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Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection, and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer’s (AD) and Huntington’s diseases (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae (S. cerevisiae). We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein, the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-synuclein clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wildtype mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-synuclein, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-synuclein in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-synuclein accumulation in transgenic mouse models of α-synuclein neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, that might yield clinical benefit for synucleinopathies including PD, Lewy body dementia, REM sleep disorder, and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.
Latrepirdine (Dimebon; dimebolin) is a neuroactive compound with antagonist activity at histaminergic, α-adrenergic, and serotonergic receptors that was associated with enhanced cognition1–4, neuroprotection5, 6, and neurogenesis7 in laboratory animals. Based on its effects on cognition in rodents and its highly favorable safety profile, the compound entered clinical trials for both Alzheimer’s disease (AD)8 and Huntington’s disease (HD)9. Related reports indicate that latrepirdine protects against the cytotoxicity associated with Aβ4210 or γ-synuclein11 by stimulating catabolism of these aggregation-prone, neurodegeneration-related proteins. Here, we sought to determine whether latrepirdine offered protection against the cytotoxicity associated with the accumulation of several neurodegeneration-related proteins including α-synuclein (α-syn), the amyotrophic lateral sclerosis (ALS)-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine (polyQ) expansion (HTT gene; htt-103Q). We report that latrepirdine improved cell viability in Saccharomyces cerevisiae (S. cerevisiae) expressing α-syn, but offered no protection to yeast strains expressing TDP43, FUS, or htt-103Q.
Parkinson’s disease (PD) and AD are the two most common forms of neurodegenerative diseases. Both PD and AD patients present with progressive cognitive decline resulting from a pathogenic accumulation of insoluble protein aggregates that precedes region-specific synaptic and neuronal loss12. Formation of intracellular Lewy bodies, which consist primarily of α-synuclein (α-syn) and ubiquitin, is associated with the pathogenesis of PD, Lewy body dementia (LBD), REM sleep disorder (REMSD), and multiple system atrophy (MSA)12. Interestingly, 30–50% of AD patients also harbor atypical α-syn pathology (in addition to canonical AD neuropathology) in the cerebral cortex13,14. Indeed, α-syn was originally identified as the non-Aβ component of AD-related senile plaques, and was later determined to represent a primary component of Lewy bodies12,15.
Recent studies suggest that α-syn exists as a stable helically folded tetramer that must undergo destabilization prior to misfolding and formation of the fibrillar aggregates that are observed in synucleinopathies16. We hypothesized that a compound that protects against the cytotoxicity associated with α-syn might provide clinical benefit to patients harboring synucleinopathies, including PD, LBD, REMSD, MSA, and in some instances of AD.
To date, the most promising regulators of α-syn degradation and protection against α-syn-induced cytotoxicity are small molecule enhancers of rapamycin (SMERs; most notably SMER-28). Recently, SMER-28 was shown to induce autophagy, improve cell viability, and promote clearance in cellular models of neurodegenerative disease-related proteins including APP metabolites (among them Aβ17; htt18, and α-syn18). In related studies, we reported that latrepirdine protected against cytotoxicity associated with Aβ42 accumulation in S. cerevisiae10, in mammalian cells, and in the brains of transgenic CRND8 mice19 via induction of mTOR- and Atg5-dependent autophagy. Here, we provide evidence to indicate that latrepirdine: (1) protects against α-syn-related cytotoxicity; (2) enhances autophagy and degradation of α-syn in three different model systems; and (3) when chronically administered, reduces α-syn levels in mouse brain in parallel with the induction of autophagy.
The synthesis and characterization of latrepirdine was described previously20. Briefly, latrepirdine was purchased from SinoChemexper (Shanghai, China) and purity of the compound was determined to be >99% or provided directly by Medivation Inc. For use in vitro, latrepirdine was dissolved directly into culture media to the desired concentration, as described in20. For administration in vivo, latrepirdine was dissolved into 0.9% saline (vehicle) at a final concentration of 3.5 mg/ml (made fresh every 2 days).
