Inefficient quality control of ribosome stalling during APP synthesis generates CAT-tailed species that precipitate hallmarks of Alzheimer's disease.

Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-ß (Aß) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aß-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. We used Drosophila models expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and ß-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. We find that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid ß peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. Our finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets.

DRD1 agonist A-68930 improves mitochondrial dysfunction and cognitive deficits in a streptozotocin-induced mouse model.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by irreversible cognitive deficits and memory dysfunction. Dopamine is the most abundant catecholaminergic neurotransmitter in the brain which regulates motivation, reward, movement, and cognition. Recently, increasing evidences have shown that dopaminergic system is disturbed in AD conditions, and pharmacological interventions targeting dopamine D1 receptor (DRD1) exhibit certain therapeutic benefits in AD models. However, the underlying link between DRD1 and AD remains elusive. This study sought to test whether the selective DRD1 agonist A-68930 could improve streptozotocin (STZ)-induced cognitive impairment in mice. Here we found that A-68930 treatment through intraperitoneal injection efficiently alleviated STZ-induced cognitive deficits in mice. Moreover, our mechanism researches revealed that the DRD1 signaling induced by A-68930 significantly rescued STZ-induced mitochondrial biogenesis deficit, mitochondrial dysfunction, Aß overexpression, and tau phosphorylation in mice hippocampus and cortex and SH-SY5Y cells, which may be mediated through stimulating AMPK/PGC-1α pathway. This study indicates that DRD1 agonist A-68930 can improve STZ-induced cognitive deficits and mitochondrial dysfunction in vivo and in vitro, and DRD1 may represent an appropriate target candidate for AD drug development.

Inhibition of heat shock proteins increases autophagosome formation, and reduces the expression of APP, Tau, SOD1 G93A and TDP-43.

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.

Microglial activation in the right amygdala-entorhinal-hippocampal complex is associated with preserved spatial learning in AppNL-G-F mice.

BACKGROUND: In Alzheimer`s disease (AD), regional heterogeneity of ß-amyloid burden and microglial activation of individual patients is a well-known phenomenon. Recently, we described a high incidence of inter-individual regional heterogeneity in terms of asymmetry of plaque burden and microglial activation in ß-amyloid mouse models of AD as assessed by positron-emission-tomography (PET). We now investigate the regional associations between amyloid plaque burden, microglial activation, and impaired spatial learning performance in transgenic mice in vivo. METHODS: In 30 AppNL-G-F mice (15 female, 15 male) we acquired cross-sectional 18 kDa translocator protein (TSPO-PET, 18F-GE-180) and ß-amyloid-PET (18F-florbetaben) scans at ten months of age. Control data were obtained from age- and sex-matched C57BI/6 wild-type mice. We assessed spatial learning (i.e. Morris water maze) within two weeks of PET scanning and correlated the principal component of spatial learning performance scores with voxel-wise ß-amyloid and TSPO tracer uptake maps in AppNL-G-F mice, controlled for age and sex. In order to assess the effects of hemispheric asymmetry, we also analyzed correlations of spatial learning performance with tracer uptake in bilateral regions of interest for frontal cortex, entorhinal/piriform cortex, amygdala, and hippocampus, using a regression model. We tested the correlation between regional asymmetry of PET biomarkers with individual spatial learning performance. RESULTS: Voxel-wise analyses in AppNL-G-F mice revealed that higher TSPO-PET signal in the amygdala, entorhinal and piriform cortices, the hippocampus and the hypothalamus correlated with spatial learning performance. Region-based analysis showed significant correlations between TSPO expression in the right entorhinal/piriform cortex and the right amygdala and spatial learning performance, whereas there were no such correlations in the left hemisphere. Right lateralized TSPO expression in the amygdala predicted better performance in the Morris water maze (ß = -0.470, p = 0.013), irrespective of the global microglial activation and amyloid level. Region-based results for amyloid-PET showed no significant associations with spatial learning. CONCLUSION: Elevated microglial activation in the right amygdala-entorhinal-hippocampal complex of AppNL-G-F mice is associated with better spatial learning. Our findings support a protective role of microglia on cognitive function when they highly express TSPO in specific brain regions involved in spatial memory.

Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains.

Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer's diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGL, which is capable of en-hancing APP expression. We observed that aging brain tissues with APP had protective effects on ZIKV infection by reducing the availability of the viruses. Also, knockdown of APP expression or blocking ZIKV-APP interactions enhanced ZIKV replication in human neural progenitor/stem cells. Finally, intracranial infection of ZIKV in APP-null neonatal mice resulted in higher mortality and viral yields. Taken together, these findings suggest that APP is a restriction factor that protects against ZIKV by serving as a decoy receptor, and plays a protective role in ZIKV-mediated brain injuries.

Amyloid precursor protein 770 is specifically expressed and released from platelets.

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. ß-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.

Increase of BACE1, Brain-Renal Risk Factor, Contributes to Kidney Damage in an Alzheimer's Disease Mouse Model.

BACKGROUND: It is believed that there is a certain correlation between the brain and kidneys, but it is poorly understood. Many findings suggested that there were previously unknown signaling pathways involving AßPP and BACE1 in the kidney. OBJECTIVE: Exploring the changes of BACE1 activity in APP23 mouse kidneys, providing evidence for the function of AßPP and BACE1 activity in the kidney. METHODS: The activity and expression of BACE1 were detected in the kidney of APP23 mice by enzymatic assay and western blotting. The protein expression levels of AßPP, claudin1, occludin, VE-cadherin, and Klotho (membrane-form klotho) were examined by using western blotting. The renal pathological changes of APP23 mice were examined by the routine renal pathological procedures. RESULTS: In this study, we found that the AßPP protein level was increased in kidneys of APP23 mice compared with wild-type (WT) mice. Additionally, the activity and expression of BACE1 were increased in kidneys of APP23 mice compared to that of WT. BACE1 was predominantly distributed on the lumen side of renal tubular epithelial cells. The protein levels of Klotho and VE-cadherin were decreased, occludin expression was also decreased, and claudin-1 expression was increased. Renal pathological damage which observed in kidneys of APP23 mice was more serious than that in kidneys of WT mice. CONCLUSION: Our findings suggest that the increase of AßPP protein levels under Thy-1 neuron promoter in the APP23 mice promoted the increase of renal BACE1 expression and enzymatic activity in the kidneys. Moreover, certain pathological damage in the kidneys of APP23 mice were observed. APP23 mice are easily affected by external risk factors compared with WT mice.

A Preliminary Study of Cu Exposure Effects upon Alzheimer's Amyloid Pathology.

A large body of evidence indicates that dysregulation of cerebral biometals (Fe, Cu, Zn) and their interactions with amyloid precursor protein (APP) and Aß amyloid may contribute to the Alzheimer's disease (AD) Aß amyloid pathology. However, the molecular underpinnings associated with the interactions are still not fully understood. Herein we have further validated the exacerbation of Aß oligomerization by Cu and H2O2 in vitro. We have also reported that Cu enhanced APP translations via its 5' untranslated region (5'UTR) of mRNA in SH-SY5Y cells, and increased Aß amyloidosis and expression of associated pro-inflammatory cytokines such as MCP-5 in Alzheimer's APP/PS1 doubly transgenic mice. This preliminary study may further unravel the pathogenic role of Cu in Alzheimer's Aß amyloid pathogenesis, warranting further investigation.

microRNAs expression correlates with levels of APP, DYRK1A, hyperphosphorylated Tau and BDNF in the hippocampus of a mouse model for Down syndrome during ageing.

Down syndrome (DS) patients are more susceptible to Alzheimer's disease (AD) due to the presence of three copies of genes on chromosome 21 such as DYRK1A, which encodes a broad acting kinase, and APP (amyloid precursor protein), leading to formation of amyloid beta (Aß) peptide and hyperphosphorylation of Tau. In this study, we investigated the association among miRNAs miR-17, -20a, -101, -106b, -199b, -26a, 26b and some of their target mRNAs such as APP, DYRK1A and BDNF, as well as the levels of hyperphosphorylated Tau in the hippocampus of a 2 and 5 months old mice model of trisomy 21 (Ts65Dn). Results indicated that increased APP expression in the hippocampus of 5 months old DS mice might be correlated with decrease in miR-17, -20a, -101 and -106b. Whereas at 2 months of age normal levels of APP expression in the hippocampus was correlated with increased levels of miR-17, -101 and -106b in DS mice. DYRK1A mRNA also increased in the hippocampus of 5 months old DS mice and it is associated with decreased levels of miR-199b. Increased levels of DYRK1A in 5-month old mice are associated with increased phosphorylation of Tau at Thr212 residue but not at Ser199-202. Tau pathology is accompanied by decreased expression of BDNF and increased miR-26a/b in mice of 5 months of age. Taken together, data indicate that miR-17, -20a, -26a/b, -101, -106b and -199b might be interesting targets to mitigate Tau and Aß pathology in DS.

