Amyloid Precursor Protein mRNA Research Articles

Abstract TP210: Small Extracellular Vesicle-derived Micrornas Differentiate Ischemic Stroke And Intracerebral Hemorrhage: A Pilot Study

Investigating the short and long-term signaling mechanisms via small extracellular vesicles (sEVs) in peripheral blood following human Ischemic Stroke (IS) and Intracerebral Hemorrhage (ICH) is of great interest. The sEV’s cargo can induce distant responses which can be used to derive potential novel therapeutic targets and biomarkers to differentiate the two brain pathologies. Thus, we sequenced the miRNA cargo derived from peripheral blood sEVs in IS (n=3), ICH (n=3), and Vascular Risk Factor-matched Control (VRFC; n=3) subjects. Subjects were divided into contrast groups (IS vs VRFC, ICH vs VRFC, ICH vs IS), and log2 transformed expression underwent Kruskal-Wallis tests to identify differentially expressed (DE; p<0.05) miRNAs. We found 55 DE miRNAs in IS vs VRFC, 38 in ICH vs VRFC, and 45 in ICH vs IS ( Fig. 1A ). The combination of these miRNAs differentiated the three groups on Principal Components Analysis ( Fig. 1B ). IS associated miRNA included miR-30a, miR-30b, and miR-144. miR-30a is involved in hematopoietic stem cell self-renewal and can impair B cell differentiation. miR-30b may be an immune suppressor via Notch1. In male mice, miR-144 is protective against atherosclerosis. ICH associated miRNA included miR-195, miR-1-3p, and miR-20b-5p. miR-195 can inhibit the pro-inflammatory roles of macrophages. miR-1-3p is involved in cardiomyocyte development, can target TLR1 (Toll-Like Receptor 1), and may regulate autophagy. miR-20b-5p reduces Amyloid Precursor Protein (APP) mRNA and protein levels; vascular accumulation of APP is one cause of Lobar ICH. We show differential expression of sEV-derived miRNAs in peripheral blood of human IS and ICH patients that are involved in relevant signaling processes. sEV cargo profiles pose a largely underexplored intercellular signaling mechanism in IS and ICH with the potential to better characterize long distance signaling from injured brain to peripheral blood leukocytes in these brain disorders.

Abstract 1397: Targeting the Iron Response Element (IRE)-mRNA of Alzheimer's Amyloid Precursor Protein for Binding to Iron Response Protein (IRP1)

Abstract 1397: Targeting the Iron Response Element (IRE)-mRNA of Alzheimer's Amyloid Precursor Protein for Binding to Iron Response Protein (IRP1)

MRNA‐Binding Protein DJ‐1 as a pivotal protein in AD pathology

AbstractBackgroundAlzheimer’s Disease (AD) is a complex neurodegenerative disorder of the synapse, characterized by the accumulation of amyloid beta plaques and neurofibrillary tangles. We have recently shown that Parkinson Protein 7, DJ‐1, is upregulated by mTORC1 activity, and is a translational hub that is predicted to coordinate the expression of a population of synaptic proteins (Niere et al., 2016). This cellular function of DJ‐1 is mediated through its RNA‐binding properties, yet many of its target mRNAs are unidentified, and the effects of this protein‐mRNA association are not well‐defined.MethodSynaptically increased DJ‐1 expression was determined by Western blotting of synaptoneurosomes from AD patients (Niere et al., 2020), and APP/PS1 and P301S mouse models of AD. DJ‐1 target mRNAs were first bioinformatically identified using a modified consensus sequence of DJ‐1 protein (van der Brug et al., 2008), and confirmed via RNA‐immunoprecipitation of DJ‐1 from WT mouse cortices. FMR1KO mouse cortices were used as a negative control.ResultSynaptic DJ‐1 expression is increased in a preclinical mouse model of AD exhibiting plaque pathology, and AD patients. Interestingly, there is no change in synaptic DJ‐1 levels in the P301S mouse model, which presents neurofibrillary tangles. We bioinformatically identified that mRNA coding for the RNA‐binding protein FMRP, and tau‐tubulin kinase 1 (TTBK1), known for phosphorylating pathogenic tau, as potential targets for DJ‐1, and confirmed it via RNA‐immunoprecipitation. Furthermore, the protein levels of FMRP is significantly decreased in the synapses of AD patients and APP/PS1 mice, while there is no change in the P301S model consistent with no change in DJ‐1 expression.ConclusionDecrease in FMRP in AD synapses where DJ‐1 expression is high suggests that DJ‐1 is a translational repressor of FMRP. FMRP binds to the mRNA of amyloid precursor protein (APP), the building block of amyloid beta plaques. Therefore, DJ‐1’s capacity to bind both Fmr1, coding for a protein which is known as a translational suppressor of APP mRNA (Borreca et al., 2016), and Ttbk1, a tau kinase involved in tau phosphorylation and aggregation (Sato et al., 2006) suggests that DJ‐1 might be at the crossroads of both AD hallmark pathologies.