Yeast cells were grown at 30°C unless specifically mentioned. Growth of yeast strains for was monitored using Bioscreen (www.bioscreen.fi). Yeast strains were pre-grown in 2% raffinose, diluted to an OD600 of 0.05 and induced with 2% galactose. OD measurements were taken every 1hr. Raw data were averaged among independent experiments and OD600 plotted over time as a growth curve. For spotting assays, yeast cells were grown overnight to mid log phase in raffinose medium. Cultures were then normalized to OD600=5.0, and 10X serially diluted and spotted onto the respective dropout plates containing 2% glucose or galactose. Pictures of plates were taken after 2–3 days of growth.
Yeast strain YTS158 (generous gift from Daniel J. Klionsky, University of Michigan) was grown in the absence or presence of different concentrations of latrepirdine (as indicated) to mid-log phase. Cells were then harvested and alkaline phosphatase activity was measured using a spectrophotometric assay as described21.
SH-SY5Y cells stably transfected with a doxycyline-inducible (“tet-off”) wild-type α-synuclein gene, and control SH-SY5Y cells over-expressing the β-galactosidase (β-gal) gene, (a gift from L. Stefanis, Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens, Athens, Greece)22 were cultured and treated with or without drug. See Supplemental Methods section for detailed methods.
All mice in this study were maintained on a hybrid C3H/He-C57BL/6 background (referred to here as “wild-type” mice). Male two-month-old littermates received 21 consecutive once-daily intraperitoneal injections of either 3.5 mg/kg latrepirdine (n=4) or 0.9% saline (vehicle; n=4). Following treatment, animals were sacrificed and transcardially perfused with ice-cold PBS (pH 7.4). Brains were dissected and snap-frozen for biochemical analysis. Animals were individually housed and maintained on a 12:12 light:dark cycle (lights on at 7am) with ad libitum access to food and water throughout the course of the entire experiment. All experimental protocols described herein were conducted within NIH guidelines for animal research and were approved by the Institutional Animal Care and Use Committee (IACUC) at Mount Sinai School of Medicine. See Supplemental Methods for detailed methods.
Integrated density of immunoreactive Western blot bands was measured using MultiGauge Software and normalized to % control (vehicle or nTg littermate, where indicated). In all instances, Shapiro-Wilk test for normality of distribution and Levene’s test for homogeneity of variance were utilized for inclusion in parametric tests (p>0.05 for Shapiro-Wilk and Levene’s tests). Independent samples t-tests (parametric design) or Mann-Whitney U tests (non-parametric design) were utilized to determine significant mean differences between two groups. Significance for t-tests and ANOVAs are reported with a p≤0.05 using two-tailed tests with an α-level of 0.05. All statistical analyses were performed using SPSS v18.0 and/or GraphPad Prism 5.
We employed an S. cerevisiae model systems to investigate whether latrepirdine could protect from α-syn or any of a panel of proteotoxic species associated with neurodegenerative diseases (Table 1). Integration of a single copy of α-syn in S. cerevisiae (1XαSyn) had no appreciable effect on cellular growth23. However, an increase in α-syn gene dosage from one to two copies (2XαSyn) resulted in growth arrest and cell death23. To test the possibility that yeast could be protected from α-syn toxicity by latrepirdine, we monitored the growth of the 2XαSyn strain and the isogenic wild type (W303α) strain in the presence of different concentrations of latrepirdine (Figure 1A–C). Latrepirdine treatment was associated with sustained viability of the 2XαSyn strain, with no effect on growth of the W303α strain. We conclude that latrepirdine treatment was associated with sustained cell viability in the face of α-syn overexpression, which may be related to an effect of the drug on protein degradation.
