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Nat Chem Biol. Author manuscript; available in PMC 2011 Jun 1.
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PMCID: PMC3018711

Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins


A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction, and oxidation of disulfides. Through a small-molecule-screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated-ER-membranes and trigger apoptotic cell death, via mitochondrial outer membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI provides a new mechanism linking protein misfolding and apoptotic cell death.


Protein folding diseases encompass a large class of neurological disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington disease (HD), and prion diseases1. Huntington disease, for example, is a polyglutamine disease caused by a mutation that expands a CAG repeat region within the huntingtin gene. This mutation leads to a polyglutamine-expanded huntingtin protein that improperly folds; ultimately, this causes cell death in the striatum and cortex2. Precisely how mutant huntingtin causes HD remains unclear; however, both humans and animal models of HD show markers of apoptotic cell death39.

Apoptosis is an elaborate cell death program essential for neuronal pruning during development, and for the clearance of cells that become dysfunctional10. The most common form of apoptosis proceeds via the intrinsic pathway through mitochondria. In this pathway, an initiation event triggers mitochondrial outer membrane permeabilization (MOMP), which is a perforation in the outer mitochondrial membrane created by oligomerized Bax or Bak protein11,12. The induction of MOMP leads to the release of proteins (e.g., cytochrome c and Smac) from the mitochondrial intermembrane space, which in turn activates caspase enzymes that degrade key structural and functional components of the cell13. Several upstream triggers of MOMP have been reported, including DNA damage, loss of cell adhesion, growth factor withdrawal, and endoplasmic reticulum (ER) stress14. The endoplasmic reticulum is an important site of protein folding, dysregulation of which can activate a cell death cascade. However, in some neurodegenerative diseases (e.g., HD and PD) the aberrant protein accumulates in the cytosol, suggesting additional mechanisms exist to monitor protein folding and to control cellular homeostasis.

We used a cell-based model of HD to screen tens of thousands of synthetic compounds and natural products for their ability to suppress cell death induced by polyglutamine-expanded huntingtin exon one. We then used Huisgen cycloaddition chemistry (or “Click-Chemistry”) to identify protein disulfide isomerase (PDI) as the molecular target of multiple active compounds. We found that in response to expression of mutant huntingtin exon one, PDI becomes concentrated at ER-mitochondrial junctions and induces MOMP. The death-suppressing compounds we identified in our screen block this cascade by inhibiting the enzymatic activity of PDI. Finally, we show that inhibiting PDI activity in normal rat brain cells suppresses the toxicity of misfolded huntingtin and APP/Aβ protein.


Small molecule inhibitors of apoptosis

To identify small molecule suppressors of polyglutamine-induced apoptosis, we adapted a PC12 cell model of HD into a high-throughput screening format15. In this system, PC12 cells were transfected with the first exon of the human huntingtin (htt) gene, containing either wild-type (Q25) or mutant (Q103) polyglutamine (polyQ) repeats, fused to EGFP; we refer to these two cell lines as “Q25” and “Q103”. Protein expression was induced by tebufenozide, an ecdysone analog that binds to the Bombyx mori ecdysone receptor. Following addition of tebufenozide to the culture medium, mutant cells accumulated peri-nuclear inclusion bodies (~12 hours) and underwent apoptosis (15–48 hours), which we quantified using Alamar Blue, a fluorescent indicator of cell viability (Fig. 1).

Figure 1
Cell-based (PC12) model of mutant huntingtin protein misfolding and cell toxicity

We screened 68,887 compounds derived from small molecule libraries containing natural products, natural product analogs, synthetic drug-like compounds, and annotated, biologically active compounds (Fig. 1d). Hit compounds from the primary screen were prioritized based on their potency in a dose-response curve. These hits were filtered to eliminate those that were likely acting through non-polyQ-dependent mechanisms (e.g., compounds that suppress htt protein expression, or compounds that act as general caspase inhibitors; Supplementary Fig. 1 and data not shown). The top five hits were prioritized based on both potency and efficacy of cell viability rescue and the compounds’ capacity to restore “normal” (uninduced) cell morphology to induced Q103-expressing cells (Fig. 2 and Supplementary Fig. 2). Interestingly, these compounds did not significantly alter the accumulation of htt-Q103 inclusion bodies (Supplementary Fig. 3).

