Caspases are cysteine proteases that mediate a variety of processes including regulation of the inflammatory response and mediating programmed cell death. Various apoptotic caspases have been shown to play a crucial role in embryonic development and tissue homeostasis 
, while deregulation of caspase activity is observed in a variety of pathological conditions. The role that caspase-6 plays in various neurodegenerative conditions is the topic of investigation by numerous groups and highlights the desire to identify selective pharmacological reagents to disrupt enzymatic activity. Several lines of evidence connect caspase-6 with Alzheimer's disease (AD) including localization in disease brains and neurofibrillary tangles 
as well as direct cleavage of proteins with known involvement in AD progression 
. Furthermore, axonal degeneration induced by APP activation of DR6 was reported to be mediated by caspase-6 activity 
. Caspase-6 is also thought to play a role in Huntington's disease as it mediates cleavage of mutant huntingtin protein to induce pathogenesis in relevant disease models 
. More recently caspase-6 has also been implicated in Parkinson's disease as the neuroprotective function of DJ-1 protein is dependent on caspase-6 proteolysis 
. Despite the allure of caspase-6 as a therapeutic target, however, there are no drug-like therapies that selectively modulate this enzyme.
Caspase-6 is classified as an executioner caspase based on its structural homology to caspase-3 and -7 and its requirement for activation by upstream initiator caspases 
, although alternative mechanisms of activation have been proposed 
. Activated caspase-6 performs proteolytic digestion of a number of substrates with an aspartic acid residue in the P1 position, with P2–P4 amino acid residues conferring substrate specificity against other caspase isoforms 
. The preferred cleavage motif as defined for caspase-6 is Valine-Glutamate-Isoleucine-Aspartate (VEID) as defined by peptidic substrate mapping 
. These generalized consensus motifs provide utility as the basis of peptide substrates that are frequently used to interrogate the activity of caspase enzymes. Despite their utility in biochemical assays, there are challenges with enzyme-substrate cross-reactivity 
. Many of these peptide substrates are processed by a host of different caspase isoforms and would preclude their use in a cellular context where numerous caspase family members are present 
. The VEID sequence is found at amino acid residues 227–230 in the helical rod region of the nuclear intermediate filament protein lamin A/C. Despite enzyme-substrate cross-reactivity, claims have been made that lamin A/C is proteolyzed only by caspase-6 at this site 
. Likewise, many of the available peptide inhibitors have served as useful tools to inhibit enzymatic activity but fail to exhibit selective caspase isoform inhibition 
. This is likely due to the high degree of active site homology as well as presence of a warhead attached to the inhibitor P1 Asp residue that covalently modifies a conserved catalytic cysteine residue 
. Peptide inhibitor polarity may also prevent their cell penetration thus precluding their utility as viable tools to assist in the development of cellular caspase assays. It is therefore no surprise that there are no published reports of assays to specifically monitor inhibition of caspase-6 in a cellular context.
Several strategies have been reported to assess pan executioner caspase activity in whole cells including the synthesis of cell penetrant substrates that rely on novel fluorescent dyes or peptide leader sequences to encourage cell uptake 
. Another more elaborate strategy to monitor cellular activity of a specific caspase isoform was the utilization of split TEV technology to transiently activate caspase-3. Cellular enzymatic activity from this system 
, or others 
, is readily monitored via engineered FRET reporters. To overcome the hurdles of complex cell engineering and liabilities of possible substrate non-specificity, we developed a whole cell ELISA assay to monitor the proteolysis of endogenously expressed lamin A/C upon induction of the endogenous caspase machinery by apoptosis. We demonstrate that this is a high throughput, robust, whole cell assay for monitoring caspase-6 activity without compromising cellular membrane integrity. The assay serves as a valuable tool for facilitating drug discovery efforts against this important target.