To test whether the protection from proteotoxicity by latrepirdine was specific to α-syn, we also studied the effect of latrepirdine on other S. cerevisiae models of proteotoxicity in neurodegenerative diseases, including the ALS-associated genes TDP43 (2XTDP43) and FUS (1XFUS), and the HD-associated htt-103Q. Despite the well-established properties of each of these proteins to form aggregates that are toxic to yeast, latrepirdine offered specific protection from only the proteotoxicity of α-syn (Figure 1A–C); i.e., the drug was unable to afford protection from the proteotoxicity associated with FUS, TDP43, or Htt-103Q (Figure 1D–F). One factor underlying the heterogeneity of proteotoxicity may be attributable to the different intracellular compartments in which the aggregates accumulate. Heterogeneity of proteotoxicity also occurs within the same polypeptide as exemplified by the prion protein (PrP) strain phenomenon, in which some aggregates of PrP are benign and well-tolerated by cells while other aggregates are toxic and pathogenic for transmissible spongiform encephalopathies24. Either or both of these phenomena might explain, at least in part, the differential rescue of yeast from some, but not all toxic aggregates. In related studies, we reported that latrepirdine protects yeast against proteotoxicity associated with Aβ42 accumulation via induction of autophagy10 as well as in mammalian cell lines and in mouse brain19. The specificity of latrepirdine for α-syn shown here, taken together with these two reports, suggests that fibrillar assemblies formed by α-syn or Aβ42 aggregates may be preferentially targeted for degradation by the autophagic pathway following latrepirdine stimulation.
We sought to determine whether latrepirdine protection of yeast from α-syn toxicity was attributable to activation of autophagy – a conserved pathway regulating the degradation of long-lived proteins, cellular organelles, and protein aggregates. Addition of rapamycin to yeast cultures strongly induced autophagy, even in a nutrient-rich medium25. Using a yeast reporter of non-specific autophagy (in which maturation of the autophagosome activated an alkaline phosphatase; YTS158 strain21), we observed a significant and dose-dependent increase in alkaline phosphatase activity following treatment with latrepirdine, suggesting that latrepirdine activates autophagy (Figure 2A). Since we observed latrepirdine-induced activation of mTOR-dependent autophagy in mammalian cell lines and mouse brain19, we sought to determine whether activation of TOR-dependent autophagy could also protect the 2XαSyn strain from α-syn toxicity. Treatment of yeast with rapamycin (10μM) was also associated with a significant increase in the viability of 2XαSyn cells, further confirming that stimulation of autophagic activity was sufficient to prevent the proteotoxicity associated with α-syn aggregation in the 2XαSyn model (Figure 2B).
ATG8 (yeast homolog of LC3) encodes a ubiquitin-like protein required for autophagosome formation. In order to test the hypothesis that latrepirdine-mediated protection from α-syn toxicity involved autophagy, we crossed the 2XαSyn strain with an atg8Δ::LEU2 strain (Atg8Δ2XαSyn; Table 1). Atg8Δ2XαSyn cultures were grown in the absence or presence of 100μM or 250μM latrepirdine, and growth was monitored by OD600 (Figure 2C). Although 100μM latrepirdine was associated with protection from α-syn toxicity in the 2XαSyn strain (Figure 1A–C), this concentration was insufficient to protect the Atg8Δ2XαSyn strain (Figure 2C). However, higher concentrations of latrepirdine (250μM) appeared to offer partial protection from α-syn toxicity in Atg8Δ (Figure 2C). This observation suggests latrepirdine may protect cells against α-syn-related toxicity through at least one pathway that involves the regulation of Atg8-dependent autophagy. In contrast to prior observations18, ablation of Atg8-dependent autophagy resulted in increased cell viability of Atg8Δ2XαSyn after 24 hours of growth, by comparison to 2XαSyn cells, although both exhibited less viability when compared to the isogenic wild-type strain at all time points (Figure 2D). We propose that a compensatory mechanism that is selective for survival of the Atg8Δ2XαSyn strain may be induced after 24 hours of growth, allowing for modest improvement in cell viability. In future studies, we plan to investigate these pathways in more detail in order to understand the mechanisms underlying improved survival after 24 hours in the autophagy-deficient Atg8Δ2XαSyn strain.
