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Latrepirdine (Dimebon™) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer’s disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine-stimulated APP catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer’s disease (AD). We utilized several mammalian cellular models to determine whether latrepirdine regulates mTOR- and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered latrepirdine prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with latrepirdine led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that latrepirdine possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying latrepirdine effects on neurogenesis, autophagy, and behavior might warranty the further study of latrepirdine as a potentially viable lead compound that might yield more consistent clinical benefit following optimization of its pro-neurogenic, pro-autophagic, and/or pro-cognitive activities.
Latrepirdine (Dimebon™; dimebolin) is a neuroactive compound with antagonist activity at histaminergic, α-adrenergic, and serotonergic receptors1. Based on its effects on cognition in rodents2-6, taken in conjunction with its highly favorable safety profile, the compound has formed the basis for clinical trials for both Alzheimer’s disease (AD)7 and Huntington’s disease (HD)8, despite a poor understanding of the molecular mechanisms underlying its putative mnemoactive properties.
Latrepirdine has been reported to possess several properties that are potentially relevant to the treatment of neurodegenerative diseases:  protection of cultured cells from the cytotoxicity of amyloid-β (Aβ) peptide9;  stabilization of mitochondrial function and calcium homeostasis1;  modulation of Aβ release from cultured cells, isolated intact nerve terminals, and from hippocampal neurons in living mouse brain10; and  promotion of neurogenesis in the murine hippocampus11. We reported that latrepirdine stimulates APP catabolism and Aβ secretion10, an unexpected result for a drug that was claimed to benefit AD. In pursuit of a parsimonious subcellular mechanism underlying this unexpected result, we considered the possibility that latrepirdine-stimulated Aβ release might occur via an unconventional secretory pathway associated with induction of macroautophagy (autophagy)12, a highly regulated process that can be activated in response to various stressful conditions13.
Several laboratories have demonstrated that autophagy plays a neuroprotective role in cell and animal models of neurodegenerative diseases, including AD, HD14 and Parkinson’s disease (PD)15. Converging data indicate that therapeutic manipulation of autophagy with rapamycin can improve behavioral function and arrest neuropathology in at least two mouse models of AD16-18. To this end, small molecule enhancers of rapamycin (SMERs; most notably SMER-28) induced autophagy, improved cell viability, and promoted clearance of neurodegenerative disease-related proteins including APP metabolites [among them Aβ19, huntingtin (htt)20, and α-synuclein20] in cellular models.
Herein, we report that:  latrepirdine modulates Atg5-dependent autophagic activity in a dose-dependent manner and via the mTOR-signaling pathway;  latrepirdine potentiates the degradation of APP metabolites in cell culture and in mouse brain; and  latrepirdine improves the memory behavior of TgCRND8 mice, while reducing the accumulation of insoluble Aβ42. Given the pressing need for effective disease-modifying treatment for symptomatic AD and the current evidence that Aβ-lowering agents might only be effective for prophylaxis21, we argue that identification of the molecular basis of the pro-cognitive and anti-neurodegeneration actions of latrepirdine remains highly valuable.
The synthesis and characterization of latrepirdine was described previously10. 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 in10. 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).
N2a cells stably transfected with APP K670N, M671L (N2a SweAPP; generous gift of Dr. Gopal Thinakaran, University of Chicago) or untransfected N2a cells, stable human cervical carcinoma (HeLa) cells expressing EGFP-LC3 (kind gift from Aviva M. Tolkovsky), and mouse embryonic fibroblasts (MEFs) derived from wildtype mice or ATG5 −/− mice (kindly provided by Noboru Mizushima) were cultured and treated with or without drug and samples were prepared for analysis as in Supplemental Methods.
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 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 specific animal ages, group assignment, and tissue preparation.
Mice were trained and tested in operant chambers on three consecutive days in the cued and contextual fear conditioning paradigm, similar to that described in _ENREF_3535. See Supplemental Methods for detailed protocol. Freezing behavior was recorded remotely and analyzed using Stoelting ANY-MAZE Fear Conditioning Software (Stoelting Co, Wood Dale, Il.).