Environmental lead exposure aggravates the progression of Alzheimer's disease in mice by targeting on blood brain barrier.

Alzheimer's disease (AD) is a neurodegenerative disease that can be induced by heavy metals such as lead. However, there is limited information on the role of blood-brain barrier (BBB) in lead induced AD-like pathology. This study investigates the potential mechanism of lead exposure aggravating the progression of Alzheimer's disease in mice through the BBB. 200 mg/L and 500 mg/L lead acetate were given to C57BL/6J and APP/PS1 mice through drinking water from a week before mating, until the offspring were 7-months-old. 8 female juvenile mice in each group were selected for this investigation. Lead exposure increased blood lead concentration which revealed the internal exposure level, accelerated Aß1-42 deposition in APP/PS1 mouse cortexes and abnormal change in Zonula Occludin-1 (ZO-1) and Claudin-5 protein. It also increased the expression of p-tau in both the C57BL/6J and APP/PS1 mice, and decreased mRNA and protein expression in low-density lipoprotein receptor (LRP-1). Additionally, it increased the mRNA and protein expression of amyloid beta precursor protein (APP) and beta secretase 1 (BACE-1). The activated astrocytes increased in the brains of APP/PS1 mice, and coalesced around the Aß1-42 deposition after lead exposure. The main vessels in deutocerebrum were attached with Aß1-42 deposition. These results offer insight into the mechanism of preventing lead induced AD through cerebrovascular pathways.

Essential Oil of Acorus tatarinowii Schott Ameliorates Aß-Induced Toxicity in Caenorhabditis elegans through an Autophagy Pathway.

BACKGROUND: Acorus tatarinowii Schott [Shi Chang Pu in Chinese (SCP)] is a traditional Chinese medicine frequently used in the clinical treatment of dementia, amnesia, epilepsy, and other mental disorders. Previous studies have shown the potential efficacy of SCP against Alzheimer's disease (AD). Nevertheless, the active constituents and the modes of action of SCP in AD treatment have not been fully elucidated. PURPOSE: The aim of this study was to investigate the protective effects of SCP on abnormal proteins and clarify its molecular mechanisms in the treatment of AD by using a Caenorhabditis elegans (C. elegans) model. METHODS: This study experimentally assessed the effect of SCP-Oil in CL4176 strains expressing human Aß in muscle cells and CL2355 strains expressing human Aß in pan-neurons. Western blotting, qRT-PCR, and fluorescence detection were performed to determine the oxidative stress and signaling pathways affected by SCP-Oil in nematodes. RESULTS: SCP-Oil could significantly reduce the deposition of misfolded Aß and polyQ proteins and improved serotonin sensitivity and olfactory learning skill in worms. The analysis of pharmacological action mechanism of SCP-Oil showed that its maintaining protein homeostasis is dependent on the autophagy pathway regulated partly by hsf-1 and sir-2.1 genes. CONCLUSION: Our results provide new insights to develop treatment strategy for AD by targeting autophagy, and SCP-Oil could be an alternative drug for anti-AD.

Kinesin light chain-1 serine-460 phosphorylation is altered in Alzheimer's disease and regulates axonal transport and processing of the amyloid precursor protein.

Damage to axonal transport is an early pathogenic event in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer's disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer's disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and in Drosophila neurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer's disease.

Mechanism of anti-dementia effects of mangiferin in a senescence accelerated mouse (SAMP8) model.

