Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models

Abstract

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Fig. 1.

Transcriptional profiling of yeast expressing α-syn revealed mitochondrial stress as a key signature of α-syn toxicity. (A) Profiles of 673 genes exhibiting differential expression in at least one experiment (compared against vector) at two and four hours post α-syn induction (p-value<0.05; >2-fold) are shown. These genes exhibited very little change in NoTox cells. By contrast, some of these genes were differentially expressed in HiTox cells as early as two hours post-induction, well before onset of toxicity (at four hours). By contrast to treatment with inactive compounds (5) and (6), treatment with active compounds (1), (2) and (4) resulted in a major reduction in transcriptional changes, indicating rescue. Differentially expressed genes were color coded red (upregulated) and green (downregulated). (B) GO annotation of differentially regulated genes (>2-fold) revealed upregulation of the expression of oxidoreductase transcripts and downregulation of the expression of mitochondrial, ribosomal, respiratory and carbohydrate transport transcripts in HiTox α-syn cells induced for four hours. The expression of transition metal ion binding transcripts was both upregulated and downregulated. Similar, more subtle changes were detected in HiTox cells after two hours of induction. Only slight downregulation of carbohydrate transport transcripts was detected in NoTox cells after four hours of induction. (C) Treatment with compounds (1) or (2) markedly reversed the HiTox-induced transcriptional changes in the ribosomal, mitochondrial, respiratory and oxidoreductase GO categories. Compound (4) showed a more modest transcriptional restoration in these categories. Compounds (5) and (6) exhibited partial reversal of the α-syn-induced transcriptional changes in these GO categories; however, the magnitude of this reversal was less complete that that seen in cells treated with compounds (1), (2) or (4).

Fig. 2.

HiTox cells exhibit mitochondrial abnormalities. (A) Thin-section EM of HiTox cells at four hours post-induction. NoTox cells (b) exhibited comparable features to those of cells expressing vector (a), whereas HiTox cells exhibited numerous defects, including vesicular accumulation, swollen and less electron dense mitochondria, and hypertrophied ER (c, and inset). M, mitochondria; n, nucleus; v, vacuole. An asterisk denotes the vesicle clusters. Scale bar: 1.0 μm. (B) ROS production was measured using CM-H₂DCFDA. Vector or NoTox cells had very little or no ROS, whereas nearly 35% of HiTox cells exhibited ROS. ROS reactivity in HiTox cells was ameliorated upon treatment with (1–4) (<15%), but not with (5) or (6) (>25%). The bar graph shows the mean ± s.e.m. from at least three independent experiments and an asterisk represents a value that significantly differed from that of HiTox, with a P-value less than 0.05 as determined by two-tailed Student’s t-test. (C) Subcellular fractionation shows little α-syn in the mitochondria. NoTox and HiTox strains were subjected to cellular fractionation to track the compartmentalization of α-syn-GFP. Western blots (left) and coomassie staining (right) of three fractions are shown: total lysate (S₄₀₀₀), crude mitochondrial pellet (P_13,000) and gradient-purified mitochondria (Mito). Western blot analysis shows the specific enrichment of mitochondria in the gradient (porin). P_13,000 fraction contains vacuole, ER and mitochondria, whereas most α-syn-GFP remains soluble in S₄₀₀₀. Only mitochondria are highly enriched by the sucrose step gradient, with most α-syn-GFP eliminated from this fraction. Quantitative western analysis with Licor Odyssey IR imaging (data not shown) revealed that only 0.11% (NoTox) and 0.02% (HiTox) of α-syn-GFP localized to mitochondria. The absolute amount of mitochondrial α-syn-GFP does not change, rather the difference between NoTox and HiTox is a function of higher total α-syn-GFP levels in the HiTox strain.

Fig. 3.

A high-throughput chemical screen identified small-molecule antagonists of α-syn toxicity. (A) Secondary screen identified four bioactive molecules that rescue at micromolar concentration, as well as two biologically inactive molecules, all representing the same chemical family. (B) Growth curve of HiTox cells (black curve), which did not grow. Bioactive compound (3) was maximally effective at 1 μM, restoring growth of HiTox cells to nearly 50% that of wild-type cells (supplementary material Fig. S5) at 48 hours post-induction. (C) Growth of HiTox cells was rescued by treatment with 1 μM of the bioactive compounds (1–4), but not by compounds (5) or (6). These differences were consistent over three different experiments, but slight differences in starting OD and room temperature and humidity prohibit averaging across experiments. (D) Propidium iodide (PI) staining for cell viability revealed ~25% toxicity in HiTox cells six hours after induction. Treating HiTox cells with 1 μM compound (3) restored viability (by reducing toxicity to <10%), whereas (5) had no significant effect. The bar graph shows the mean ± s.e.m. from at least three independent experiments and an asterisk represents a value that significantly differed from that of HiTox, with a p-value less than 0.05 as determined by two-tailed Student’s t-test.

