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

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

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

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