Inhibitory Effect of Hexahydrocurcumin on Memory Impairment and Amyloidogenesis in Dexamethasone-Treated Mice.

A high dose of dexamethasone induces neurodegeneration by initiating the inflammatory processes that lead to neural apoptosis. A dexamethasone administration model induces overproduction of amyloid-β (Aβ) and tau protein hyperphosphorylation and shows abnormalities of cholinergic function similar to Alzheimer's disease (AD). This study aimed to investigate the protective effect of hexahydrocurcumin on the brain of dexamethasone-induced mice. The results showed that hexahydrocurcumin and donepezil attenuated the levels of amyloid precursor protein and β-secretase mRNA by reverse transcription polymerase chain reaction, decreased the expression of hyperphosphorylated tau, and improved synaptic function. Moreover, we found that hexahydrocurcumin treatment could decrease interleukin-6 levels by attenuating p65 of nuclear factor kappa-light-chain-enhancer (NF-κB) of activated beta cells. In addition, hexahydrocurcumin also decreased oxidative stress, as demonstrated by the expression of 4-hydroxynonenal and thereby prevented apoptosis. Therefore, our finding suggests that hexahydrocurcumin prevents dexamethasone-induced AD-like pathology and improves memory impairment.

High intake of chicken and pork proteins aggravates high-fat-diet-induced inflammation and disorder of hippocampal glutamatergic system

High-fat diets have been associated with neurodegenerative diseases, which are also largely related to the type and amount of dietary proteins. However, to our knowledge, it is little known how dietary proteins affect neurodegenerative changes. In this study, we investigated the effects of dietary proteins in a high-fat diet on hippocampus functions related to enteric glial cells (EGCs) in Wistar rats that were fed either 40% or 20% (calorie) casein, chicken protein or pork protein for 12 weeks (n=10 each group). Inflammatory factors, glutamatergic system, EGCs, astrocytes and nutrient transporters were measured. A high-chicken-protein diet significantly increased the levels of systemic inflammatory factors, Tau protein and amyloid precursor protein mRNA level in the rat hippocampus. The type and level of dietary proteins in high-fat diets did not affect the gene expression of glial fibrillary acidic protein and α-synuclein (P>.05), indicating a negligible effect on astrocyte activity. However, the high-protein diets up-regulated glutamate transporters compared with the low-protein diets (P<.05), while they reduced the γ-aminobutyric acid content in high-chicken and -pork-protein diets (P<.05). Thus, compared with a low-protein diet (20%), a high-chicken or -pork-protein diet (40%) under a high-fat background could alter the balance between glutamatergic system and neurotransmitter and have a stronger effect on the interactions between hippocampal glutamatergic system and EGCs.

Ginkgo biloba Alleviates Cisplatin-Mediated Neurotoxicity in Rats via Modulating APP/Aβ/P2X7R/P2Y12R and XIAP/BDNF-Dependent Caspase-3 Apoptotic Pathway

