Acute dosing of latrepirdine (Dimebon), a possible Alzheimer therapeutic, elevates extracellular amyloid-beta levels in vitro and in vivo

Abstract

Background: Recent reports suggest that latrepirdine (Dimebon, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-beta (Abeta) peptide in the brain, and Abeta-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Abeta using in vitro and in vivo experimental systems.

Results: We evaluated extracellular levels of Abeta in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 muM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 muM or 10 muM). Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Abeta in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 muM) and led to more modest increases in extracellular Abeta(x-42 )levels (+10%; p = 0.001); of note, however, was the observation that extracellular Abeta(x-40 )levels did not change.

Conclusions: Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Abeta as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Abeta levels must now be determined.

Figure 1

Comparison of latrepirdine 1D NMR proton spectra in DMSO from two sources. Latrepirdine was custom synthesized by either Nanosyn Inc or SinoChemexper and purity was determined to be ~97.3% or >99.0%, respectively. 1D NMR proton spectra are compared here for latrepirdine synthesized by SinoChemexper (top) or Nanosyn (bottom, reprinted with permission from Wu et al, 2008 [16]). Aliquots of latrepirdine from both sources were tested in each protocol. Identical effects were observed regardless of source of compound, and data were pooled for overall analysis.

Figure 2

Effect of latrepirdine on the levels of APP metabolites from cell lysates and conditioned media of mouse N2a cells. Cells were treated for 6 hours in the presence of vehicle or increasing concentrations of latrepirdine (as labeled). (a) Representative western blot of total intracellular and secreted metabolites of APP from at least 3 independent experiments, each performed in duplicate. (b) Quantification of western blot band densitometry represented as mean percent of vehicle +/- S.E.M. 5 nM latrepirdine produced an approximately half-maximal ~37% increase (SD = 0.33, t(10) = 2.75, p = 0.02), and 500 nM produced a maximal significant ~64% increase (SD = 0.49, t(10) = 3.16, p = 0.01) in extracellular Aβ compared to vehicle. The highest concentration (5 μM) also caused a significant ~59% increase (SD = 0.52, t(10) = 2.78, p = 0.02) in extracellular Aβ levels, and an increase in extracellular Aβ levels was observed with 50 nM latrepirdine, which approached significance on a two-tailed test (SD = 0.54, t(4) = 2.62, p = 0.059). Incubation with 5 nM or 500 nM latrepirdine resulted in a significant ~34% (SD = 0.35, t(10) = 2.33, p = 0.04) and ~27% (SD = 0.14, t(10) = 4.87, p = 0.0006) increase in sAPPα accumulation, respectively, in conditioned media compared to vehicle. An increase in extracellular sAPPα levels was observed with 50 nM latrepirdine, which approached significance on a two-tailed test (SD = 0.53, t(4) = 2.31, p = 0.081). No significant increases in sAPPα levels were distinguished between vehicle and either 500 pM or 5 μM latrepirdine. No significant accumulation of holoAPP of APP-CTFs (C83-CTF or C99-CTF) was observed following 6 h incubation in the absence (vehicle) or presence of varying concentrations of latrepirdine (as indicated). (c) Vehicle and all latrepirdine concentrations were indistinguishable by mean Aβ_x-42/Aβ_x-40 ratio, quantified by Aβ species-specific sandwich ELISA (mean Aβ_x-42/Aβ_x-40, n = 3 independent experiments, each performed in duplicate). Absolute Aβ_x-40 and Aβ_x-42 levels in the media were ~380-490 pM and ~14-20 pM, respectively. *Value represents a significant mean difference from vehicle by independent samples t-test, two-tailed α = 0.05, #p < 0.10; *p < 0.05; **p < 0.01.

Figure 3

Secreted Aβ_x-40 and Aβ_x-42 levels in the releasates of synaptoneurosomes following incubation with latrepirdine. Post-natal day 10-14 TgCRND8 cortical synaptoneurosomes were incubated 0 (baseline), 1, 3, 5, or 10 minutes in the presence of 1 μM (n = 5) or 10 μM (n = 6) latrepirdine. (a) 1 μM latrepirdine stimulates an increase in secretion of Aβ_x-42, but not Aβ_x-40, from isolated synaptoneurosomes following 3 minutes (SD = 0.06, t(4) = 2.81, p = 0.048) of incubation with the drug, and a decrease (~6%) is observed at 10 minutes (SD = 0.01, t(5) = 9.61, p = 0.0007), likely representing a depletion of available Aβ_x-42 (mean % baseline +/- S.E.M). (b) 10 μM latrepirdine stimulates an increase in secretion of Aβ_x-42, but not Aβ_x-40, following 1 (SD = 0.038, t(5) = 6.73, p = 0.001), 3 (SD = 0.028, t(5) = 7.35, p = 0.0007), and 5 (SD = 0.056, t(5) = 5.29, p = 0.0029) minutes of incubation with the drug (mean % baseline +/- S.E.M). (c) An immediate ~4.1% increase (SD = 0.13, t(5) = 3.205, p = 0.024) in Aβ_x-42/Aβ_x-40 ratio was observed following 1 minute of incubation with 10 μM latrepirdine (mean Aβ_x-42/Aβ_x-40). (Note: TgCRND8 mice generate ~50%/50% Aβ_x-42/Aβ_x-40 under normal conditions and the increase in Aβ_x-42/Aβ_x-40 ratio may be due to the large stimulation of Aβ secretion at this time point). *Value represents a significant mean difference between baseline and time-point by paired t-test, two-tailed α = 0.05, *p < 0.05, **p < 0.01.

Figure 4

Acute administration of latrepirdine increases ISF Aβ_x-40 levels 9-10 hours following treatment. A single i.p. treatment of latrepirdine (SD = 30.94, t(4) = 2.997, p = 0.04), but not vehicle (SD = 5.68, t(4) = 0.775, p = 0.48), produces an increase in ISF Aβ_x-40 levels from baseline at 9-10 hours post-treatment (a). This increase in ISF Aβ_x-40 level was also determined to be significantly higher than ISF Aβ_x-40 levels observed in vehicle treated animals at 9-10 hours post-treatment (SD = 30.94, t(5) = 3.31, p = 0.01; b). Data are represented as mean percent of baseline +/- S.E.M. *Value represents a significant difference as determined by paired samples t-test (a) or independent samples t-test (b), two-tailed α = 0.05, *p < 0.05.