We tested latrepirdine altered α-syn aggregation using a stably expressing, inducible tet-off system to overexpress wild-type human α-syn in differentiated SH-SY5Y neurons. In this system, α-syn expression is off in the presence of doxycyline (dox) and switched on in cells deprived of dox. We analyzed the levels of monomeric and oligomeric α-syn in total lysates, Triton X-100 soluble, and insoluble protein fractions. Total levels of α-syn protein were higher than in control cells maintained in the α-syn off (+ dox) state. We observed that treatment with 10 nM latrepirdine by 14 days decreased the levels of all forms of α-syn, including aggregates and soluble monomer in the Triton X-100 soluble, total, and insoluble fractions (Figure 3A). Similar results in Western blots were obtained using the LB509 anti-α-syn antibody (data not shown). However, no significant differences were found in the levels of α-syn mRNA measured by quantitative RT-qPCR at 14 days of culture (Figure 3D), suggesting that latrepirdine regulated α-syn levels via a mechanism that was probably post-translational. This is consistent with a role for protein degradation (e.g., autophagy) in α-syn catabolism.
We observed that differentiated SH-SY5Y neurons grown in the absence of dox for 14 days (overexpressing α-syn), exhibited increased necrotic cell death (Figure 3B,C). This conclusion was supported by the observation of a ~36% rise in levels of LDH released into the culture medium in cells over-expressing α-syn as compared to cells maintained in medium containing dox (Figure 3C). In addition, we determined the viability of the remaining non-necrotic cell by measuring the capacity of these cells to metabolize the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) into a soluble formazan. Reduction in the capacity of MTS metabolism is an indicator of cell injury. We found that latrepirdine treatment decreased cell death of α-syn overexpressing cells as measured by LDH release (Figure 3C) and increased their viability as indicated by their capacity to reduce MTS (Figure 3B). This cytoprotective effect of latrepirdine treatment was observed with concentrations of latrepirdine as low as 0.1 nM. As a control, we also measured cell death and viability in SH-SY5Y differentiated cells overexpressing β-galactosidase in the absence (with or without dox) or presence of latrepirdine. These cells did not display significant changes in cell death or viability (Figure 3E).
In a related paper19, we provided evidence that chronic latrepirdine (3.5 mg/kg/day) administration enhanced autophagy, reduced accumulation of Aβ42, and improved behavioral impairment in an AD mouse model. We next investigated whether chronic administration of latrepirdine to wild-type mice for 3 weeks was sufficient to activate autophagy and/or to alter α-syn levels in their brains. Male wild-type mice received 21 once-daily intraperitoneal (i.p.) injections of either latrepirdine (3.5 mg/kg; n=4) or vehicle (0.9% saline; n=4). Following treatment, mice were sacrificed and whole-brain lysates were analyzed for autophagy markers, including LC3 and the polyubiquitin binding protein p62/SQSTM1 (p62), and α-syn levels by Western blot (Figure 4). LC3 is converted from its cytosolic precursor (LC3-I) to its membrane-associated form (LC3-II) by the addition of phosphatidylethanolamine, which allows for the recruitment of LC3-II to the outer membrane of the autophagosome26. When autophagy is induced, autophagosomes accumulate and mature until terminal degradation upon fusion with the lysosome26.
Following 21 days of latrepirdine treatment, we noted a significant decrease in α-syn monomer in the brains of WT mice, and this was associated with significantly elevated LC3-II accumulation but without accumulation of p62. These results support the hypothesis that latrepirdine stimulates α-syn degradation in vivo in the WT mouse brain, which is likely via induction of autophagy, an observation that dovetails with our recently reports10, 19. Furthermore, the current results suggest that the turnover of α-syn monomer may be determined, at least in part, by autophagic stasis, raising the possibility that enhanced autophagic activity might mitigate the toxicity associated with cellular accumulation of α-syn. Ongoing experiments with latrepirdine in cellular and animal models expressing human α-syn will help to elucidate the specific mechanisms of latrepirdine action on regulation of the autophagic pathway with regard to clearance of pathological α-syn.
