Novel 5' untranslated region directed blockers of iron-regulatory protein-1 dependent amyloid precursor protein translation: implications for down syndrome and Alzheimer's disease

Abstract

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5' untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5'UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the 'ninth' small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5'UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer's disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5'UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5'UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer's disease.

Figure 1. Alignment of human and mouse APP 5′UTRs with human PrP 5′UTR sequences relative to the L- and H-ferritin Iron-responsive elements (IREs).

Panel A: The human and mouse APP 5′UTR specific IRE-like RNA stem loops, the human PrP 5′UTR, and the human and mouse SNCA specific IRE –like stem loops each aligned adjacent to the ferritin L- and H IRE RNA stem loops. Shown, the L- and H-mRNAs encode canonical IRE RNA stem loops whereas the APP IRE in non canonical although fully iron responsive . The α-synuclein IRE (SNCA IRE) represents a non canonical IRE traversing the central splice junction of exon-1 and exon-2 (the CAGUGN loop/splice site sequences) of SNCA mRNA . Typical IRE stem loops fold to exhibit an apical AGU pseudotriloop which is depicted in red lettering at the apex of the H-ferritin and SNCA IREs relative to an analogous AGA from the IRE–like stem loop encoded by APP mRNA . Panel B: Maps of the 5′UTRs encoding by the human and mouse APP mRNAs, PrP mRNA, SNCA mRNA, and the mRNAs for L- and H-ferritin (IRE stem loops are displayed as lollipops). Panel C: Relative alignment of the sequences that encode the 5′UTR specific IRE-like stem loops in human APP mRNA, PrP mRNA, SNCA mRNA, and the L- and H-ferritin mRNAs. Panel D: Screen and counter-screening Constructs : The human APP 5′UTR cassette was subcloned in front of the luciferase reporter gene in the dicistronic pCD(APP) reporter construct. The same-sized and related PrP 5′UTR was subcloned in an identical format into the pCD(PrP) reporter construct for the purpose of counter-screening, as described in the materials and methods section.

Figure 2. Relative capacity of thirteen APP 5′UTR translation blockers to reduce Aβ levels in the conditioned medium of SH-SY5Y cells.

Following 48 h treatment (1 µM) for each inhibitor, the histogram shows reduction of total Aβ levels confirmed after averaging five independent samplings from the following:- JTR-009 treated

Figure 3. The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein (SNCA).

Panels A and B: Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and SNCA levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). Right Panel: Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. Panel C: Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p<0.01), 0.1 µM (** = p<0.015), and 1 µM (*** = p<0.01) analyzed by ANOVA (N = 5). Dotted line: Representative LDH assay parallel to Aβ determination for SH-SY5Y cells treated for 72 h at concentrations up to 100 µM of JTR-009 (N = 4). Panel D: MTS assay for cellular mitochondrial viability after treatment of SH-SY5Y cells with JTR-005 and JTR-009 at the concentrations shown. Y axis: Percent of maximal viability ± SEM after treatment of the cells with JTR-009 and JTR-005 (N = 3)). Shown are the relative trend-lines for the dose-responsive viability of JTR-005 and JTR-009 compared to untreated cells (‘poly’ = non linear polynomial regression of the data).

Figure 4. Evaluation of the potency and selectivity of APP blocker-9.

Panel A: Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC₅₀ = 0.1 µM; posiphen: IC₅₀ = 5 µM, N = 4). Panel B: Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. Panel C: Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). Panel D: Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein (SNCA) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). Panel E: Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. Panel F: Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).

Figure 5. RNA pulldown assay to measure the dose-dependent capacity of the cyclic benzimidazole JTR-009 to substitute for IRP1 binding to APP 5′UTR sequences in SH-SY5Y cells: correlated repression of APP translation.

RNA pulldown assays were conducted as ilustrated in Figure 6 and as described by Cho et al., 2010 Panel A and B: Representative RNA binding assays in which recovered beads measured the dose-responsive capacity of JTR-009 (0 µM 0.3 µM, 3 µM and 30 µM) to inhibit IRP1 binding to 30 base biotinylated probes encoding the APP 5′UTR. In Panel B Western blots measured relative levels of IRP1 and IRP2 bound to biotinylated RNA probes for APP IRE sequences after recovery in steptavidin bead fractions. Densitomteric quantitation of bead-specific IRP1 is shown in Panel A. Panel C: Measurement of the dose-dependent off-target action of JTR-009 to suppress H-ferritin IRE binding to SH-SY5Y specific IRP1 and IRP2 (bead fraction). Panel D and E: Dose-dependent decrease of APP levels in response to JTR-009 measured in the supernatants of bead fractions (experimental<control set (p<0.00 1). Panel E: Western blots of lysate supernatants showing APP as measured using the N terminal specific 22C11 and C-terminal specific A8717 anitibodies. Panel D: Densitometric quantitation of the data in Panel E to measure the extent to which JTR-009 dose dependently repressed APP expression in SH-SY5Y cells (Dunnetts test, p = 0.03). Data from 5 separate trials, each in triplicate.

Figure 6. RNA binding assay to measure the capacity of JTR-009 to replace IRP1 binding to biotinylated probes encoding core APP IRE sequences compared to IRP1 binding to the H-ferritin IRE sequences (N = 4).

Panel A: Top Panel: Cartoon representation of the protocol employed to detect RNA binding between IRE probes and IRP1 in SH-SY5Y cell lysates. Bottom Panel: Effect of JTR-009 treatment of SH-SY5Y cells (24 h, 10 µM) to alter ferritin-H IRE binding to IRP1 compared to that of the APP IRE. Panel B: The calcein assay for iron levels in SH-SY5Y cells in response to treatment with JTR-009. Cells were treated with either DMSO (negative control), extracellular iron chelator (DPTA), or JTR-009 at 10 µM for 48 hours. Panel C: The anti-amyloid-Aβ-42 efficacy of the APP 5′UTR inhibitors JTR-005 and JTR-009, as measured in conditioned medium from SH-SY5Y neuronal cells (Chemiluminescent BetaMark x-42 ELISA assay from Covance, inc). Data shows the mean values for the reduction of levels of Aβ-42 ± SEM (N = 3) after 72 h treatment of the cells with JTR-009 compared to JTR-005 at 10 µM (p<0.01), analyzed by ANOVA.

Figure 7. Model for the action of the bezimidazole JTR-009 to act as an inhibitor of APP translation by irreversibly replacing the iron dependent translation repressor IRP1 from interacting with APP 5′UTR sequences.

Binding of JTR-009 selectively targeted APP 5′UTR sequences and then was found to repress APP levels leading to reduced amyloid levels without perturbing cellular iron uptake.