Mangiferin (2-ß-d-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one), a xanthanoid, is one of the major compounds isolated from mango leaves and bark fruit. Previous studies have identified several properties of mangiferin, such as preventing microbial growth, reducing oxidative stress and helping reduce risk of diabetes. The aim of the present study is to explore the potential anti-dementia effects of Mangiferin in a senescence-accelerated mouse prone 8 (SAMP8) mouse model. Morris water maze (MWM) test showed that mangiferin significantly improved the learning and memory retention in SAMP8 mice. In addition, mangiferin reduced the damage in hippocampal neurons and mitochondria, and decreased the expression of amyloid-ß (Aß1-40 and Aß1-42); however, no influence on the expression of amyloid precursor protein (APP) within the brain of SAMP8 mice. Moreover, Mangiferin inhibited lipid peroxidation (LPO). In conclusion, we provided evidences to show that mangiferin significantly restored the learning and memory impairment in the SAMP8 mouse model, and reduced the pathological injury in hippocampal by modulating lipid oxidation and amyloid-ß deposition in the brain.

Mitophagy Failure in APP and Tau Overexpression Model of Alzheimer's Disease.

Mitochondrial alterations and oxidative stress are common features of Alzheimer's disease brain and peripheral tissues. Moreover, mitochondrial recycling process by autophagy has been found altered in the sporadic form of the disease. However, the contribution of the main proteins involved in this pathology such as amyloid-ß protein precursor (AßPP) and tau needs to be achieved. With this aim, human unmodified fibroblasts were transduced with lentivectors encoding APP and Tau and treated with CCCP to study the mitophagy process. Both AßPP and tau separately increased autophagy flux mainly by improving degradation phase. However, in the specific case of mitophagy, labeling of mitochondria by PINK1 and PARK2 to be degraded by autophagy seemed reduced, which correlates with the long-term accumulation of mitochondria. Nevertheless, the combination of tau and AßPP was necessary to cause a mitophagy functional impairment reflected in the accumulation of depolarized mitochondria labeled by PINK1. The overexpression of Tau and APP recapitulates the mitophagy failure previously found in sporadic Alzheimer's disease.

Interferon beta ameliorates cognitive dysfunction in a rat model of Alzheimer's disease: Modulation of hippocampal neurogenesis and apoptosis as underlying mechanism.

Neuronal apoptosis and impaired hippocampal neurogenesis are major players in cognitive/memory dysfunctions including Alzheimer's disease (AD). Interferon beta (IFNß) is a cytokine with anti-apoptotic and neuroprotective properties on the central nervous system (CNS) cells which specifically affects neural progenitor cells (NPCs) even in the adult brain. In this study, we examined the effect of IFNß on memory impairment as well as hippocampal neurogenesis and apoptosis in a rat model of AD. AD model was induced by lentiviral-mediated overexpression of mutant APP in the hippocampus of adult rats. Intranasal (IN) administration of IFNß (0.5 µg/kg and 1 µg/kg doses) was started from day 23 after virus injection and continued every other day to the final day of experiments. The expression levels of APP, neurogenesis (Nestin, Ki67, DCX, and Reelin) and apoptosis (Bax/Bcl-2 ratio, cleaved-caspase-3 and seladin-1) markers were evaluated by immunohistochemistry, real-time PCR, immunofluorescence and western blotting. Moreover, thioflavin T and Nissl stainings were used to assess Aß plaque levels and neuronal degeneration in the hippocampus, respectively. Our results showed that IFNß treatment reduced APP expression and Aß plaque formation, and concomitantly ameliorated spatial learning and memory deficits examined in Y-maze and Morris water maze tests. Moreover, in parallel with reducing apoptosis and neural loss in the hippocampal subfields, IFNß decreased ectopic neurogenesis in the CA1 and CA3 regions of the AD rat hippocampus. However, IFNß increased neurogenesis in the dentate gyrus neurogenic niche. Our findings suggest that IFNß exerts neuroprotective effects at least partly by inhibition of apoptosis and modulation of neurogenesis. Taken together, IFNß can be a promising therapeutic approach to improve cognitive performance in AD-like neurodegenerative context.

