Targets of Caspase-6 Activity in Human Neurons and Alzheimer Disease

Jennifer Hammond,Guy Klaiman,Tracy L. Petzke,Andréa C. LeBlanc

doi:10.1074/mcp.m800007-mcp200

Jennifer Hammond, Guy Klaiman

Open Access

https://doi.org/10.1074/mcp.m800007-mcp200

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Abstract

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and alpha-Tubulin. A neoepitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.

Highlights

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals
In Alzheimer disease (AD) brains, Csp6 is strongly activated in neuropil threads, neuritic plaques, and neurofibrillary tangles in the absence of classical apoptotic features [3]
The absence of active Csp6 in the nuclei of neurons combined with its presence in neurofibrillary tangles, neuritic plaques, and neuropil threads suggests that Csp6 is involved in neurodegeneration in AD

Summary

EXPERIMENTAL PROCEDURES

An in-gel digest with trypsin, which cuts C-terminal to a Lys or Arg residue, was done on the proteins, and the resulting peptides were extracted using acidic and basic conditions. All of these above steps were performed robotically (ProGest digestion robot, Genomic Solutions, Ann Arbor, MI). Digestion of in Vitro Translated (IVT) or Purified Proteins with Caspases—Proteins were digested for 4 h at 37 °C in Stennicke’s buffer with RCsp Western Blot Analyses—The rabbit neoepitope antiserum against Csp3-cleaved ␤-actin (Fractin) was kindly provided by Dr Greg Cole (Department of Medicine, University of California, Los Angeles, CA) [41]. Total no. of peptides includes additional peptides that did not reach an expectation score of Ͻ10Ϫ3

Membrane and lipid binding

RESULTS

Topoisomerase I

DISCUSSION