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Human Molecular Genetics, 2004, Vol. 13, No. 4 437-446
DOI: 10.1093/hmg/ddh045

A screen for drugs that protect against the cytotoxicity of polyglutamine-expanded androgen receptor

Federica Piccioni1, Benjamin R. Roman1, Kenneth H. Fischbeck1 and J. Paul Taylor1,2,*

1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1250, USA and 2University of Pennsylvania School of Medicine, Department of Neurology, Philadelphia, PA 19104, USA

Received October 21, 2003; Accepted December 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinobulbar muscular atrophy is a neurodegenerative disorder caused by expansion of a CAG triplet repeat sequence encoding a polyglutamine tract in the androgen receptor. It has been shown that the mutant protein is toxic in cell culture and triggers an apoptotic cascade resulting in activation of caspase-3. We developed an assay of caspase-3 activation in cells expressing the mutant androgen receptor. This assay was used to screen 1040 drugs, most of which are approved for clinical use. Drugs that inhibit polyglutamine-dependent activation of caspase-3 were subjected to follow-up screens to identify compounds that reproducibly prevent polyglutamine-induced cytotoxicity. Four drugs satisfied these criteria. Three of these (digitoxin, nerifolin and peruvoside) are structurally and functionally related compounds of the cardiac glycoside class and known inhibitors of Na+K+-ATPase. The fourth compound, suloctidil, is a calcium channel blocker.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Spinobulbar muscular atrophy (SBMA), also known as Kennedy's disease, is an X-linked disorder characterized by progressive degeneration of motor neurons in the brainstem and spinal cord (1). The cause of SBMA is trinucleotide (CAG) repeat expansion in the first exon of the androgen receptor (AR) gene (2). Eight other diseases have the same kind of mutation: Huntington's disease (HD), dentatorubro-pallidoluysian atrophy and six forms of spinocerebellar ataxia (3,4). All of these polyglutamine diseases likely share a common molecular mechanism involving a toxic gain of function in the mutant protein caused by the expansion. Currently, there is no therapy for any of the polyglutamine diseases. Treatment developed for any one of these diseases may be applicable to all.

A variety of pathological changes has been described in model systems of polyglutamine disease including transcriptional interference, altered axonal transport, impaired proteasomal function, and induction of apoptosis (5–7). While apoptosis may not be a direct target of expanded polyglutamine, caspases may contribute to the generation of toxic, polyglutamine-containing fragments, thus initiating or amplifying the disease. For example, the cytotoxicity of AR with expanded polyglutamine is associated with cleavage by caspase-3, and elimination of the caspase-3 cleavage sites ameliorates the toxicity of mutant AR (8,9). Intervention upstream of caspase-3 activation may be beneficial in polyglutamine disease. Here, we describe the use of a simple cell-based assay to identify compounds that inhibit polyglutamine-induced caspase-3 activation. The assay is based on the transient expression in human embryonic kidney (HEK) 293T cells of AR with an expanded polyglutamine tract and measurement of caspase-3 activation using a specific fluorogenic substrate. We identified four drugs that block polyglutamine-induced caspase-3 activation and protect against polyglutamine cytotoxicity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Assay development and validation
Expression of truncated androgen receptor with a 112-glutamine repeat (AR112) in HEK 293T cells results in accumulation of insoluble polyglutamine aggregate detectable by western blot, the appearance of intracellular inclusions, and cell death within ~72 h (10). Polyglutamine-induced cytotoxicity is accompanied by robust activation of caspase-3, which can be demonstrated with the fluorogenic substrate acetyl-aspartate-glutamate-valine-aspartate-7-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC; Fig. 1). At the concentration used in this assay, Ac-DEVD-AFC is cleaved predominantly by caspase-3, although a minor contribution by other caspases may be detected as well. The specificity of caspase-mediated cleavage was confirmed by inhibition with benzyloxy-valine-alanine-aspartate-O-methyl-fluoromethylketone (z-VAD-fmk). To develop a cell-based drug screening assay, cell lysis, substrate incubation and fluorescence quantitation were adapted to a 48-well format. After optimization of cell number, quantity of transfected DNA and incubation period, the assay was found to yield a signal-to-noise ratio of 3.6±0.4 when comparing positive control (cells treated with the caspase inhibitor z-VAD-fmk) to negative control (cells treated with the vehicle dimethylsulfoxide, DMSO). To determine the utility of this assay in our screening protocol, we determined the Z' factor, a value that represents the robustness of the assay after taking into account the dynamic range of the assay signal and the data variation associated with the signal measurements (11). Z' was calculated by the following equation, where values greater than 0.2 are considered acceptable (SD is standard deviation):