Neurotoxicity is an obvious adverse effect in Patients encountering a complete course of chemotherapy. The present work is conducted to evaluate the neuroprotective effect of Ginkgo biloba (Ginkgo) against the neurotoxicity induced by Cisplatin (Cis) in rats. Forty male Wistar albino rats were arranged into four groups: (1) Control group, rats were given saline; (2) Cis group, rats were injected by Cis 2 mg/kg body weight i.p., twice a week starting on the fifth day for thirty days; (3) Ginkgo group, rats were administered Ginkgo (50 mg/kg orally) daily for thirty days; and (4) Ginkgo+Cis group, rats received Ginkgo (50 mg/kg orally) daily and on the fifth day, rats were injected with Cis (2 mg/Kg body weight i.p.) twice a week for thirty days. Cis significantly increased Gamma glutamyltransferase (GGT) and Acetyl Cholinesterase (CHE) as compared to the control group and also disturbed cerebral oxidative/antioxidant redox. Co-administration of Ginkgo and Cis reversed the adverse effect of Cis on the brain tissue. Moreover, co-administration of Ginkgo and Cis ameliorated Cis induced brain damage by reducing Amyloid precursor protein (APP), amyloid β (Aβ), P2Y12R and P2X7R mRNA expressions and proteins. Furthermore, Ginkgo regulated XIAP/BDNF expressions with a consequent decrease of caspase-3 and DNA fragmentation%. The current results concluded that concurrent treatment with Ginkgo can mitigate neurotoxicity mediated by Cis in experimental animals through exhibiting antioxidant effect by restoring cerebral oxidative/antioxidant redox and anti-apoptotic effect via regulating cerebral APP/Aβ/P2Y12R/P2X7R and XIAP/BDNF signaling pathways.

Clopidogrel combats neuroinflammation and enhances learning behavior and memory in a rat model of Alzheimer's disease

Background and aimAlzheimer's disease (AD) is a progressive neurodegenerative disease. Multiple molecular mechanisms have been employed in its pathogenesis such as Amyloid β (Aβ) formation, tau protein hyperphosphorylation, reduced acetylcholine (ACh) level, and neuroinflammation. This study aimed to assess the possible neuroprotective effect of clopidogrel in AD model induced by aluminum chloride (AlCl3) in rats. MethodsSixty adult male Sprague-Dawley rats were divided into four different groups: Control, AlCl3, AlCl3 + donepezil, and AlCl3 + Clopidogrel. AlCl3 and the drugs were given orally once/day for 42 days. The spatial learning and memory and recognition memory were evaluated using Morris Water Maze (MWM) and Novel Object Recognition (NOR) tests, respectively. After euthanasia, hippocampal acetylcholinesterase (AChE) activity, tumor necrosis factor-alpha (TNF-α), and interleukin-1β (IL-1β) levels were biochemically assessed. Moreover, amyloid precursor protein (APP) mRNA gene expression was analyzed in the hippocampi of all rats. Histopathology for amyloid plaques was done. ResultsClopidogrel co-treatment significantly ameliorated the cognitive deficits induced by AlCl3 in rats. Besides, clopidogrel significantly reduced AChE activity, TNF-α and IL-1β concentrations, and APP mRNA gene expression in the hippocampi of rats compared to AlCl3 rats. The decrease of hippocampal TNF-α and IL-1β concentrations by clopidogrel was significant compared to donepezil co-treated rats. Clopidogrel co-treatment lessened amyloid plaque deposition in the hippocampal tissues of rats compared to AlCl3 rats. ConclusionThese findings demonstrate that clopidogrel could alleviate learning and memory deficit induced by AlCl3 in rats and significantly reduced AChE activity. The neuroprotective outcome of clopidogrel might be assigned to its anti-inflammatory effect.

MiR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells.

Curcumin exerts a neuroprotective effect on Alzheimer's disease; however, it is not known whether microRNAs are involved in this protective effect. This study was conducted using swAPP695-HEK293 cells as an Alzheimer's disease cell model. swAPP695-HEK293 cells were treated with 0, 0.5, 1, 2, 5, and 10 μM curcumin for 24 hours. The changes in miR-15b-5p, miR-19a-3p, miR-195-5p, miR-101-3p, miR-216b-5p, miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction. The mRNA and protein levels of amyloid precursor protein, amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction, western blot assays and enzyme-linked immunosorbent assays. swAPP695-HEK293 cells were transfected with miR-15b-5p mimic, or treated with 1 μM curcumin 24 hours before miR-15b-5p inhibitor transfection. The effects of curcumin on amyloid precursor protein, amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay. Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3'-untranslated region of amyloid precursor protein. The results show that amyloid precursor protein and amyloid-β expression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells. Curcumin suppressed the expression of amyloid precursor protein and amyloid-β and up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells. In addition, we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-β levels in the curcumin-treated cells. Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3'-untranslated region of amyloid precursor protein. These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-β in swAPP695-HEK293 cells, which was partially mediated by miR-15b-5p via targeting of the 3'-untranslated region of amyloid precursor protein.

Targeting Renin-Angiotensin System Against Alzheimer's Disease.