Integrated density of immunoreactive Western blot bands or spots were measured using MultiGauge Software and normalized to % control (vehicle or nTg littermate, where indicated). Absolute concentrations of monomeric Aβ40 and Aβ42 or oAβ were normalized to initial tissue weight. In all instances, Shapiro-Wilk test of normality 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. Oneway ANOVA (parametric analysis) or Kruskal-Wallis test (non-parametric analysis) were used to compare 3 or more groups, depending on whether data fit the assumptions of parametric analysis (see above). Two-way ANOVAs were used to analyze CHX time-course experiments with regard to effects of time and treatment, and interactions. Bonferroni’s or Dunn’s correction for multiple comparisons were utilized depending on whether data fit the assumptions of parametric or non-parametric design, respectively. 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 sought to determine whether latrepirdine might regulate autophagy as one mechanism of its reported anti-neurodegeration activity. We treated HeLa cells stably expressing LC3 fused with EGFP (eGFP-LC3)22 for 3 or 6 hours in the absence or presence of 50μM latrepirdine. Treatment with latrepirdine for 3 (data not shown) or 6 hours (Figure 1A) markedly enhanced the number of eGFP-LC3 punctae, indicating that latrepirdine induced formation of autophagosomes.
Next, mouse N2a neuroblastoma cells were treated in the absence (vehicle) or presence of 5nM, 500nM, or 50μM latrepirdine for 3 or 6 hours in order to determine the effects of acute drug treatment on the regulation of autophagy. We observed a significant and dose-dependent increase in LC3-II levels in N2a cells following 3- or 6-hour treatment with either 500nM or 50μM latrepirdine (Figure 1). Because the inhibition of autophagic clearance also results in accumulation of LC3-II, we chose to assay levels of p62, which is readily degraded by autophagy and which accumulates when autophagic clearance is inhibited (see Supplemental Figure 1). The p62 levels did not increase in the presence of 500nM or 50μM latrepirdine; instead, 50μM latrepirdine caused a trend toward reduction of p62 levels at both 3- and 6-hour treatment (Figure 1A,C), suggesting that LC3-II accumulation was associated with increased autophagosome formation and activity.
We next examined the mTOR signaling pathway, which is a key regulator of autophagic activity23. mTOR kinase activity can be monitored by the levels of phosphorylated-mTOR (p-mTOR; serine 2448) and the levels of the phosphorylated-form of its substrate, ribosomal S6 protein kinase (p70S6K; p-S6K)24. We observed a significant decrease of p-mTOR and p-S6K from N2a cells treated with 50μM latrepirdine for 3 hours, whereas the total mTOR and p70S6K levels remained relatively constant (Figure 2). Based on this result, we conclude that latrepirdine likely induced autophagy via inhibition of the mTOR signaling pathway. The specific molecular target of latrepiridine within this pathway remains unknown.
We treated ATG5 +/+ (WT) or ATG5 −/− MEFs for 3 hours in the absence or presence of 50μM latrepirdine (Figure 2). Three-hour latrepirdine treatment of WT MEFs was associated with significant decreases in p62 and p-S6K in WT MEFs (Figure 2A). No accumulation of LC3-II was noted in this cell line following 3 hours of latrepirdine treatment, which may be related to differences cell line-specific regulation of LC3-II turnover. By comparison to untreated WT MEFs, untreated ATG5 −/− MEFs displayed significant ~2-fold accumulation of both p62 and APP-CTFs, suggesting that inhibition of autophagy by deletion of ATG5 alone is sufficient to cause accumulation of APP-CTFs (see also Supplemental Figure 1, Supplemental Table 1). In contrast, incubation of WT MEFs with 50μM latrepirdine for 3 hours resulted in a trend toward decreased APP-CTFs, suggesting that the accumulation of APP-CTFs and a resultant pool of Aβ may be a result of autophagic activity that is partially relieved following addition of latrepirdine. In ATG5 −/− MEFs, incubation with 50μM latrepirdine for 3 hours did not result in any change in APP-CTF or p62 accumulation, confirming that the latrepirdine-related stimulation of clearance of APP metabolites and p62 was Atg5-dependent.