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  		Figure 1. Polyglutamine-induced caspase-3 activation. HEK 293T cells were transfected with truncated forms of wild-type (AR16) and mutant (AR112) androgen receptor. Caspase-3 is not activated by transfection with androgen receptor with normal CAG repeat length, whereas the expression of androgen receptor with elongated polyglutamine tract results in activation of caspase-3. z-VAD-fmk, a caspase inhibitor, was used as a positive control.

 
The Z' factor (Table 1) ranged from 0.32 to 0.71, indicating that the assay is of sufficient quality to generate reliable screening data. Assay variability was evaluated by determining intra-plate (well-to-well), and inter-plate coefficients of variance. These values were less than 18% over three experiments with respect to signal–noise ratio and reproducibility (Table 1).


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  		Table 1. Screen validation. The Z' factor >=0.2 indicates a robust assay. The coefficients of variance indicate good well-to-well and plate-to-plate reproducibility
 
Primary screening and retesting
To identify compounds that inhibit polyglutamine-induced activation of caspase-3, we screened the NINDS Custom Collection, which consists of 1040 drugs. The majority of these drugs have already been approved for use in humans by the US Food and Drug Administration (FDA), and the additional drugs were selected because of known or anticipated activity in the central nervous system. The complete list of drugs may be found at www.ninds.nih.gov/funding/neurodegenerationlninds_drug_screening.htm. While this library of drugs is relatively small, it is enriched in compounds that have a satisfactory safety profile and are biologically active at achievable doses in humans. This screen was conducted as part of a 26-laboratory consortium sponsored by the National Institute of Neurological Disorders and Stroke (12).


The screening was performed blind, such that the identity of the drugs was not revealed until primary screening was completed. The primary screen consisted of testing each drug in a single well at a dose of 10 µM and comparing the caspase-3 signal to the average signal of three negative control wells from the same plate. The activities of all 1040 drugs are shown in Figure 2A. The average activity was 6% inhibition of caspase-3 activation, with a standard deviation of 33% (Fig. 2B). Seventy-six drugs were found to inhibit caspase-3 activation >=39% (mean+1SD) and 21 drugs inhibited caspase-3 activation >=71% (mean+2 SD; Fig. 2C). For comparison, the average inhibition by the positive control (z-VAD-fmk) was 59±6%. The data variability of the primary screen was evaluated by comparing the control wells across all plates. The coefficients of variance for positive controls (10.6%, n=26) and negative controls (11.3%, n=26) were both low, and the Z' factor was over 0.2, indicating good quality assay data (11). Of note, the only compound in the NINDS Custom Collection predicted to inhibit caspase activation (to our knowledge) was zVAD-fmk, which was also used as our positive control. As expected, this compound was among the 76 hits in the primary screen.



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  		Figure 2. Screening of 1040 compounds. (A) Scatter plot 1040 compounds tested shown with percentage Vcaspase-3 inhibition (two drugs are off the scale). (B) Histogram of the number of compounds with percentage inhibition of caspase-3 activation. The average percentage inhibition was 6% with a standard deviation of 33%. (C) Summary of compound activities in caspase-3 assay. Seventy-six drugs were found to inhibit caspase-3 activation >=39% and 21 drugs inhibited caspase-3 activation >=71%.

 
The 76 drugs that showed the greatest inhibition of caspase-3 were retested twice in triplicate, leading to the identification of 20 drugs that consistently showed inhibition greater than 71% (mean+2SD; Table 2). These were largely the same drugs that exhibited greatest activity in the primary screen, thus corroborating those results. Of these, 15 drugs showed variability on retesting of less than 10% and were chosen for further evaluation (Table 2). The identified drugs do not directly inhibit caspase activity. Unlike z-VAD-fmk, these drugs do not reduce Ac-DEVD-AFC cleavage when added directly to the assay reaction (not shown). Thus, these drugs prevent upstream caspase-3 activation rather than directly inhibiting caspase-3 activity.