Renin Angiotensin System (RAS) is a hormonal system that regulates blood pressure and fluid balance through a coordinated action of renal, cardiovascular, and central nervous systems. In addition to its hemodynamic regulatory role, RAS involves in many brain activities, including memory acquisition and consolidation. This review has summarized the involvement of RAS in the pathology of Alzheimer’s disease (AD), and the outcomes of treatment with RAS inhibitors. We have discussed the effect of brain RAS in the amyloid plaque (Aβ) deposition, oxidative stress, neuroinflammation, and vascular pathology which are directly and indirectly associated with AD. Angiotensin II (AngII) via AT1 receptor is reported to increase brain Aβ level via different mechanisms including increasing amyloid precursor protein (APP) mRNA, β-secretase activity, and presenilin expression. Similarly, it was associated with tau phosphorylation, and reactive oxygen species generation. However, these effects are counterbalanced by Ang II mediated AT2 signaling. The protective effect observed with angiotensin receptor blockers (ARBs) and angiotensin converting enzyme inhibitors (ACEIs) could be as the result of inhibition of Ang II signaling. ARBs also offer additional benefit by shifting the effect of Ang II toward AT2 receptor. To conclude, targeting RAS in the brain may benefit patients with AD though it still requires further in depth understanding.

MiR-373-3p promotes lung adenocarcinoma cell proliferation via APP.

Previous studies have indicated that lung adenocarcinoma (LUAD) is one of the common human malignancies, and its incidence keeps rising. With the help of microarray technology, downregulation of miR-373-3p was observed in LUAD tissues compared with normal lung tissues. Notably, the present study demonstrated that the expression of amyloid precursor protein (APP) mRNA in LUAD tissues was overexpressed compared with adjacent tissues. Bioinformatic analysis demonstrated that miR-373-3p may interact with the 3′ untranslated region of APP mRNA, and then western blot and dual-luciferase reporter gene assays were employed to verify the interaction. Finally, CCK-8 assays were used to measure the tumor-suppressing effect of miR-373-3p on A549 and it was demonstrated that overexpression of miR-373-3p may more effectively inhibit the proliferation of A549 compared with APP si-RNA. Overall, the findings suggest that miR-373-3p downregulation partly accounts for APP overexpression and leads to a promotion of cell growth in LUAD. miR-373-3p may therefore act as a valuable target in potential anticancer strategies to treat LUAD.

Potential Neuroprotective Effect of Clopidogrel on Aluminum Chloride-Induced Alzheimer Disease in Rats.

Background and aim: Alzheimer’s disease (AD) is a common progressivedisease characterized by neurodegeneration. Multiple molecular mechanismssuch as amyloid β (Aβ)formation, tau protein hyperphosphorylation,reducedcholinergic neurotransmission, oxidative stress, and neuroinflammationare involved in the disease pathophysiology. The purpose of thisstudywas to assess potential neuroprotective effect of clopidogrel in ADmodelinduced by aluminum chloride (AlCl3) in ratsMethods: Fortyadult male Sprague-Dawley rats were haphazardly separated into fourgroupsof ten rats each: Control, AlCl3 (100 mg/kg orally), AlCl3 + Memantine(10 mg/kg orally), and AlCl3 + Clopidogrel groups (20 mg/kg orally).AlCl3and drugs were administrated once every day for 42 days. The spatiallearningand memory were evaluated using Morris Water Maze (MWM) test.Aftereuthanization, hippocampal acetylcholinesterase (AChE) activity, tumornecrosis factor alpha (TNF-α),and interleukin-1beta (IL-1β)concentrationswere biochemically assessed in all groups. Moreover, amyloid precursorprotein (APP) mRNA gene expression was analyzed in thehippocampusof all groups with Real-time quantitative polymerase chain reaction(qPCR). Histopathology for amyloid plaques was done in all groups’hippocampususing hematoxylin and eosin and Congo stain.Results: AlCl3 and clopidogrel co-treatment significantly ameliorated thecognitive deficits induced by AlCl3 in rats. Moreover, AlCl3 and clopidogrelco-treatment significantly reduced AChE activity, TNF-α and IL-1β concentrations,and APP mRNA gene expression in the rat hippocampi comparedtoAlCl3-treated rats. AlCl3 and clopidogrel co-treatment significantly reducedTNF-α and IL-1β concentrations in the rat hippocampi compared toAlCl3and memantine co-treated rats. In addition, AlCl3 and clopidogrel cotreatmentalleviated amyloid plaque deposition in the rat hippocampal tissuesstained with hematoxylin and eosin and Congo stains compared toAlCl3-treatedrats.Conclusion:Theseresults showed that clopidogrel could improve cognitivedeficitstriggered by AlCl3 in rats. The neuronal protection influence of clopidogrelin AlCl3-triggered AD might be mediated through its antiinflammatoryeffect as demonstrated by its ability to decrease hippocampalTNF-α and IL-1β concentrations. It might also be mediated through its loweringeffect on AChE activity and/or decreasing mRNA gene expression ofAPPgene in the hippocampus