Figure 2
Dose-response curves for hit compounds that suppress Q103-induced apoptosis

Identifying small molecule targets

A covalent interaction between a small molecule and its target protein can increase the facility of target identification. Considering that alpha-chloromethylketone-like moieties are frequently used for irreversibly inhibiting proteases, we speculated that compound 16F16, with its related chloroacetyl moiety, was covalently binding its target protein. In support of this hypothesis, we found that an analog of 16F16 lacking the chloro substituent was inactive. Additional structure-activity studies of 16F16 revealed that changing the ester from methyl to ethyl did not affect its activity. However, modifications at this site that incorporated biotin or fluorescein affinity tags resulted in a complete loss of activity (data not shown). Therefore, we identified a site on 16F16 that could tolerate small structural changes; however, incorporation of traditional large affinity handles was not compatible with activity(see Supplementary Methods).

To overcome this problem, we adapted the copper-mediated Huisgen 1,3,-dipolar cycloaddition of an azide to an alkyne; this reaction has been shown to be bio-orthogonal and facile in the presence of cell lysates1620. The advantage in using this approach was that we could introduce a minimal structural modification to 16F16 (e.g., a terminal propargyl alkyne) without losing biological activity. Once the alkyne-derivatized-16F16 was covalently bound to its target protein, we could subsequently attach a tag (e.g., fluorescein-azide or rhodamine-azide) via modified Huisgen cycloaddition reaction (Fig. 3a).

Figure 3
Identification of small molecule target proteins using Huisgen cycloaddition chemistry

Following synthesis, we tested the alkyne analog of 16F16 (16F16A) for its ability to suppress Q103-induced apoptosis and found that the alkyne modification did not disrupt target binding. We then coupled azidofluorescein to PC12-Q103 lysates treated with 16F16A, and purified the resulting fluorescein-tagged proteins with an anti-fluorescein antibody. By mass spectrometry we identified two isoforms of protein disulfide isomerase (PDIA1 and PDIA3) as specific targets of 16F16A which we then confirmed by competitive binding assays, labelling purified PDI, and a Western blot of affinity purified target proteins (Fig. 3b–d, Supplementary Tables 1, 2, and 3).

In performing the competitive binding assays, we discovered that in addition to 16F16, the other four active hits from the screen (Fig. 2) also showed competitive inhibition of 16F16A binding to PDI, whereas inactive analogs did not (Fig. 3c). Given the ability of PDI to function as a disulfide reductase, we tested whether cystamine, a simple organic disulfide that has been shown to delay onset of neuropathological sequellae in animal models of HD and PD, could compete for 16F16A binding to PDI2124. Cystamine was able to compete with 16F16A for binding to PDI, whereas hypotaurine, an inactive cystamine analog, did not have this effect (Fig. 3c). Given the results of the PDI binding experiments, we went on to test these hit compounds, along with cystamine, for their ability to inhibit the enzymatic activity of PDI in vitro. Here, we observed a striking correlation between the PDI binding assays (Fig. 3c), inhibition of PDI enzymatic activity, and rescue of polyQ-induced cell death (Fig. 4).

Figure 4
Compounds that bind to PDI and rescue htt-Q103-induced toxicity inhibit PDI reductase activity in vitro

PDI within mitochondrial-associated-membranes induces MOMP

In humans, protein disulfide isomerases (EC constitute a family of at least 17 enzymes of the thioredoxin superfamily that function primarily in the ER as chaperone proteins and facilitate disulfide bond rearrangements via catalysis of thiol-disulfide-exchange25,26. In addition to their well-described function in the ER, PDI proteins have been reported in the cytosol and on mitochondria, where their physiological functions are less clear25,27,28. We examined the localization of PDI over time in subcellular fractions of induced Q25 and Q103 cells and found that following induction, Q103 cells differentially accumulated PDI in the mitochondrial fraction (2.8 fold over Q25 at 24 hours; Fig. 5a–c). Inhibiting PDI activity with 16F16 further increased mitochondrial accumulation of PDI in Q103 cells (5.3 fold over Q25 at 24 hours). The latter result suggested that by inhibiting the enzymatic activity of PDI, we were allowing levels of the mitochondrial-associated PDI to increase beyond a point that would otherwise induce apoptosis. To identify more precisely the localization of mitochondrial-associated PDI we performed protease-shaving analysis of the ER/microsomal and crude (sucrose gradient purified) mitochondrial fractions. These experiments showed that the mitochondrial-associated PDI was similar to the ER/microsomal PDI in that it was protected from trypsin digestion (Supplementary Fig. 4). Mitochondria are known to contact the ER through mitochondrial-associated-membranes (MAM; Fig. 5d)29. This membrane fraction, which is an extension of the endoplasmic reticulum (ER) that contacts mitochondria, has been shown to function as an important regulatory connection between the ER and mitochondria3032. When we purified MAMs from PC12 mitochondria via Percoll gradient sedimentation, we found that the mitochondrial-associated PDI was predominantly localized to the MAM compartment (Fig. 5e).