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  		Table 2. Retesting of drugs identified from the primary screen. The 76 drugs that showed the greatest inhibition of caspase-3 were retested twice in triplicate leading to the identification of 20 drugs. The 15 drugs showing inhibitory activity greater than 71% and variability less than 10% were selected for further evaluation
 
Secondary screening
Since rapid induction of cell death after drug addition could lead to a falsely positive low caspase activity signal, we first examined whether these 15 drugs were inherently cytotoxic. Transfected cells were incubated in 10 µM drug for 48 h, followed by a measurement of the reduction rate of the tetrazolium salt XTT (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) to a water-soluble formazan product, which is indicative of cytoplasmic dehydrogenase activity and correlates with cell viability (13). With eight drugs, the XTT reduction rate was decreased by more than 30% relative to the control, and these drugs were considered toxic (Table 3). The other seven drugs were non-toxic by this test using the arbitrary cut-off of 30% decrease in the XTT assay. We next evaluated the ability of the 15 drugs to protect against polyglutamine-induced cytotoxicity by examining the ability of cells to exclude the fluorescent vital dye propidium iodide. To count large numbers of cells, and to eliminate examiner bias, these measurements were made with a fluorescence-activated cell sorter-analyzer (FACS-analyzer). HEK 293T cells were transfected with AR112 and treated for 48 h with the selected compounds or the negative control DMSO. As expected, the drugs identified as toxic in the XTT assay enhanced cell death when compared to control. Of the remaining seven drugs, four were found to protect cells from polyglutamine cytotoxicity (Table 3), reducing polyglutamine-induced cell death by 30–40%. Suloctidil is a calcium channel blocker that has been previously investigated for stroke prevention (14). The other three drugs (digitoxin, nerifolin and peruvoside) are structurally and functionally related compounds of the cardiac glycoside class and known inhibitors of Na+K+-ATPase. Examination of the NINDS Custom Collection revealed four additional cardiac glycosides (oaubain, digoxin, lanatoside C and strophanthidin). Three of these (ouabain, digoxin and lanatoside C) were among the 76 drugs identified in the primary screen, but were subsequently excluded by arbitrary cut-offs. Subsequent testing showed that they also potently inhibited caspase-3 activation by polyglutamine (data not shown). The last cardiac glycoside, strophanthidin, was identified as a false positive hit in the primary screen due to inherent drug cytotoxicity. This drug showed no inhibitory activity across a wide range of doses and was cytotoxic above 10 µM (Table 3 and data not shown).


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  		Table 3. Secondary screening. XTT assay results for each drug are represented as a percentage compared with control cells (set at 100%). Eight drugs were found to reduce cell metabolic activity by >=30% and were considered to be toxic (shown with asterisk). Four drugs (digitoxin, nerifolin, peruvoside, suloctidil) reduced polyglutamine-induced cell death by 30–40% (shown in bold). For FACS analysis, the results were expressed either as a percentage of PI-positive (non-viable) cells relative to total cells
 
We also evaluated these drugs in a green fluorescent protein (GFP)-based FACS viability assay. The use of an AR112-GFP fusion protein allowed evaluation of polyglutamine-induced cytotoxicity specifically in the transfected population and also allowed precise monitoring of transgene expression levels. This assay confirmed the ability of cardiac glycosides to prevent polyglutamine cytotoxicity across a range of doses and demonstrated that transgene expression levels were not influenced by drug treatment at doses that inhibited polyglutamine cytotoxicity (Table 4). Western blots were performed to examine transgene expression levels and showed that the steady state levels of AR112 were not reduced by effective concentrations of these drugs, corroborating the results of the FACS assay (not shown). Dose–response curves and the IC50 values (drug concentration that produces 50% of the maximal inhibition) for these four drugs are shown in Figure 3. Suloctidil was found to have an IC50 of 5.5 µM. Nerifolin, peruvoside and digitoxin were effective at lower concentrations, with IC50s of 24, 28 and 37 nM, respectively. For comparison, the therapeutic serum level of digitoxin when used as a cardiac inotrope is ~15–30 nM.