Amyloid β1-43 Accumulates in the Lens Epithelium of Cortical Opacification in Japanese Patients.

We investigated the accumulation of amyloid β (Aβ1-40, Aβ1-42, Aβ1-43) in the lens epithelium of patients with opacification of five different types (cortical cataract [COR]; nuclear cataract [NUC]; posterior subcapsular cataract [PSC]; retrodots [RD]; and water clefts [WC]). Samples were collected from Japanese patients taken during cataract surgery; Aβ levels and mRNA expression were determined by ELISA and a real-time RT-PCR method, respectively. Levels of Aβ1-40 and Aβ1-42 in the lens epithelium of patients with COR, NUC, PSC, RD, and WC showed no significant differences in comparison with transparent lens epithelium. Levels of Aβ1-43 in the lens epithelium of patients with PSC and WC were not detected, and NUC and RD were slightly elevated. In contrast to the results in these cataract types, high Aβ1-43 levels were observed in the lens epithelium of patients with COR, and a close relationship was observed between Aβ1-43 levels and the degree of lens opacification (R = 0.8229, n = 6). The levels of Aβ1-43 were also higher in the lens epithelium of patients with mixed-cataract showing cortical opacification, and the Aβ1-43 levels in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in that of mixed-cataract patients without cortical opacification. In addition, the level of an amyloid precursor protein mRNA in the lens epithelium of mixed-cataract patients with cortical opacification was significantly higher than in transparent lens and mixed-cataract patients without cortical opacification. We found high levels of Aβ1-43 accumulation in the lens epithelium of Japanese patients with cortical opacification.

Genistein, the Isoflavone in Soybean, Causes Amyloid Beta Peptide Accumulation in Human Neuroblastoma Cell Line: Implications in Alzheimer's Disease.

The isoflavone, genistein, present in soybean is being actively investigated for its potential beneficial effect against Alzheimer's disease. Our data, however, show that in SHSY5Y cells genistein causes increased expression (mRNA and protein) of amyloid precursor protein (APP), increased mRNA expression and activity of β-secretase and diminished level of insulin degrading enzyme (IDE) which also degrades amyloid beta peptide. These effects of genistein lead to enhanced accumulation of amyloid beta peptide (Aβ42) in SHSY5Y cells. The results do not support the view that genistein could be a putative drug against AD and instead strengthen the epidemiological study which implies that genistein content of soybean food product (Tofu) leads to cognitive impairment.

Fragile X mental retardation protein expression in Alzheimer's disease.

The FMR1 protein product, FMRP, is an mRNA binding protein associated with translational inhibition of target transcripts. One FMRP target is the amyloid precursor protein (APP) mRNA, and APP levels are elevated in Fmr1 KO mice. Given that elevated APP protein expression can elicit Alzheimer’s disease (AD) in patients and model systems, we evaluated whether FMRP expression might be altered in Alzheimer’s autopsy brain samples and mouse models compared to controls. In a double transgenic mouse model of AD (APP/PS1), we found no difference in FMRP expression in aged AD model mice compared to littermate controls. FMRP expression was also similar in AD and control patient frontal cortex and cerebellum samples. Fragile X-associated tremor/ataxia syndrome (FXTAS) is an age-related neurodegenerative disorder caused by expanded CGG repeats in the 5′ untranslated region of the FMR1 gene. Patients experience cognitive impairment and dementia in addition to motor symptoms. In parallel studies, we measured FMRP expression in cortex and cerebellum from three FXTAS patients and found reduced expression compared to both controls and Alzheimer’s patient brains, consistent with animal models. We also find increased APP levels in cerebellar, but not cortical, samples of FXTAS patients compared to controls. Taken together, these data suggest that a decrease in FMRP expression is unlikely to be a primary contributor to Alzheimer’s disease pathogenesis.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

