</u>alpain-<u>r</u>esistant (tau^CR) form of tau for expression in <i>Drosophila</i>. PCR was used to create a second tau mutant (tau^17kD) in which only the amino acids corresponding to the reputed toxic 17 kD fragment (aa 44-230) are expressed in <i>Drosophila</i>.</p