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  		Table 4. GFP-based FACS viability assay. HEK 293 T cells were transfected as described in Materials and Methods. The use of GFP-tagged protein allowed evaluation of cytotoxicity specifically in the transfected population and allowed monitoring of the transgene expression levels. The results are expressed as the relative number of GFP/PI doubly positive cells (transfected, dead) relative to total GFP positive cells (all transfected cells)
 


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  		Figure 3. Dose–response curves. IC50s (drug concentration that produces 50% of maximal inhibition of caspase-3 activation) were very similar for the three cardiac glycosides. The activity of suloctidil showed a sharp threshold. This has been described previously and is probably an intrinsic property of the drug (37).

 
Na+K+-ATPase inhibition by low K+
Na+K+-ATPase is a membrane-bound enzyme that maintains inward Na+ and outward K+ electrochemical gradients. Nerifolin, peruvoside and digitoxin all belong to a class of cardioactive steroids that share the property of being potent and highly specific inhibitors of the Na+K+-ATPase (15). The Na+K+-ATPase is also strongly inhibited by incubating cells in K+-depleted (<1 mM) media (16,17). To examine the relationship of Na+K+-ATPase activity and polyglutamine toxicity, we transfected HEK 293T cells with AR112 and 24 h later replaced the growth media with either complete media (containing 5 mM KCl) or K+-depleted media (containing 2.5, 1.0 or 0.1 mM KCl) and incubated for an additional 24 h. We found no inhibition of polyglutamine-induced caspase-3 activation despite reduction of extracellular K+ to as low as 0.1 mM. In contrast, addition of 1 µM ouabain 24 h post-transfection reduced polyglutamine-induced caspase-3 activation by 40%. These findings indicate that inhibition of the Na+K+-ATPase alone is not sufficient to prevent polyglutamine toxicity.

Inhibition of Bax-induced activation of caspase-3
In order to determine whether the protective effect of the drugs we identified was limited to polyglutamine toxicity, we investigated their effects on the activation of caspase-3 by Bax, a proapoptotic member of the Bcl-2 family of proteins that is a critical integrator of apoptotic signals. Overexpression of Bax results in altered permeability of the outer mitochondrial membrane, release of pro-apoptotic contents to the cytosol, and induction of a caspase cascade that ultimately results in activation of caspase-3 (18). We found that caspase-3 activation by Bax is potently inhibited by the cardiac glycosides nerifolin, peruvoside and digitoxin, at 1 µM concentration (Fig. 5). This result suggests that cardiac glycosides work at a point downstream of Bax in the apoptotic cascade, before activation of caspase-3. In contrast, suloctidil had no effect on caspase-3 activation by Bax, suggesting that this drug works at a point upstream from Bax (Fig. 5).



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  		Figure 5. Bax activation of caspase-3 is blocked by the cardiac glycosides, but is not blocked by suloctidil. The results are expressed as percentage caspase-3 activity relative to control (set at 100%).

 
Caspase-3 inhibition in a motor neuronal cell line
Transient transfection of a truncated form of androgen receptor with an expanded polyglutamine tract in the mouse motor neuron-neuroblastoma hybrid cell line MN-1 results in cellular toxicity (10). Since SBMA is primarily a disease of motor neurons, we investigated whether cardiac glycosides were able to inhibit polyglutamine-induced caspase-3 activation in MN-1 cells. We found inhibition of caspase-3 activation in MN-1 cells of 56±11% by nerifolin, 53±3% by peruvoside, and 53±4% by digitoxin (Fig. 6). The potency of the cardiac glycosides in MN-1 cells was similar to that observed in HEK-293T cells. Suloctidil did not show inhibition (data not shown).



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  		Figure 6. The cardiac glycosides block polyglutamine toxicity in the motor neuronal cell line MN-1. The results are expressed as percent inhibition relative to control (set at 100%).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We describe the use of a cell-based drug screening assay to identify drugs that protect against the toxicity of expanded polyglutamine. This assay was based on our observation that the cytotoxicity of AR with expanded polyglutamine is associated with induction of apoptosis in cultured cells, including robust activation of caspase-3. A role for apoptosis in the pathogenesis of polyglutamine disease was initially suggested by pathological studies that found markers of apoptosis in the brains of HD patients (19–21). There is a report suggesting that mutant huntingtin may directly activate caspase-8 (22). However, the more compelling evidence suggests that caspases may contribute to initiation and amplification of disease by cleaving disease proteins to generate toxic polyglutamine-containing fragments (8,9). Thus, drugs that intervene upstream of caspase-3 activation might be beneficial in polyglutamine disease. While the precise role of apoptosis in pathogenesis remains to be elucidated, for our purposes caspase-3 activation is a useful surrogate marker of polyglutamine cytotoxicity.