The effects of dietary restriction and aging on amyloid precursor protein and presenilin-1 mRNA and protein expression in rat brain

The objective of this study was to examine the effects of aging and long-term dietary restriction (DR) on the level of amyloid precursor protein (APP) and presenilin-1 (PS-1), proteins that are critically involved in Alzheimer's disease. Changes in mRNA and protein expression were assessed by real-time PCR and western blot analysis during aging and long-term DR in the cortex and hippocampus of 6-, 12-, 18-, and 24-month-old rats. Prominent regional changes in expression were observed in response to aging and DR. Although the hippocampus displayed significant alterations in APP mRNA and protein expression and no significant changes in PS-1 expression, an opposite pattern was observed in the cortex. DR counteracted the age-related changes in APP mRNA expression in both structures of old animals. The observed DR-induced increase in mRNA levels in the hippocampus was accompanied by an increase in the level of full-length protein APP. These results indicate that although both structures are very sensitive to aging, a specific spatial pattern of changes in APP and PS-1 occurs during aging. Furthermore, these findings provide evidence that DR can affect APP and PS-1 expression in a manner consistent with its proposed 'antiaging' effect.

Icariin, a phoshphodiesterase-5 inhibitor, improves learning and memory in APP/PS1 transgenic mice by stimulation of NO/cGMP signalling

Phosphodiesterase-5 (PDE5) inhibitors are predominantly used in the treatment of erectile dysfunction, and have been recently shown to have a potential therapeutic effect for the treatment of Alzheimer's disease (AD) through stimulation of nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling by elevating cGMP, which is a secondary messenger involved in processes of neuroplasticity. In the present study, the effects of a PDE5 inhibitor, icarrin (ICA), on learning and memory as well as the pathological features in APP/PS1 transgenic AD mice were investigated. Ten-month-old APP/PS1 transgenic mice overexpressing human amyloid precursor protein (APP695swe) and presenilin 1 (PS1-dE9) were given ICA (30 and 60 mg/kg) or sildenafil (SIL) (2 mg/kg), age-matched wild-type (WT) mice were given ICA (60 mg/kg), and APP/PS1 and WT control groups were given an isovolumic vehicle orally twice a day for four months. Results demonstrated that ICA treatments significantly improved learning and memory of APP/PS1 transgenic mice in Y-maze tasks. The amyloid precursor protein (APP), amyloid-beta (Aβ1-40/42) and PDE5 mRNA and/or protein levels were increased in the hippocampus and cortex of APP/PS1 mice, and ICA treatments decreased these physiopathological changes. Furthermore, ICA-treated mice showed an increased expression of three nitric oxide synthase (NOS) isoforms at both mRNA and protein levels, together with increased NO and cGMP levels in the hippocampus and cortex of mice. These findings demonstrate that ICA improves learning and memory functions in APP/PS1 transgenic mice possibly through the stimulation of NO/cGMP signalling and co-ordinated induction of NOS isoforms.

Excessive hydrogen peroxide enhances the attachment of amyloid β1–42 in the lens epithelium of UPL rats, a hereditary model for cataracts

Several studies have reported that hydrogen peroxide (H2O2) is related to the toxicity of amyloid β (Aβ), and that the accumulation of Aβ in the lenses of humans causes lens opacification. In this study, we investigate the accumulation of Aβ1–42 in the lenses of UPL rats, which then leads to lens opacification. In addition, we demonstrate the effect of disulfiram eye drops (DSF), a potent radical scavenger, on Aβ1–42 accumulation in the lenses of UPL rats. The H2O2 levels in 46- to 60-day-old UPL rat lenses are significantly higher than in normal rats, and the Aβ1–42 levels in 53- and 60-day-old UPL rats are also increased only in lens epithelium containing capsules (capsule-epithelium), not in the lens cortex and nucleus. However, no increases in amyloid precursor protein (APP), β- or γ-secretase mRNA were observed in lenses of the corresponding ages. It has been thought that Aβ1–42 that accumulates in the lenses of UPL rats is actually produced in another tissue containing neuronal cells, such as brain or retina. Aβ1–42 levels in the brain and retina rise with aging, and the levels of APP, β- and γ-secretase mRNA in the retinas of 53-day-old UPL rats with opaque lenses are significantly higher than in 25-day-old UPL rats with transparent lenses. In contrast to the results in retinas, the levels of APP, β- and γ-secretase mRNA in the brains of 25- and 53-day-old UPL rats are similar. On the other hand, in an in vitro study, Aβ1–42 attachment in the lens capsule-epithelium of UPL rats was found to increase in H2O2. In addition, in an in vivo study, the inhibition of H2O2 by DSF was found to attenuate the increase in Aβ1–42 in the lens capsule-epithelium of 60-day-old UPL rats. Taken together, we hypothesize that excessive H2O2 in the lens enhances the attachment of Aβ1–42 in the lens capsule-epithelium of UPL rats, and that the instillation of DSF has the ability to attenuate the attachment of Aβ1–42 by inhibiting H2O2 production in lens. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

