Cyclopamine modulates γ-secretase-mediated cleavage of amyloid precursor protein by altering its subcellular trafficking and lysosomal degradation.

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-ß (Aß), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aß is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aß generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aß generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.

Drosophila lilliputian is required for proneural gene expression in retinal development.

BACKGROUND: Proper neurogenesis in the developing Drosophila retina requires the regulated expression of the basic helix-loop-helix (bHLH) proneural transcription factors Atonal (Ato) and Daughterless (Da). Factors that control the timing and spatial expression of these bHLH proneural genes in the retina are required for the proper formation and function of the adult eye and nervous system. RESULTS: Here we report that lilliputian (lilli), the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of ato and da in the developing fly retina. We find that lilli controls ato expression at multiple enhancer elements. We also find that lilli contributes to ato auto-regulation in the morphogenetic furrow by first regulating the expression of da prior to ato. We show that FMR2 regulates the ato and da homologs MATH5 and TCF12 in human cells, suggesting a conservation of this regulation from flies to humans. CONCLUSIONS: We conclude that lilliputian is part of the genetic program that regulates the expression of proneural genes in the developing retina.

Association of the Protein-Quality-Control Protein Ubiquilin-1 With Alzheimer's Disease Both in vitro and in vivo.

Alzheimer's disease (AD) belongs to a class of diseases characterized by progressive accumulation and aggregation of pathogenic proteins, particularly Aß proteins. Genetic analysis has identified UBQLN1 as an AD candidate gene. Ubiquilin-1 levels reduce with AD progression, suggesting a potential loss-of-function mechanism. The ubiquilin-1 protein is involved in protein quality control (PQC), which plays essential roles in cellular growth and normal cell function. Ubiquilin-1 regulates γ-secretase by increasing endoproteolysis of PS1, a key γ-secretase component. Presently, the effects of ubiquilin-1 on cellular physiology as well as Aß-related events require further investigation. Here, we investigated the effects of ubiquilin-1 on cellular growth and viability in association with APP (amyloid-ß protein precursor), APP processing-related ß-secretase (BACE1, BACE) and γ-secretase using cell and animal-based models. We showed that loss-of-function in Drosophila ubqn suppresses human APP and human BACE phenotypes in wing veins and altered cell number and tissue compartment size in the wing. Additionally, we performed cell-based studies and showed that silencing UBQLN1 reduced cell viability and increased caspase-3 activity. Overexpression of UBQLN1 significantly reduced Aß levels. Furthermore, pharmacological inhibition of γ-secretase increased ubiquilin-1 protein levels, suggesting a mechanism that regulates ubiquilin-1 levels which may associate with reduced Aß reduction by inhibiting γ-secretase. Collectively, our results support not only a loss-of-function mechanism of ubiquilin-1 in association with AD, but also support the significance of targeting ubiquilin-1-mediated PQC as a potential therapeutic strategy for AD.

Characterization of human lysophospholipid acyltransferase 3.

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K0.5 (5 microM), a monounsaturated acyl-CoA having the highest apparent Vmax (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.

Amyloid-ß interrupts canonical Sonic hedgehog signaling by distorting primary cilia structure.

BACKGROUND: Primary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. These small organelles serve as sensory structures sampling the extracellular environment and reprogramming the transcriptional machinery in response to environmental change. Primary cilia are decorated with a variety of receptor proteins and are necessary for specific signaling cascades such as the Sonic hedgehog (Shh) pathway. Disrupting cilia structure or function results in a spectrum of diseases collectively referred to as ciliopathies. Common to human ciliopathies is cognitive impairment, a symptom also observed in Alzheimer's disease (AD). One hallmark of AD is accumulation of senile plaques composed of neurotoxic Amyloid-ß (Aß) peptide. The Aß peptide is generated by the proteolytic cleavage of the amyloid precursor protein (APP). We set out to determine if Aß affects primary cilia structure and the Shh signaling cascade. METHODS: We utilized in vitro cell-based assays in combination with fluorescent confocal microscopy to address our study goals. Shh signaling and cilia structure was studied using two different cell lines, mouse NIH3T3 and human HeLa cells. To investigate how Aß levels affect Shh signaling and cilia structure in these cells, we utilized naturally secreted Aß as well as synthetic Aß. Effects on Shh signaling were assessed by luciferase activity while cilia structure was analyzed by fluorescent microscopy. RESULTS: Here, we report that APP localizes to primary cilia and Aß treatment results in distorted primary cilia structure. In addition, we demonstrate that Aß treatment interrupts canonical Shh signal transduction. CONCLUSIONS: Overall, our study illustrates that Aß can alter primary cilia structure suggesting that elevated Aß levels, like those observed in AD patients, could have similar effects on neuronal primary cilia in the brain. Additionally, our study suggests that Aß impairs the Shh signaling pathway. Together our findings shed light on two novel targets for future AD therapeutics.