Intranasal interferon beta improves memory and modulates inflammatory responses in a mutant APP-overexpressing rat model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by progressive cognitive decline. According to the critical role of inflammation in pathogenesis of AD and memory deficits, a cytokine with anti-inflammatory properties like interferon beta (IFNß), currently used to slow down disease progression and protect against cognitive disturbance in multiple sclerosis, might be also an effective treatment in AD condition. This study aimed to answer if the intranasal (IN) administration of IFNß with high CNS accessibility can alleviate memory impairments in a mutant APP-overexpressing rat model of AD through modulating inflammatory responses. To address this question, the lentiviruses carrying human amyloid protein precursor (APP) with the Swedish and Indiana mutations (LV-APPSw/Ind) were bilaterally injected in the hippocampus of adult rats. Memory performance was assessed using passive avoidance task on days 49 and 50 after injection. Moreover, the expression of glial markers (GFAP and Iba1) and pro-inflammatory (TNF-α, IL-1ß and IL-6) and anti-inflammatory cytokines (IL-10) were evaluated in the hippocampus. Therapeutic effects of IN-administered IFNß (0.5 µg/kg and 1 µg/kg doses, every other day from day 23 to 50 after lentivirus injection) were examined in the LV-APP-injected rats. Our results showed that over-expression of mutant human APP gene in the hippocampus led to learning and memory deficits concomitant with gliosis and pro-inflammatory responses. Interestingly, treatment of AD-modeled rats with IFNß ameliorated memory impairments possibly through suppressing gliosis and shifting from pro-inflammatory toward anti-inflammatory status, suggesting that IFNß may be a promising therapeutic agent to improve cognitive functions and modulate inflammatory responses in an AD-like neurodegenerative context.

APP processing and metabolism in corneal fibroblasts and epithelium as a potential biomarker for Alzheimer's disease.

Alzheimer's disease (AD) primarily affects the brain and is the most common form of dementia worldwide. Despite more than a century of research, there are still no early biomarkers for AD. It has been reported that AD affects the eye, which is more accessible for imaging than the brain; however, links with the cornea have not been evaluated. To investigate whether the cornea could be used to identify possible diagnostic indicators of AD, we analyzed the proteolytic processing and isoforms of amyloid precursor protein (APP) and evaluated the expression of AD-related genes and proteins in corneal fibroblasts from wild-type (WT) corneas and corneas from patients with granular corneal dystrophy type 2 (GCD2), which is related to amyloid formation in the cornea. Reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the expression of AD-related genes, i.e., APP, ADAM10, BACE1, BACE2, PSEN1, NCSTN, IDE, and NEP. RT-PCR and DNA sequencing analysis demonstrated that isoforms of APP770 and APP751, but not APP695, were expressed in corneal fibroblasts. Moreover, the mRNA ratio of APP770/APP751 isoforms was approximately 4:1. Western blot analysis also demonstrated the expression of a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), beta-site APP-cleaving enzyme 1 (BACE1), nicastrin, insulin degradation enzyme, and neprilysin in corneal fibroblasts. Among these targets, the levels of immature ADAM10 and BACE1 protein were significantly increased in GCD2 cells. The expression levels of APP, ADAM10, BACE1, and transforming growth factor-beta-induced protein (TGFBIp) were also detected by western blot in human corneal epithelium. We also investigated the effects of inhibition of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems (UPS) on APP processing and metabolism. These pathway inhibitors accumulated APP, α-carboxy-terminal fragments (CTFs), ß-CTFs, and the C-terminal APP intracellular domain (AICD) in corneal fibroblasts. Analysis of microRNAs (miRNAs) revealed that miR-9 and miR-181a negatively coregulated BACE1 and TGFBIp, which was directly associated with the pathogenesis of AD and GCD2, respectively. Immunohistochemical analysis indicated that APP and BACE1 were distributed in corneal stroma cells, epithelial cells, and the retinal layer in mice. Collectively, we propose that the cornea, which is the transparent outermost layer of the eye and thus offers easy accessibility, could be used as a potential biomarker for AD diagnosis and progression.

Astaxanthin Ameliorates Lipopolysaccharide-Induced Neuroinflammation, Oxidative Stress and Memory Dysfunction through Inactivation of the Signal Transducer and Activator of Transcription 3 Pathway.