P4‐225: Therapeutic action of translation blockers on expression of the amyloid precursor protein as a central ferroxidase

It is possible to repress amyloid precursor protein (APP) mRNA translation as both an anti-amyloid pharmacotherapy for Alzheimer's disease (AD) and also to tone oxidative ferroxidase activity from APP over-expression.This therapeutic model is consistent with cases where AD pathology is the genetic consequence of duplications of the APP gene. Our pharmacological goal is to reduce APP expression. The antioxidant N-acetyl cysteine selectively reduced APP translation in neural cells while maintaining compensatory APLP-1 /-2 levels, then this drug also limited Aβ production in vivo.Secondly, posiphen is the + enantiomer of the well-tested anti-cholinesterase phenserine, which entered phase II clinical trials for its capacity to limit amyloid in AD subjects via its action as an APP 5'UTR translation blocker. We screened two molecular compound libraries at Harvard and Columbia Universities. From our screens, thirteen top leads shared chemical features pertinent to potently blocking APP 5'UTR directed translation (1nM). Most of our lead APP translation blockers were found to be highly potent intercalators of the RNA stem loop folded from APP 5'untranslated region. All were planar and had either a benzimidazole, benzothiazole or benzoxazole ring in its backbone. Top APP 5'UTR inhibitors were 50-fold more potent blockers of Aβ precursor translation than NAC or posiphen. The most active lead was the ninth compound in the series, APP blocker-9 (“JTR-009”). Critically, JTR-009 effectively reduced APP levels in primary mouse E-18 cortical neurons at single nanomolar concentrations similar to serum concentrations of effective clinical drugs. JTR-009 was anti-amyloid and our calcein assays demonstrated it adjusted cellular iron content to promote cellular viability. Our top APP translation blocker, JTR-009, is a planar tricyclic benzimidazole that intercalates selectively into the single 146 base APP 5'UTR specific RNA stem loop. Confirming specificity and potency, we are checking how JTR-009 blocks ribosome scanning of the APP 5'UTR to impede translation (Aβ output) while not interacting with the 5'UTRs of APP-like protein (APLP-1/-2) mRNAs. This APP translation blocker adjusted the intrinsic levels of APP with its concomitant ferroxidase activity and iron export properties, thus to optimally promote neural cell viability.

Effects of Nerve Growth Factor and Nitric Oxide Synthase Inhibitors on Amyloid Precursor Protein mRNA Levels and Protein Stability

We determined previously that nitric oxide (NO) modulates the nerve growth factor (NGF)-mediated increases in amyloid precursor protein (APP) levels in PC12 cells. To elucidate potential mechanisms, the effects of NGF and NO synthase (NOS) inhibitors on APP mRNA levels and protein stability were evaluated. Surprisingly, treatment of PC12 cells with NGF resulted in decreased levels of APP695 and APP751/770 mRNA. Therefore, the effect of NGF on APP protein stability was examined using the translation inhibitor, cycloheximide. Under these conditions, NGF did not alter the rate of APP degradation, suggesting that NGF may be enhancing the translation rate of APP. Since NOS inhibitors attenuate the NGF-mediated increase in APP levels, their effect on APP mRNA levels and protein stability was also assessed. S-methylisothiourea (S-MIU), selective for inducible NOS, decreased both APP695 and APP751/770 mRNA levels while the non-selective NOS inhibitor, Nω-nitro-L-arginine methylester (L-NAME) had no effect. In both control and NGF-treated PC12 cells, S-MIU increased the half-life of APP, with the greatest effect observed with the APP695 isoform. Based on these data we propose that in PC12 cells, NGF increases APP levels through enhanced translation rate and that NO, which modulates the NGF-induced increase in APP protein, also regulates APP mRNA levels and could play a role in APP processing.