Figure 5
PDI accumulates at MAM contacts and induces MOMP

Inhibiting PDI in Q103-expressing cells suppresses mitochondrial release of cytochrome c and subsequent activation of caspases (Fig. 5f and Supplementary Fig. 1). In light of finding increased levels of PDI in association with mitochondria isolated from Q103-expressing cells, we hypothesized that PDI may engage MOMP by interacting with proapoptotic proteins associated with the outer mitochondrial membrane at a critical concentration ratio. We tested this hypothesis in vitro using a “MOMP assay”, whereby we added purified PDI to isolated mitochondria and monitored the mitochondrial release of cytochrome c. Here, we found that under physiological conditions, there is a threshold concentration of PDI that can induce MOMP and that small molecule PDI-inhibitors suppressed this activity (Fig. 5g). Finally, overexpressing PDIA1 or PDIA3 in PC12 cells simulated the MOMP assay in a cellular context and induced apoptosis that could be rescued by blocking the catalytic activity of PDI with small molecule inhibitors (Fig. 5h–i).

To further characterize and validate the mechanism of PDI-induced apoptosis, we evaluated the PDI-induced MOMP mechanism in two additional model systems. The induction of MOMP is primarily mediated by the formation of homo-oligomeric channels of Bax or Bak in the mitochondrial outer membrane12. Therefore, we tested whether PDI could induce MOMP in mitochondria isolated from wild type or Bax/Bak double-knockout MEF cells33. These experiments showed that PDI could induce MOMP in wild-type MEF mitochondria, but mitochondria from Bax/Bak double-knockout cells were resistant (Fig. 6a). The mechanism governing the assembly of Bax/Bak homo-oligomeric complexes is not well understood; however, it has been shown that Bax can form an active oligomeric complex via oxidation of conserved cysteine residues34. We investigated the possibility that PDI could induce MOMP by oxidizing Bax or Bak and found that wild-type MEF mitochondria undergoing MOMP had disulfide-linked oligomers of Bak, but not Bax (Fig. 6a and Supplementary Fig. 7). However, the combined knockdown of Bak and Bax protein in PC12-Q103 cells showed an additive rescue of apoptosis suggesting that both Bax and Bak function as downstream cell death effectors (Supplementary Fig. 5). In addition, overexpressing Bcl-2 protein in PC12-Q103 cells also suppressed apoptosis, suggesting that Bcl-2 can antagonize PDI-induced activation of Bax and Bak (Supplementary Fig. 5).

Figure 6
Characterization of PDI-induced MOMP and validation in model systems

We next tested the protective effects of PDI inhibitors in a corticostriatal brain slice model for mutant htt-exon 1 (htt-N90Q73)-induced neurotoxicity3537. Of three PDI inhibitors tested, all showed dose-dependent rescue of htt-N90Q73-induced neurotoxicity in medium spiny neurons (MSNs) in the striatal region of these brain slices (Fig. 6b–e). We also confirmed the mechanism of PDI-induced toxicity in MSNs by rescuing htt-N90Q73 neurotoxicity with PDIA3 shRNA (Fig. 6f and Supplementary Fig. 6).

To broaden the scope of our study, we repeated these experiments in rat brain slices expressing amyloid precursor protein (APP), which is processed in situ to Aβ peptides, the causative agents central to the amyloid cascade hypothesis of Alzheimer’s disease38,39. Similar to the results obtained for mutant huntingtin, we observed a dose-dependent rescue of Aβ toxicity in pyramidal neurons when PDI was inhibited by small molecules or shRNA knockdown (Fig. 6g–j).