We tested whether latrepirdine altered the accumulation of intracellular APP metabolites using stable SweAPP (APPK670N,M671L)-overexpressing N2a cells (the generous gift of Dr. Gopal Thinakaran). N2a SweAPP cells were treated for 3 hours in the absence or presence of 50μM latrepirdine. Cell lysates and conditioned media were separately analyzed for APP metabolites by Western blot. We observed a significant accumulation of LC3-II and without accumulation of p62, indicating enhanced autophagy. Moreover, we noted parallel changes in levels of both intra- and extra-cellular APP metabolites (Figure 2F-L), in which the induction of autophagy was associated with decreased intracellular Aβ (IC-Aβ) accumulation and a trend toward increased secreted Aβ(sAβ). These changes are consistent with our previous report that latrepirdine stimulates secretion of APP metabolites10, suggesting that latrepirdine may reduce intracellular APP/Aβ accumulation through stimulating both secretion of APP metabolites and intracellular catabolism of APP/Aβ via autophagy.
Unexpectedly, following a 24-hour treatment of MEFs or N2a SweAPP cells with 50μM latrepirdine, we observed significant hyperaccumulation of LC3-II, p62, and APP metabolites, a profile consistent with an inhibition of autophagy, potentially via inhibition of end-stage lysosomal protease activity. This effect was observed in the both the presence (N2a SweAPP cells or WT MEFs) or absence (ATG5 −/− MEFs) of an intact autophagic pathway and was therefore Atg5-independent. While latrepirdine inhibited mTOR signaling following brief drug treatments, no regulation of the mTOR pathway could be detected following 24-hour exposure of cells to the drug (Figure 3A,E). This effect appeared to be distinct from the regulation of autophagic activity observed with short-term exposure to latepirdine.
A related report demonstrated that some histamine-related compounds were associated with an off-target and receptor-independent vacuolar-ATPase-dependent inhibition of lysosomal protonation when employed at high concentrations and with long incubation times (similar to the conditions employed here)25. We propose that this effect may underlie our observation that 24-hour incubation with 50μM latrepirdine potentiates the accumulation of typical p62 and APP-CTFs, even in the absence of an intact autophagic pathway (Figure 3E-K).
An aging-dependent decrease in basal neuronal turnover of brain autophagic substrates has been identified as a feature of the molecular neuropathology associated with the progression of AD. Here, we hypothesized that autophagic/lysosomal failure leads to the hyperaccumulation of typical autophagic substrates, including APP metabolites, α-syn, LC3, and p62. Along this line, it is relevant that cortical accumulation of α-syn, a known autophagic substrate, is observed in 30-50% of AD patients26. Here, we utilized the TgCRND8 (APP K670N/M671L/V717F) mouse model of Alzheimer’s cerebral amyloidosis27. Male TgCRND8 mice or their non-transgenic littermates (nTg) were sacrificed at 3 or 4 months of age, and cerebral cortices were dissected for analysis of levels of APP metabolites, α-syn, p62, and LC3 in both soluble and insoluble fractions (for schematic, see Supplemental Figure 2). By 3 months of age, TgCRND8 mice are impaired in acquisition and learning reversal in the reference memory version of the Morris water maze task27.