To develop a cell-based drug screen, we adapted our caspase-3 activation assay to a 48-well format. This assay is based on cleavage of the fluorogenic substrate Ac-DEVD-AFC and quantitation using a plate-reading fluorimeter. After optimization, the assay was found to yield a strong signal-to-noise ratio, a good Z' factor, and low well-to-well and plate-to-plate variability. This assay was used as a primary screen to identify candidate drugs from the NINDS Custom Collection. Importantly, most of these drugs are approved for use in humans by the FDA, thus potentially accelerating the rate at which hits may be translated from the laboratory to the clinic.

After the primary screen, we chose a low stringency cut-off and selected the 76 drugs that showed >=39% inhibition of caspase-3 activation (mean+1SD) for retesting. These 76 drugs were retested twice in triplicate, and the 15 drugs with the strongest and most consistent inhibition were chosen for secondary screening. The secondary screening consisted of an XTT metabolic assay to exclude toxic drugs and a FACS-based viability assays to select drugs that prevent polyglutamine-induced cell death. These assays identified four drugs: digitoxin, nerifolin, peruvoside and suloctidil.

Digitoxin, nerifolin, and peruvoside are structurally and functionally related compounds of the cardiac glycoside class and known inhibitors of Na+K+-ATPase. The IC50s of these drugs in the caspase-3 assay were found to be similar (ranging from 24–37 nM), which is similar to the therapeutic range of these drugs in human serum (15–30 nM). The NINDS Custom Collection contained four additional cardiac glycosides (ouabain, digoxin, lanatoside C and strophanthidin). Three of these (ouabain, digoxin and lanatoside C) were subsequently found also to potently inhibit caspase-3 activation by polyglutamine. Strophanthidin, however, showed no inhibitory activity across a wide range of doses and was cytotoxic at 10 µM.

Since William Withering's 1785 monograph on the efficacy of the leaves of the foxglove plant (Digitalis purpurea), the cardiac glycosides have played a prominent role in medicine. These drugs exert a positive inotropic effect on cardiac muscle and have been useful in the treatment of congestive heart failure. All cardiac glycosides are highly specific inhibitors of the Na+K+-ATPase (the ‘sodium pump’), and their potency as positive inotropes correlates closely with their ability to inhibit this enzyme (23). Cardiac glycosides are composed of a steroid nucleus containing an unsaturated lactone at the C17 position and (typically) a sugar moiety attached to C3 of the steroid ring. Many analogs have been generated with substitutions at various positions of the steroid ring, including different numbers and categories of carbohydrates at C3 as well as variations in the saturation and size of the lactone ring. The lactone ring plays an important role in the inhibition of the Na+K+-ATPase, while the sugar moiety on C3 is not required for biological activity, but rather appears to be important for kinetics of action. The cardiac glycosides present in the NINDS Custom Collection are shown in Figure 7. The positive inotropic effect of the cardiac glycosides is an indirect function of the increased intracellular [Na+] that results from pump inhibition (23). Increased intracellular [Na+] results in increased intracellular [Ca2+], probably through Na+–Ca2+ exchange, which increases myocardial contractility and activates a Ca+-mediated signaling cascade (24,25). Increased intracellular [Ca2+] has pleiotropic effects on cell signaling, including promotion of cell survival mediated through the activity of protein kinase B and Akt (26) or the transcription factor CREB (27). It is possible that the ability of the cardiac glycosides to prevent polyglutamine cytotoxicity is related to inhibition of the Na+K+-ATPase, perhaps mediated by Ca2+. Indeed, an anti-apoptotic activity of cardiac glycosides was previously described in vascular smooth muscle cells and rat cerebellar granule cells (17,28). The antiapoptotic effect of ouabain in a porcine renal proximal tubular cell line has also been described and attributed to activation of P13 kinase (29). However, the role of the Na+K+-ATPase in regulating programmed cell death is poorly understood and likely to be complex since this enzyme is found to have opposing influences on cell death in different systems (30).