Suppressing caspase activation or blocking the release of caspase cleavage products has been shown to be effective in delaying disease progression in transgenic mouse models of ALS, HD, and AD4045. In our screen for suppressors of polyQ-induced cell death, we identified several compounds that act upstream of caspase activation by targeting the A1 and A3 isoforms of PDI. As chaperone proteins, PDIs are involved in the recognition and repair of aberrant protein assembly and show increased expression in patients with ALS, PD, and Creutzfeldt-Jakob disease (human prion disease)4648. The initial upregulation of PDIs constitutes a defense mechanism to repair misfolded proteins and restore normal cellular homeostasis49. However, as we have shown here in brain slice assays for mutant htt-N90Q73 or APP/Abeta-induced neurodegeneration, ultimately limiting increases in PDI activity and/or expression with small molecule compounds or shRNA, respectively, provides net neuroprotective benefit. Thus, analogous to expression of p53, which accumulates to repair damaged DNA, but induces apoptosis at extreme levels of DNA damage, PDI is also capable of engaging a cell death cascade when it accumulates at high levels in response to misfolded proteins.

The PDI apoptosis pathway appears to be specific for misfolded proteins, since other apoptotic stimuli (e.g., chemical inducers and serum withdraw) are not suppressed by inhibiting PDI (Supplementary Fig. 9). Furthermore, the ability of PDI to induce MOMP in isolated mitochondria and its inhibition-mediated rescue of apoptosis in cells depleted of BiP/GRP78, a key regulator of the unfolded protein response, suggests that the PDI pathway operates independently of factors that regulate ER stress-induced apoptosis (Supplementary Fig. 10). From our analysis of PDI-induced MOMP in isolated mitochondria, PDI can oligomerize Bak in the outer mitochondrial membrane by inter-molecular oxidation of Bak cysteine residues. Although we have not excluded the involvement of Bak/Bax-activating intermediates, structural properties and conserved PDIA1/A3 residues required for oxidative activity may explain why A1 and A3 were the two PDI isoforms identified in our screen26.

Complete inhibition of PDI catalytic activity, as observed with high concentrations of inhibitors and knockdown with shRNA, is toxic in cultured cell lines such as PC12. Despite the narrow therapeutic index observed in PC12 cells, our experiments using rat brain slice explants demonstrate protective efficacy against polyglutamine and APP/Aβ toxicity in neurons. In vivo, cystamine has been shown in several studies to extend survival and to improve motor performance in HD and PD animal models; however, its mechanism of action has remained unclear2124,50. In our study, we show that cystamine competitively binds to PDI and inhibits its enzymatic activity in vitro. In cells, cystamine suppresses intrinsic apoptosis induced by polyQ-expanded huntingtin and partially blocks PDI-induced MOMP in purified mitochondria. These results suggest that the protective effect of cystamine in HD and PD model mice may be through inhibition of PDI.

In summary, we used a novel strategy to identify PDI as a target of several compounds that suppress polyglutamine-induced and APP/Aβ-induced neuronal degeneration. Furthermore, we have identified a cell death pathway regulated by PDI, a chaperone protein important for quality control in protein folding. A comprehensive understanding of how the PDI cell death pathway is controlled may aid in discovering therapeutics to treat protein misfolding diseases.


Cell culture and transfection

We used a PC12 model of HD for screening15. Briefly, PC12 cells transfected with pBWN, an ecdysone-responsive expression vector containing DNA inserts encoding exon-1 of human huntingtin (including proline-rich segment and either 25 or 103 mixed CAG/CAA repeats, EGFP, and neomycin resistance gene) were propagated (37°C, 9.5% CO2) in complete media: DMEM high glucose, 25 mM HEPES (Mediatech #15-018-CV), 10% Cosmic calf serum (HyClone #SH30087.03), penicillin and streptomycin, 2 mM glutamine, and 500 μg/ml active geneticin (G418). For transgene expression, tebufenozide (gift from Lynne Moore, Gage lab, Salk Institute) was added to the above media (200 nM final concentration from 1 mM stock in 85% ethanol). Wildtype and Bax/Bak double-knockout MEF cells (generously provided by Craig B. Thompson, U. Penn) were grown in RPMI/penicillin and streptomycin and 10% fetal bovine serum (Gibco #26140-079).