At 3 months of age, TgCRND8 mice exhibited deposition of Aβ42-immunoreacive cortical and hippocampal plaques, as well as accumulation of insoluble p62, which was recovered along with Aβ42 in the 70% formic acid fraction of extracts of TgCRND8 brain; however, neither LC3 nor α-syn were ever detectable in the formic acid fractions. When 3-month-old mice were studied, no significant difference was observed between genotypes in the levels of soluble α-syn, LC3-I, LC3-II, or p62 (Figure 4C), suggesting that autophagic clearance of these substrates is functionally intact at that age in TgCRND8 mice. However, by 4 months of age, we observed a significant increase in accumulation of soluble α-syn, total LC3 (LC3-I and LC3-II), and p62, as well as insoluble p62 (Figure 4C), suggesting the development of an aging-dependent impairment of autophagic clearance among TgCRND8 mice. Taken together, these results indicate that accumulation of insoluble Aβ42 and p62 temporally precedes the impairment of autophagic clearance, and may lead to autophagic/lysosomal failure. In future studies, we plan to investigate the temporal relationship between insoluble Aβ42 and p62 accumulation and alterations in autophagic/lysosomal stasis. Regardless of cause and effect, these data indicate that autophagic/lysosomal failure may lead to the hyperaccumulation of autophagic substrates, accelerating disease progression.
Based on the total daily intake of latrpeirdine described by Doody and colleagues7, an equivalent single i.p. bolus in an adult mouse was determined to be 3.5mg/kg/day, and we previously reported that acute administration of this dose increased secretion of Aβ40 into the interstitial fluid of the hippocampus of living Tg2576 mice10. The dosing regimen for the current study was chosen based on two primary considerations: (1) in vitro studies (herein) indicating that sustained high levels of latrepirdine may inhibit autophagic clearance and (2) the observation that 21 consecutive (once daily) injections of 3.5 mg/kg latrepirdine were sufficient to enhance (not inhibit) autophagy in young TgCRND8 mice (Supplemental Figures 3 and 4). In related studies in S. cerevisiae, we observed that latrepirdine enhanced autophagy and promoted the clearance of Aβ4228 or α-syn29. Chronic latrepirdine administration also promoted autophagy and the degradation of α-syn in the brains of wildtype mice29. Based on these results, we postulated that a once-daily i.p. bolus of 3.5 mg/kg latrepirdine for 31 days might enhance autophagy and stimulate the clearance of autophagic substrates, while avoiding the potential confound of prolonged exposure to high concentrations of latrepirdine (Figure 3).
Male, 90-day-old TgCRND8 mice or their wild-type littermates (nTg) received 31 consecutive once daily i.p. injections of either 3.5 mg/kg latrepirdine or 0.9% saline (vehicle). At the culmination of treatment, mice were tested for cued and contextual fear conditioning using a paradigm that has been widely accepted for evaluating learning and memory deficits in APP transgenic mice30. No difference was noted for cued or contextual memory between vehicle- or latrepirdine-treated nTg mice. We observed a significant increase in cued memory only among latrepirdine-versus vehicle-treated TgCRND8 mice (p=0.01; Figure 5). A weak, non-significant trend toward an improvement in contextual memory among latrepirdine-versus vehicle-treated mice (p=0.099) was also observed. Neither performance in the cued nor contextual memory tasks by latrepirdine-treated TgCRND8 mice was sufficient to correct the animals to the level of performance observed for their nTg littermates. However, because TgCRND8 mice are known to be impaired in related tasks at 3 months of age27, we suggest that the effect of improved cued memory may be related to arrest of disease progression, rather than improved cognition per se.
Brain extracts were analyzed for soluble and insoluble APP metabolites, LC3, p62, and α-syn by Western blot. No differences were observed between treatment groups for levels of soluble APP metabolites (including Aβ, Figure 5). We further assayed the brain extracts for concentrations of soluble oligomeric oAβ (oAβ) subtypes that were dimeric or larger in structure. We observed a trend toward decreased mean soluble oAβ levels among latrepirdine- (1225 pg/g) versus vehicle-treated (1318 pg/g) TgCRND8 mice that did not reach statistical significance (p=0.0851). We also noted a significant decrease in soluble α-syn monomer (p=0.0042) in the brains of TgCRND8 mice, which was associated with significantly elevated LC3-II accumulation (p=0.0095) and without accumulation of soluble p62 (Figure 5), confirming our results from pre-depositing 3 month-old TgCRND8 mice (Supplemental Figures 3 and 4).
