c-Abl tyrosine kinase down-regulation as target for memory improvement in Alzheimer’s disease

BackgroundGrowing evidence suggests that the non-receptor tyrosine kinase, c-Abl, plays a significant role in the pathogenesis of Alzheimer’s disease (AD). Here, we analyzed the effect of c-Abl on the cognitive performance decline of APPSwe/PSEN1ΔE9 (APP/PS1) mouse model for AD.MethodsWe used the conditional genetic ablation of c-Abl in the brain (c-Abl-KO) and pharmacological treatment with neurotinib, a novel allosteric c-Abl inhibitor with high brain penetrance, imbued in rodent’s chow.ResultsWe found that APP/PS1/c-Abl-KO mice and APP/PS1 neurotinib-fed mice had improved performance in hippocampus-dependent tasks. In the object location and Barnes-maze tests, they recognized the displaced object and learned the location of the escape hole faster than APP/PS1 mice. Also, APP/PS1 neurotinib-fed mice required fewer trials to reach the learning criterion in the memory flexibility test. Accordingly, c-Abl absence and inhibition caused fewer amyloid plaques, reduced astrogliosis, and preserved neurons in the hippocampus.DiscussionOur results further validate c-Abl as a target for AD, and the neurotinib, a novel c-Abl inhibitor, as a suitable preclinical candidate for AD therapies

Principles of self-assembly of helical pores from dendritic dipeptides

The self-assembly of the dendritic dipeptides (4-3,4-3,5)nG2-CH -Boc-L-Tyr-L-Ala-OMe and their achiral dendritic alcohol (4-3,4-3,5)nG2-CHOH precursors, both with n = 1-16, where n represents the number of methylenic units in the alkyl groups of the dendron, are reported. All chiral dendritic dipeptides and achiral dendritic alcohols self-assemble into helical porous columns that are stable in both solution and solid state. The pore diameter (D) of the columns self-assembled from dendritic dipeptides is ≈ 10 Å larger than that of structures assembled from dendritic alcohols. The increase of the D at the transition from dendritic alcohol to dendritic dipeptide is accompanied by a decreased solid angle of the building block. This trend is in agreement with previous pore size-solid angle dependences observed with different protective groups of the dipeptide and primary structures of the dendron. However, within the series of dendritic alcohols and dendritic dipeptides with various n, the D increases when the solid angle increases. The results of these investigations together with those of previous studies on the role of dipeptide stereochemistry and protective groups on this self-assembly process provide the molecular principles required to program the construction of supramolecular helical pores with diameter controlled at the Å level from a single dendritic dipeptide architecture. These principles are expected to be valid for libraries of dendritic dipeptides based on dendrons and dipeptides with various primary structures

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism

Nuclear localization of RPS14 persists during infection and emetine treatment in low-density cells.

<p><b>A)</b> Cells were seeded at 0.5 million/4-well chamber slide, infected and treated with either emetine (75 nM) or GCV (5μM) for 24 h. Cells were stained with IE1/2 (Alexa 555:Red) for evidence of infection and RPS14 (FITC: Green) and nuclear DAPI. Stained slides were subjected to confocal microscopy and nuclear localization was quantified using NIS elements software (Nikon). <b>B)</b> The same procedure was repeated at 72 hpi. The images shown are a representative of two independent experiments. Percent nuclear localization is represented as mean ± SD of at least 25 cells per condition from two different fields in the slide.</p

The anti-HCMV activity of emetine is reduced in low-density HFFs.

<p><b>A)</b> Cells were seeded at indicated densities in a 96-well plate, infected with pp28-luciferase HCMV Towne (MOI = 1) and treated with emetine (75 nM) or GCV (5 μM). Luciferase activity was measured at 72 hpi and normalized to the activity of infected untreated cells. Data represent mean ± SE of triplicate determinations from a representative of two independent experiments. <b>B)</b> Same cell lysates as in A were collected for Western blotting at 72 hpi and expression of viral proteins UL44 and pp65 was determined. <b>C)</b> Supernatants from each well as in A were used to infect cells (1 million cells in a 96-well plate) in the corresponding well and luciferase activity was measured at 72 hpi. <b>D)</b> Cells were seeded into 12-well plates at 0.5 and 2 million cells/plate, infected with 100 PFU/well of HCMV Towne or MCMV, and treated with emetine (75 nM) or GCV (5 μM). After 10 days (for HCMV) or 3 days (for MCMV) plaques were stained with crystal violet and the number of plaques enumerated. Data shown are average of 2 wells (±SD) for a representative experiment from two different experiments.</p

Emetine achieves high tissue concentrations and is efficacious against MCMV replication.

<p><b>A)</b><i>In vivo</i> pharmacokinetics of emetine. Plasma, liver, lung and spleen samples were collected at the indicated time points after single oral administration of emetine at 0.1 mg/kg in male BALB/c mice. Concentrations were measured using UPLC-MS/MS methods. For plasma, the concentrations observed were mostly below the level limit of quantification. <b>B)</b> Quantitative real-time PCR of viral gB was performed on DNA extracted from blood at day 14 post infection. Plaque assay was performed from salivary glands <b>C)</b> or liver <b>D)</b> collected at day 14 post infection. Emetine was administered orally starting 24 hpi or 72 hpi at 0.1 or 1.0 mg/kg every 3 days. GCV dose was 10 mg/kg/dose administered intraperitoneally twice daily.</p