Cycloaddition reactions and in-gel fluorescence scanning

Cell lysates were prepared from 24-hour-induced Q103 cells by swelling (23 × 106 cells/ml in sodium phosphate buffer, pH 8.1, 5 min, 4°C), passing through a 30-gauge needle (10 strokes), and clarified (14,000 g, 20 min, 4°C). Clarified crude cell lysate (43 μl, 2×106 cell eq.) was incubated with alkyne-derivatized probe (16F16A or 16F16A-DC, 7 μmol, 1hr, RT) and Cu(I)-catalyzed Huisgen cycloaddition reactions (using RhN3 or FcN3 tags) were carried out as described20. Labeled lysates were prepared for SDS-PAGE analysis and visualized in-gel using Molecular Dynamics Fluorimager 595 and appropriate excitation and emission filters for the fluorphore. Competitive binding assays were performed as described above, except that cell lysates were pre-incubated for 1 hr at RT with 20× molar excess of the tested compound (competition controls were incubated with inactive analogs or equivalent addition of DMSO or water). Cystamine (C121509), hypotaurine (H1384) and bovine PDI (P3818) were purchased from Sigma-Aldrich.

Affinity purification and MS sequence identification

Uncoupled FcN3 tag was removed from 16F16A-fluorescein-labeled lysates using Zebra desalting columns (Pierce #89882). Labeled proteins were affinity purified over anti-fluorescein-coupled affinity matrix (5 mg monoclonal anti-fluorescein coupled to NHS-activated sepharose, Amersham #17-0906-01) and eluted with 100 mM glycine (pH 2.8). Affinity purified proteins were analyzed by SDS-PAGE and fluorescently-tagged targets (major doublet at 53–57 kDa) were excised from the gel and submitted to Taplin Biological Mass Spectrometry Facility (Harvard Medical School) for protein identification. Tandem samples were run on SDS-PAGE and transferred to PVDF membranes for Western blot analysis using anti-PDI antibody.

Brain slice assays for HD and AD

Coronal brain slices (250 μm-thick) containing both cortex and striatum were prepared from CD Sprague-Dawley rat pups (Charles River) at postnatal day 10 using a Vibratome (Vibratome Co.). Brain slices were placed in 6-well plate transwell inserts (BD Biosciences) under which culture medium was added, containing 15% heat-inactivated horse serum, 10 mM KCl, 10 mM HEPES, 100 U/ml penicillin/streptomycin, 1 mM MEM sodium pyruvate, and 1 mM L-glutamine in Neurobasal A (Invitrogen). A biolistic device (Helios Gene Gun; Bio-Rad) was then used to transfect brain slices according to manufacturer’s instructions; 1.6 μm gold particles were used with the device set at 95–105 psi and an aperture distance of ~2.5 cm. As the co-transfection linkage rate of biolistics approaches 100%, multiple DNAs can be reliably co-expressed in neurons by coating the gold particles with mixtures of independent expression plasmids. Thus, labeling of striatal or cortical neurons was done by mixing a separate plasmid driving YFP expression together with either an Htt or APP expression construct as described below, and a third plasmid expressing shRNA was added to the mixture for the RNAi knockdown studies. In all cases, total DNA load was balanced in control conditions where necessary by the addition of the corresponding empty vectors. Compounds were added to the culture wells at the time of slice preparation and transfection, to a final DMSO concentration of 0.1%; the slices were then placed in humidified incubators under 5% CO2 at 32 deg. C.

For the HD brain slice model, YFP co-transfected medium spiny neurons (MSNs) were identified after 4 days of incubation by their location within the striatum and by their characteristic dendritic arborization using Leica MZFLIII fluorescent stereomicroscopes. MSNs exhibiting normal-sized cell bodies, even and continuous expression of YFP within all cell compartments, and >2 discernable primary dendrites > 2 cell bodies long were scored as “healthy”. The htt plasmid used to induce neurodegeneration of MSNs included human htt exon-1 containing a 73 polyglutamine repeat and was constructed based on clones and sequences that were generous gifts of Dr. Chris Ross (Johns Hopkins) and the Hereditary Disease Foundation. This htt sequence was cloned into the gWiz expression vector (Genlantis). Positive control KW+CGS was KW-6002 (50 μM) and CGS-21680 (30 μM); Boc-D-FMK (Sigma-Aldrich, Inc; 100μM).