Formic acid fractions (Triton X-100-“insoluble” material) were analyzed by Aβ40- or Aβ42-specific sandwich ELISA, and a significant decrease in the levels of insoluble Aβ42 were observed when latrepirdine-treated mice were compared with vehicle-treated mice (p=0.046; Figure 5). Western blot analysis of insoluble p62 levels revealed a modest, but statistically significant, decrease among latrepirdine-versus vehicle-treated mice (p=0.0334). These results suggest that the cognitive benefits of latrepirdine were associated with decreases in insoluble Aβ42 and p62, and may be due to increased autophagic substrate clearance.
Protein aggregates are resistant to degradation and thus inherently longer-lived31; therefore, maintenance of efficient intracellular and extracellular proteolysis is required in order to prevent accumulation of toxic aggregates. Following logically from this consideration is the notion that maintenance or restoration of optimal levels of autophagic flux may be of therapeutic benefit to several neurodegenerative diseases32. Indeed, recent reports indicate that activation of autophagy reduces intracellular accumulation of protein aggregates and improves cognition in three different mouse models of AD16-18, 33, 34. At the cellular level, the work presented here, and through collaborative works28,29, indicates that activation of mTOR-dependent autophagy by rapamycin neutralizes the proteotoxicity induced in S. cerevisiae by α-syn29 or Aβ4228. When taken together with a wealth of published evidence, the principle emerges that pharmacological regulation of proteostasis in general, and of autophagy in particular, represents attractive potential interventions for treatment of some neurodegenerative diseases.
Given the evidence that latrepirdine has biological activity in TgCRND8 mice that includes activation of autophagy (reported herein), and that mTOR modulation of autophagy is much more effective as prophylaxis than as therapy for established AD in mouse models17, then (as appears to be the case with other therapies directed at Aβ21) latrepirdine might be most useful in presymptomatic AD or in cases of mild cognitive impairment. However, if the current data are relevant to the clinical benefit observed in the Russian trial of latrepirdine in mild-to-moderate AD, that would imply that symptomatic AD subjects might improve or have their disease progression arrested.
It is worth noting that we have not investigated the recently reported pro-neurogenic actions of latrepirdine11, so we cannot comment on whether the autophagy modulation that we have observed plays a role in the drug’s reported pro-neurogenesis properties, although examination of this possible connection would be worth undertaking. However, since we are contemplating the treatment of neurodegenerative disorders, the putative pro-neurogenic action only makes the drug a more intriguing target for further investigation, as does its clean safety profile and blood-brain-barrier (BBB)-penetrability. The evidence reported herein suggest that, despite mixed results in clinical trials, latrepirdine possesses potentially relevant biological activities and, as such, the drug may yet be useful as a lead scaffold for developing clinically safe, BBB-penetrating, pro-autophagic, pro-neurogenic drugs for treatment and/or prevention of cerebral proteinopathies, including α-synucleinopathies and Alzheimer’s cerebral amyloidosis.
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 and CGG are members 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), the McCusker Alzheimer’s Research Foundation (RM, GV); Conicyt (PFB-16 to SB); the Fidelity Biosciences Research Initiative (SJ, JL, DR, GAP); Cure Alzheimer’s Fund (SG; CGG); the US Department of Veterans Affairs (SG); the NIH (P01AG10491 to SG; U01AG01483 to PS; NS045283 to CGG, R01NS060123; U54RR022220 to ZY; P30 NS061777 and S10 RR022415 to RW; and P50AG05136 to Mary Sano), the Canadian Institutes of Health Research and Alzheimer Society of Ontario (PF), and Baxter Healthcare (PS and NR). The authors would also like to thank Rosilyn Kazanjian for the gift in memory of Powel Kazanjian that supported the purchase of the Luminex xMAP100/200 system. 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.
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. P.S. and N.R. hold research grant support from Baxter Healthcare, Inc (both P.S. and N.R.) and Pfizer (N.R.). N.R. is a consultant for Bristol Meyer Squibb and Eisai Research. G.A.P. is on the scientific advisory boards of Amicus Pharmaceuticals and Neurophage, Inc.
Supplementary information is available at Molecular Psychiatry’s website.