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  		Figure 7. Structure of the cardiac glycosides and suloctidil. The first three cardiac glycosides (nerifolin, peruvoside and digitoxin) inhibited polyglutamine-induced caspase-3 activation and protected against polyglutamine toxicity in secondary screens. The next three (ouabain, digoxin and lanatoside c) were subsequently also found to inhibit polyglutamine-induced caspase-3 activation. Strophanthidin had no inhibitory activity. Suloctidil, a calcium channel blocker, inhibited caspase-3 activation at higher concentrations than the cardiac glycosides. *Identified in our screen.

 
Alternatively, since strophanthidin inhibits the Na+K+-ATPase with potency comparable to the other cardiac glycosides, but showed no inhibition of caspases-3 activation in our assays, it is possible that the protective effect of the cardiac glycosides might reflect an activity unrelated to the Na+K+-ATPase. This notion is supported by the observation that inhibiting the Na+K+-ATPase by depleting extracellular K+ had no inhibitory effect on polyglutamine-induced caspase-3 activation. Strophanthidin differs from the other cardiac glycosides in that it has no sugar moiety on C3 of the steroid ring, a feature that does not have an effect on its potency as a pump inhibitor (Fig. 7). If there is a novel target for the cardiac glycosides that is responsible for the protective effect against polyglutamine, perhaps the presence of a carbohydrate side-group at C3 is important for that activity.

Whatever the target for the cardiac glycosides, it seems likely that they interrupt the polyglutamine-induced apoptotic cascade at a relatively distal point. This is inferred from the ability of these drugs to inhibit caspase-3 activation in response to overexpression of the pro-apoptotic protein Bax. Bax is an integrator of multiple pro- and anti-apoptotic signals, and when activated, translocates to the mitochondria where it mediates a transition resulting in increased mitochondrial membrane permeability, loss of the proton gradient, and release of pro-apoptotic factors such as cytochrome c into the cytoplasm (31). Upon release, cytochrome c initiates a protease cascade by forming a complex with Apaf-1 leading to its polymerization, which serves to activate procaspase-9 by cleavage to caspase-9. Caspase-9 in turn activates procaspase-3 by cleavage to caspase-3, an effector caspase that cleaves further cellular substrates and culminates in cell death (18,32). This distal component of the apoptotic cascade is under substantial regulation by pro- and anti-apoptotic modifiers such as the inhibitor of apoptosis (IAP) family of proteins and chaperones and provides a plethora of candidate targets for cardiac glycoside intervention (33,34). In contrast, suloctidil had no effect on caspase-3 activation by Bax, and thus appears to interrupt polyglutamine toxicity at a more proximal point.

There is already substantial clinical experience in humans with the drugs identified here. Despite a narrow therapeutic window, cardiac glycosides are in widespread clinical use. Suloctidil showed efficacy in treatment of peripheral vascular disease and secondary prevention of stroke (35). However, this drug was associated with hepatic toxicity and development was discontinued in favor of drugs with fewer side effects. Based on the results of our assay, these drugs may be considered promising candidates for evaluation in clinical trials in polyglutamine disease. However, further investigation in transgenic animal models is needed before proceeding to human studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture, plasmids, transfection and drug treatment
HEK 293T (human embryonic kidney) and MN-1 cells were cultured in 75 cm2 vented tissue culture flasks at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's minimal essential medium (Gibco BRL) supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin/100 µg/ml streptomycin and 2 mM L-glutamine. Expression plasmids pAR16, pAR112, pAR112-GFP, and pBax have been previously described (10,36). For the caspase-3 activation screening assay, cells were seeded in 48-well assay plates 1 ml/well at a density of 120 000 cells/well 24 h before transfection and transfection with 0.2 µg of AR112 plasmid DNA was performed using Lipofectamine Plus following the manufacturer's protocol (Invitrogen Life Technologies). For IC50 determination, HEK 293T cells were seeded in six-well culture dishes at a density of 7x105 cells/well 24 h prior to transfection. MN-1 cells were seeded at a density of 4x105 cells/well in six-well culture dishes and transfected with 1 µg of plasmid DNA using the FuGENE-6 transfection reagent (Roche Diagnostics) following the manufacturer's protocol. The NINDS Custom Collection consists of 1040 biologically active compounds dissolved in DMSO (MicroSource Discovery Systems). Sixteen hours after transfection, each drug was added to a single well to make a final concentration of 10 µM drug and 0.5% DMSO. This concentration of DMSO has no affect on the caspase-3 activation assay (data not shown). The transfected cells were incubated with the test drugs for an additional 48 h, and then harvested for analysis.

