APP mRNA

Figure 18.14 Evidence for an iron-responsive element in the 5 -UTR of APP mRNA. APP 5 -UTR sequences were computer-folded to generate the predicted RNA stem. (From Rogers et al., 2002. Reproduced by permission of the Journal of Biological Chemistry.)...

For example, the brains of senescent rats exposed to lead during early development exhibited delayed overexpression of p-amyloid precursor protein (APP) mRNA, altered activities of several signal-dependent and development-specific transcription factors, and elevated concentrations of both APP and the amyloidogenic P-amyloid (AP) product of APP. The effects included a substantial elevation in one of the regulators (Spl) of the APP gene. 

In a mice model of Parkinson s disease, catechins are thought to chelate metal ions such as copper (II) and iron (II) and therefore prevent the generation of potentially damaging free radicals, to suppress the translation of APP mRNA, to reduce holo-APP, and to decrease Ap levels. After the oxidation of catechins by free radicals, a dimerized product is formed with an increased iron-chelating potential and ability to scavenge superoxide anions [56-58]. 

This article focuses on how the marked increase in the steady-state levels of metals (iron, copper and zinc) in the AD brain contributes to gene expression with deleterious consequences for neuronal survival [3]. Certainly APP mRNA translational control by iron (Rogers et al., 2002) and APP gene transcriptional control by copper [1] each provide new genetic support for the model that APP is a metalloprotein with an integral role in metal metabolism. 

IL-l-and iron-responsive sequences were demonstrated to be active RNA regulatory elements in the 5 untranslated region of the Alzheimer s APP transcript. A novel IL-1 responsive acute-box RNA enhancer was immediately in front of the start codons of both APP and ferritin mRNAs [47]. Since this finding our purpose has been to define the function of the newly discovered iron regulatory translational enhancer in APP mRNA in the context of lL-1-dependent APP translation (Figure 3). Like ferritin, APP is a metalloprotein [47,48]. 

Figure 3. An Iron responsive Element (Type II) in the 5 VTR of APP mRNA. Top Panel The APP 5 UTR was predicted to fold into the stable RNA stemloop similar to the 5 UTRspeciflc IRE in H-ferritin transcript ("APP 5 UTR" AG = -54 kCal/mol) (Zuker et al, 1989). Bottom Panel The APP mRNA IRE (Type II) was identified after alignment with the ferritin Iron-responsive Element (shown in two clusters of > 70% sequence similarity)(Adapted from Rogers et al., 2002).

This Iron-Responsive Element (Type II) in the 5 of APP mRNA was fully functional as assessed by multiple separate transfection assays [48]. RNA gel-shift experiments showed that the mutant version of the APP 5 UTR cRNA probe no longer binds to Iron-regulatory Proteins (IRP) (shaded box in Figure 3) [48]. Using RNA electrophoretic mobility shift assays (REMSA) we performed many controls to demonstrate that IRP-1 specifically binds to the stemloop that is predicted to fold from APP 5 untranslated region sequences [48]. Our preliminary data also confirmed that IRP-2 selectively interacted with Ae APP 5 UTR to the same extent as originally observed for IELP-1. 

Addition of serum growth factors overrides the action of another 3 UTR element, a 29-base sequence (2285-2313), that normally destabilizes APP mRNA in endothelial cells (and peripheral blood lymphocytes) [95] (Figure 4). 

Ribosome attachment to APP mRNA and translation o APP holoprotein is regulated by both 5 UTR and 3 UTR sequences. IRP-l/IRP-2 interacts with APP mRNA in vitro. 

Figure 4. Post-Transcriptional regulatory domains mapped to the APP Transcript. The 3 kb APP transcript is controlled at the level of message translation by the action ofS UTR regulatory domains (lL-1 (47), iron (48)). A TGPP transcriptional regulatory element was reported to be in DNA sequences encoded by the APP S UTR (Lahiri et al., 2003). The 3 untranslated region is alternatively polyadenylated, and the longer APP transcript is translated more efficiently than the shorter transcript (97). A29nt RNA destabilizing element was mapped to the 3 UTR of APP mRNA (95).

Daily intramuscular injection of the intracellular Fe chelator, desferrioxamine, was also shown to decrease the cognitive decline in a large cohort of AD patients [41]. Desferrioxamine (DFO) was at first thought to chelate aluminium, which was considered a risk factor for AD [112]. However desferrioxamine is commonly used as an iron chelator in the treatment of Sickle Cell disease [113], and for treatment of patients suffering from poisoning during acute iron overload [114-116]. Thus the mechanism of action of desferrioxamine in the treatment of AD is likely associated ivith iron chelation rather than aluminum chelation. Desferrioxamine appears to specifically suppresses APP mRNA translation (Figure 4) [102]. 

Figure 5, Primary screen far drugs targeted to the APP S untranslated region. In a screen of a library of 1,200 FDA-pre-approved compounds phenserine(ACHEi) and the potent intracellular iron chelator, desferrioxamine, were the validated positive control drugs that suppressed APP mRNA translation through APP 5 UTR sequences. In this transfection based screen several lead APP-5 UTR directed drugs were identified to limit luciferase gene expression driven by the APP 5 UTR. As an internal selectivity control for this screen, downstream dicistronic GFP gene expression (at the translational level by a viral internal ribosome entry site (IRES)) was unresponsive to drug action. Several leads (i,e, dimercaptopropanol) were identified to be chelators as described.

Payton, S., et al., Alzheimer s Disease Drug Discovery Targeted to the APP mRNA 5 Untranslated region paroxetine and dimercaptopropanol are drug hits. Journal of molecular Neuroscience, 2003. 20 p. 267-275. 

---