Protein aggregates are resistant to degradation and therefore inherently longer-lived, allowing for sustained toxicity associated with the buildup of these cytotoxic assemblies27. Based on this principle, a wealth of studies have recently investigated the potential of protein catabolism pathway as therapeutic targets for several neurodegenerative diseases, including AD, HD and PD. Evidence indicates that α-syn is a substrate of autophagy – a conserved catabolic pathway for the degradation of long-lived proteins, cellular organelles, and protein aggregates. Studies of α-syn clearance indicate that α-syn is primarily degraded by the ubiquitin-proteasome system (UPS) under normal conditions in vivo; however, autophagy may be the primary clearance pathway in cases of α-syn overexpression or for the degradation of oligomeric α-syn, which may be too large to fit into the narrow pore of the UPS28. Cellular studies also indicate that α-syn catabolism can be regulated by SMERs, which non-specifically increase Atg5-dependent autophagy18. Another report suggests that conditional deletion of ATG7 results in the accumulation of α-syn aggregates in dopaminergic neurons of mice29.
Herein, we report that latrepirdine, a neuroactive compound with a compelling clinical safety profile, protects S. cerevisiae and differentiated SH-SY5Y neurons against α-syn-induced (or Aβ42-induced10) cytotoxicity. Interestingly, S. cerevisiae expressing TDP43, FUS, or htt-103Q were not protected by latrepirdine in these assays and these findings (specifically regarding htt-103Q) are partially validated by the recent failure of latrepirdine in a phase II trial for HD9. Taken together, we suggest that the latrepirdine-related protection against α-syn cytotoxicity are likely due to promotion of a conserved catabolic pathway (e.g., autophagy). We provide evidence here (Figures 2 and and4),4), and elsewhere10, 19, that latrepirdine stimulates autophagy, resulting in the degradation of α-syn and increased cell viability yeast, in differentiated SH-SY5Y neurons, and in vivo in mouse brains. Based on these findings, we propose that latrepirdine may represent a potentially viable lead compound that might yield clinical benefit for synucleinopathies following optimization of its pro-autophagic and/or pro-neurogenic activity.
Recently, latrepirdine failed in a US-based phase II replication trial30 of a prior successful Russian phase II trial of mild-to-moderate AD8. We speculate that this may, in part, have occurred due to a lack of understanding of the underlying molecular mechanism(s) of latrepirdine. Given our observations reported herein, any disparity in the contribution of α-syn to the neuropathology in the Russian vs US latrepirdine studies might also explain, at least in part, the inconsistency of the cognitive benefit in the two trials. If this speculation were correct, then one would predict that latrepirdine might be more beneficial in treating synucleinopathies such as PD, LBD, REMSD. and/or MSA by comparison to AD, in which an unpredictable and lower percentage (30–50%) of the clinical population harbors α-syn neuropathology13–15. Since animal models of these diseases are now available, one important direction of this research will be to assess the potential of latrepirdine to improve the neuropathology and/or clinical manifestations of synucleinopathy. These experiments are now underway.
The work in this manuscript was used in a dissertation by JWS as partial requirement for the fulfillment of the PhD degree. JWS is a trainee in the Integrated Pharmacological Sciences Training Program supported by grant T32GM062754 from the National Institute of General Medical Sciences. MLL was supported by the Deutsche Forschungsgemeinschaft. SG is a member of the Oligomer Research Consortium of the Cure Alzheimer’s Fund.
The authors acknowledge the generous support of the NH&MRC (APP1009295 to RM, GV, SG), McCusker Alzheimer’s Research Foundation (RM, GV); Fidelity Biosciences Research Initiative (SJ, JL, DR, GAP); Cure Alzheimer’s Fund (SG); the US Department of Veterans Affairs (SG); and the NIH (P01AG10491 to SG; P50AG05138 to Mary Sano; P30 NS061777 and S10 RR022415 to RW; R01NS060123 and U54RR022220 to ZY). The authors would also like to thank Rosilyn Kazanjian for her gift in memory of Powel Kazanjian. The authors would like to thank Loren E. Khan and Justine Bonet for technical assistance in animal colony management and Dr. Yun Zhong for technical support.
Supplementary information is available at Molecular Psychiatry’s website.
Potentially Competing Financial Interests: A.P. is Vice President of Preclinical Development for Medivation, Inc. S.G. holds research grant support from Amicus Pharmaceuticals and is a consultant to the Pfizer-Janssen Alzheimer’s Immunotherapy Alliance. G.A.P. is on the scientific advisory boards of Amicus Pharmaceuticals and Neurophage, Inc.