For the AD brain slice model, cortical pyramidal neurons were assayed after 3 days of incubation and were readily discerned and unambiguously identified based on their characteristic positioning and orientation within the cortex, and by their striking morphological features, most notably the extension of a single, prominent apical dendrite radially towards the pial surface. These key features were used to determine the numbers of healthy cortical pyramidal neurons, namely 1) a robust and brightly labeled cell body positioned within the pyramidal neuronal layers of the cortex; 2) the retention of a clear apical dendrite extending radially towards the pial surface the slice; 3) the extension directly from the cell soma of >2 clear basal dendrites >2 cell body diameters long; and 4) clear and continuous cytoplasmic labeling with the YFP visual marker in the cell soma as well in all dendrites and the axon. In this brain slice assay, transfection of the normal human neural APP sequence (695 aa form) produces sufficient Aβ to induce neurodegeneration of cortical neurons, and this neurodegeneration could be blocked be either β- or γ-secretase inhibitors (Braithewaite et al., submitted). As above, this APP sequence was cloned into the gWiz expression vector.


Primary screening parameters were adjusted (e.g., cell density and incubation time) to yield the best Z′ calculation between induced Q103 and Boc-D-FMK rescued cells. Mean and standard deviations for viability assays (run as biological triplicates) were plotted using Cricket Graph (interpolation function). For the In vitro PDI assay and drug/serum withdrawal apoptosis assay, non-linear regression was used to fit curves to the mean and standard deviations (N=3) calculated with GraphPad Prism software. For Western blot quantification, one representative sample of three or more experiments is shown. Statistical significance of shRNA rescue in PC12 cells was determined by ANOVA and Dunnett’s post hoc comparison test at the 0.01 confidence level. For the cortical-striatal brain slice assay, averages and SEM were calculated by counting 100 YFP positive MSNs from 20–22 slice assays. Statistical significance was determined by ANOVA and Dunnett’s post hoc comparison test at the 0.05 confidence level.

Supplementary Material


Supplementary Table 1: 56–60 kDa 16F16A tagged proteins




Supplementary Table 2: 50–56 kDa 16F16A tagged proteins



We thank E. Schweitzer for the transfected PC12 cell lines, C. Ross for Htt DNAs upon which the constructs used here were based, I. Smukste for assistance with organic synthesis, A. Bauer for assembling the BBB library, D. Dunn for additional brain slice experiments, and C. Thompson for the Bax/Bak double-knockout MEF cells. We are grateful to A. Speers, J. Alexander, and B. Cravatt for reagents and advice on the cycloaddition reactions; E. Miller, E. Signer, A. Tobin, N. Wexler, C. Johnson, R. Pacifici, and M. Finn for useful discussions; E. Miller and S. Hoffschmidt for editorial comments on the manuscript. This research was supported by grants from the NIH (NIGMS 1RO1GM085081; B.R.S. and NINDS R21NS048181; D.C.L.), the Hereditary Disease Foundation (D.C.L., B.R.S.), the High Q Foundation (B.R.S.), CHDI Foundation, Inc. (D.C.L., B.R.S.), the Arnold and Mabel Beckman Foundation (B.R.S.), the Training Program in Molecular Biophysics T32GM008281 (A.K), and a Burroughs Wellcome Fund Career Award at the Scientific Interface (B.R.S.). B.G.H. was supported in part by postdoctoral fellowship from the High Q Foundation. Brent R. Stockwell is an Early Career Scientist of the Howard Hughes Medical Institute.



B.G.H. and B.R.S designed the experiments. B.G.H. performed the experiments and analyzed the data with B.R.S. A.K. assisted with the library screening, characterization of hit compounds, analysis of synthesized analogs, validation studies, and structural identification of securinine. R.L. performed the PDI and Bcl-2 overexpression experiments. R.S., G.J.T., and D.C.L. provided the cortical-striatal brain slice data. B.G.H. and B.R.S. wrote the manuscript.

The authors declare no competing financial interests.


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