Astaxanthin (AXT), a xanthophyll carotenoid compound, has potent antioxidant, anti-inflammatory and neuroprotective properties. Neuroinflammation and oxidative stress are significant in the pathogenesis and development of Alzheimer's disease (AD). Here, we studied whether AXT could alleviate neuroinflammation, oxidative stress and memory loss in lipopolysaccharide (LPS) administered mice model. Additionally, we investigated the anti-oxidant activity and the anti-neuroinflammatory response of AXT in LPS-treated BV-2 microglial cells. The AXT administration ameliorated LPS-induced memory loss. This effect was associated with the reduction of LPS-induced expression of inflammatory proteins, as well as the production of reactive oxygen species (ROS), nitric oxide (NO), cytokines and chemokines both in vivo and in vitro. AXT also reduced LPS-induced ß-secretase and Aß1⁻42 generation through the down-regulation of amyloidogenic proteins both in vivo and in vitro. Furthermore, AXT suppressed the DNA binding activities of the signal transducer and activator of transcription 3 (STAT3). We found that AXT directly bound to the DNA- binding domain (DBD) and linker domain (LD) domains of STAT3 using docking studies. The oxidative stress and inflammatory responses were not downregulated in BV-2 cells transfected with DBD-null STAT3 and LD-null STAT3. These results indicated AXT inhibits LPS-induced oxidant activity, neuroinflammatory response and amyloidogenesis via the blocking of STAT3 activity through direct binding.

Emergence of synaptic and cognitive impairment in a mature-onset APP mouse model of Alzheimer's disease.

The synaptic changes underlying the onset of cognitive impairment in Alzheimer's disease (AD) are poorly understood. In contrast to the well documented inhibition of long-term potentiation (LTP) in CA3-CA1 synapses by acute Aß application in adult neurons from rodents, young amyloid precursor protein (APP) transgenic mouse models often, surprisingly, show normal LTP. This suggests that there may be important differences between mature-onset and developmental-onset APP expression/ Aß accumulation and the ensuing synaptic and behavioural phenotype. Here, in agreement with previous studies, we observed that developmental expression of APPSw,Ind (3-4 month old mice from line 102, PLoS Med 2:e355, 2005), resulted in reduced basal synaptic transmission in CA3-CA1 synapses, normal LTP, impaired spatial working memory, but normal spatial reference memory. To analyse early Aß-mediated synaptic dysfunction and cognitive impairment in a more mature brain, we used controllable mature-onset APPSw,Ind expression in line 102 mice. Within 3 weeks of mature-onset APPSw,Ind expression and Aß accumulation, we detected the first synaptic dysfunction: an impairment of LTP in hippocampal CA3-CA1 synapses. Cognitively, at this time point, we observed a deficit in short-term memory. A reduction in basal synaptic strength and deficit in long-term associative spatial memory were only evident following 12 weeks of APPSw,Ind expression. Importantly, the plasticity impairment observed after 3 weeks of mature-onset APP expression is reversible. Together, these findings demonstrate important differences between developmental and mature-onset APP expression. Further research targeted at this early stage of synaptic dysfunction could help identify mechanisms to treat cognitive impairment in mild cognitive impairment (MCI) and early AD.

Dysregulation of Amyloid Precursor Protein Impairs Adipose Tissue Mitochondrial Function and Promotes Obesity.

Mitochondrial function in white adipose tissue (WAT) is an important yet understudied aspect in adipocyte biology. Here, we report a role for amyloid precursor protein (APP) in compromising WAT mitochondrial function through a high-fat diet (HFD)-induced, unconventional mis-localization to mitochondria that further promotes obesity. In humans and mice, obese conditions significantly induce APP production in WAT and its enrichment in mitochondria. Mechanistically, a HFD-induced dysregulation of signal recognition particle subunit 54c is responsible for the mis-targeting of APP to adipocyte mitochondria. Mis-localized APP blocks the protein import machinery, leading to mitochondrial dysfunction in WAT. Adipocyte-specific and mitochondria-targeted APP overexpressing mice display increased body mass and reduced insulin sensitivity, along with dysfunctional WAT due to a dramatic hypertrophic program in adipocytes. Elimination of adipocyte APP rescues HFD-impaired mitochondrial function with significant protection from weight gain and systemic metabolic deficiency. Our data highlights an important role of APP in modulating WAT mitochondrial function and obesity-associated metabolic dysfunction.