Fig. 4.

Validation of lead compounds in higher order model systems. (A-C) C. elegans model with α-syn overexpression in DA neurons. A blinded assay identified compounds (1–4) as active, and compounds (5) and (6) as inactive. (A,B) Bar graphs showing the number of worms with all four intact cephalic DA (CEP) neurons in each treatment for (A) embryo and (B) adult worms. The bar graph shows the mean ± s.d. from at least three independent experiments and an asterisk represents a value that significantly differed (p-value<0.05; two-tailed Student’s t-test) from that of α-syn worms receiving only the DMSO vehicle. (C) Photos of GFP-tagged neurons in each type of treatment. Importantly, these compounds exerted rescue in adult worms that have been overexpressing α-syn for some time (B). (D,E) Primary midbrain culture model, in which cells were transfected with lentivirus encoding the α-syn A53T mutation. (D) Immunofluorescence (IF) images of midbrain cultures. Transduction with A53T lentivirus led to abnormal neuronal morphology, including loss of bipolar neuronal processes and shrunken cell bodies. These effects are partially reversed in cultures treated with (1), but not (5). Red, MAP2 marker; green, TH marker; yellow, overlap. Scale bar: 20 μm. (E) A blinded assay carried out in cultures transduced with α-syn-A53T lentivirus identified compounds (1), (2), (3) and (6) as suppressors. The concentrations of these compounds on the bar graph were as follows: (1) 0.5 μg/ml; (2) 1.0 μg/ml; (3) 0.25 μg/ml; (4) 0.125 μg/ml; (5) 0.05 μg/ml; (6) 1.0 μg/ml. The bar graph shows the mean ± s.e.m. from at least four independent experiments and an asterisk represents a significant difference with respect to the value obtained for cells expressing α-syn-A53T in the absence of compound (p-value<0.05; one-way ANOVA with Newman-Keuls post-test).

Fig. 5.

The bioactive compounds restored α-syn membrane localization, ER-to-Golgi trafficking and mitochondrial morphology. (A) Microscopy studies with GFP-tagged α-syn at four hours post-induction showed α-syn foci in HiTox cells. Localization of α-syn was restored to the plasma membrane upon treatment with the bioactive compounds. By contrast, bright foci were retained in cells treated with inactive compounds. (B) CPY maturation assay. The bar graph depicts the percentage of CPY that exits the ER and enters the Golgi or the vacuole in various treatments. At 4.5 hours post-induction, HiTox cells exhibited severe trafficking defects, as the majority of CPY was retained in the ER (~65%; DMSO and data not shown). This defect was suppressed by cells treated with 1 μM of the bioactive compounds (1–4), but not with the inactive compounds (5,6). Gel slices are shown on the right and the bar graph shows the mean ± s.e.m. (left). (C) Treatment with 1 μM (1–4) (a-d), but not with (5) or (6) (e,f), restored the ER and most of the mitochondria to normal morphology, although some vesicular accumulation persisted. m, mitochondria; n, nucleus; v, vacuole. An asterisk denotes the vesicle clusters. Scale bar: 1.0 μm.

Fig. 6.

Effect of lead compounds on rotenone neurotoxicity. (A) Immunofluorescence (IF) images of the primary midbrain cultures. Treatment with (1) dramatically restored the neuronal morphology of rotenone-treated neurons. Red, MAP2 marker; green, TH marker; yellow, overlap. Scale bar: 20 μm. (B) Exposure to 100 nM rotenone resulted in >50% death of DA neurons, whereas compound (1) antagonized rotenone-induced DA cell death in a concentration-dependent manner. The bar graph shows the mean ± s.e.m. from at least three independent experiments and asterisks represent a significant difference with respect to the value obtained for cells exposed to rotenone alone (p-value<0.001; one-way ANOVA with Dunnett’s post-test).