Caspase-3 assay
After incubation, the cells were harvested and lysed in 100 µl of buffer consisting of 10 mM Tris pH 7.3, 10 mM NaH2PO4, 150 mM NaCl and 1% Triton X-100 and stored at –80°C until used for analysis. Caspase-3 activity was determined by incubating 100 µg of lysate with 50 µM of the fluorogenic substrate Ac-DEVD–AFC (Biosource International) in a total volume of 200 µl of assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.2% CHAPS, 20% glycerol, DTT 10 mM) in the dark for 2 h at 37°C. Substrate cleavage was detected using Cytofluor II Fluorescence multiwell plate reader (Perspective Biosystems) with excitation and emission wavelengths of 420 and 520 nm, respectively. The specificity of the caspase-3 activity was confirmed by inhibition with z-VAD-fmk (PharMingen) at 20 µM. Drug activity was defined as percentage inhibition=[1–(sample well/vehicle control well mean)]x100. IC50 values were determined using Prism software (GraphPad Software Inc.).

Assessment of cell viability
Inherent drug cytotoxicity was evaluated with an XTT viability assay (Cell Proliferation Kit II, Roche Diagnostics). This assay is based on the ability of metabolically active (live) cells to reduce the tetrazolium salt XTT {sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate} to a water-soluble formazan product. Cells were plated at a density of 3x104 cells/well in a 96-well tissue culture plate and transfected as described above. Sixteen hours after transfection, the appropriate drugs were added in a volume of 0.1 ml. Sixty hours after transfection, XTT reagent was added to a final concentration 0.15 mg/ml and incubated for 4 h at 37°C, 5% CO2. The formazan product was quantitated by absorbance spectroscopy at a wavelength of 450 nM using an Anthos HT III microplate spectrophotometer. XTT reduction was expressed as a percentage relative to vehicle control cells which were set to 100%. To examine the ability of drugs to protect cells from polyglutamine cytotoxicity we used a fluorescence-activated cell sorting (FACS)-based survival assay described previously (10). Briefly, 60 h post-transfection (48 h after drug addition), cells were harvested with trypsin, gently pelleted by centrifugation and resuspended in PBS with 1% serum on ice. The cells were stained with propidium iodide (P1) 1 µg/ml (Sigma), gently vortexed, and incubated for 5 min at room temperature in the dark. 10 000 non-gated events were acquired for each sample (Beckman Coulter XL instrument and CellQuest software used for analysis). The results were expressed either as a percentage of PI-positive (non-viable) cells relative to total cells or as GFP/PI double positive (transfected non-viable) cells relative to total GFP positive cells (all transfected cells).


   ACKNOWLEDGEMENTS
 
The authors wish to thank Addis Taye and George Harmison for technical advice. J.P.T. was a Howard Hughes Medical Institute post-doctoral fellow and was supported by K22-NS44125-01.



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  		Figure 4. Inhibition of the Na+K+-ATPase with K+-depleted media does not block polyglutamine toxicity. Twenty-four hours after transfection, the growth media was replaced with either complete medium (control), medium with 1 µM ouabain, medium with 20 µM z-VAD-fmk or K+-depleted media (containing 0.1, 1.0 or 2.5 mM KCl) for an additional 24 h before assay. The results are expressed as percentage caspase-3 activity relative to control (set at 100%).

 

   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Neurology, University of Pennsylvania School of Medicine, 3 West Gates Building, 3400 Spruce St, Philadelphia, PA 19104, USA. Tel: +1 2155731147; Fax: +1 2155731153; Email: jpt{at}mail.med.upenn.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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