Mechanisms that synergistically regulate η-secretase processing of APP and Aη-α protein levels: relevance to pathogenesis and treatment of Alzheimer's disease.

The pathophysiology of Alzheimer's disease (AD) is characterized by the formation of cerebral ß-amyloid plaque from a small peptide amyloid-ß (Aß). Aß is generated from the canonical amyloid-ß precursor protein (APP) proteolysis pathway through ß- and γ-secretases. Decreasing Aß levels through targeting APP processing is a very promising direction in clinical trials for AD. A novel APP processing pathway was recently identified, in which η-secretase processing of APP occurs and results in the generation of the carboxy-terminal fragment-η (CTF-η or η-CTF) (Wang et al., 2015) and Aη-α peptide (Willem et al., 2015). η-Secretase processing of APP may be up-regulated by at least two mechanisms: either through inhibition of lysosomal-cathepsin degradation pathway (Wang et al., 2015) or through inhibition of BACE1 that competes with η-secretase cleavage of APP (Willem et al., 2015). A thorough characterization of η-processing of APP is critical for a better understanding of AD pathogenesis and insights into results of clinical trials of AD. Here we further investigated η-secretase processing of APP using well-characterized cell models of AD. We found that these two mechanisms act synergistically toward increasing η-secretase processing of APP and Aη-α levels. Furthermore, we evaluated the effects of several other known secretase modulators on η-processing of APP. The results of our study should advance the understanding of pathophysiology of AD, as well as enhance the knowledge in developing effective AD treatments or interventions related to η-secretase processing of APP.

Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

A central event in Alzheimer's disease is the accumulation of amyloid ß (Aß) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aß generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aß levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aß levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region) at residues 3008-3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

The role of ubiquitin-proteasome in the metabolism of amyloid precursor protein (APP): implications for novel therapeutic strategies for Alzheimer's disease.

The proteasome serves as a major protein quality control system inside the cell. Dysfunction of this system has been associated with Alzheimer's disease (AD) pathogenesis. Amyloid ß accumulation is considered as the central molecular event in the development of AD. This accumulation can result from dysregulated proteolysis of its precursor, APP. Here, we will review the evidence that links proteasome dysfunction to altered APP metabolism, and summarize recent progress in identifying the regulators of APP ubiquitination. Pinpointing UPS components that are APP-specific could lead new pharmaceutical strategies to combat AD.

Development and characterization of an aged onset model of Alzheimer's disease in Drosophila melanogaster.

The biggest risk factor for developing Alzheimer's disease (AD) is age. Depending on the age of onset, AD is clinically categorized into either the early-onset form (before age 60years old), or the late-onset form (after age 65years old), with the vast majority of AD diagnosed as late onset (LOAD). LOAD is a progressive neurodegenerative disorder that involves the accumulation of ß-amyloid (Aß) plaques and neurofibrillary tangles in the brains of elderly patients. Affected individuals often experience symptoms including memory loss, confusion, and behavioral changes. Though many animal models of AD exist, very few are capable of analyzing the effect of older age on AD pathology. In an attempt to better model LOAD, we developed a novel "aged AD" model using Drosophila melanogaster. In our model, we express low levels of the human AD proteins APP (amyloid precursor protein) and BACE1 (ß-site APP cleaving enzyme BACE) specifically in the fly's central nervous system. Advantages of our model include the onset of behavioral and neuropathological symptoms later in the fly's lifespan due to a gradual accrual of Aß within the central nervous system (CNS), making age the key factor in the behavioral and neuroanatomical phenotypes that we observe in this model.

Synaptic abnormalities in a Drosophila model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by memory loss and decreased synaptic function. Advances in transgenic animal models of AD have facilitated our understanding of this disorder, and have aided in the development, speed and efficiency of testing potential therapeutics. Recently, we have described the characterization of a novel model of AD in the fruit fly, Drosophila melanogaster, where we expressed the human AD-associated proteins APP and BACE in the central nervous system of the fly. Here we describe synaptic defects in the larval neuromuscular junction (NMJ) in this model. Our results indicate that expression of human APP and BACE at the larval NMJ leads to defective larval locomotion behavior, decreased presynaptic connections, altered mitochondrial localization in presynaptic motor neurons and decreased postsynaptic protein levels. Treating larvae expressing APP and BACE with the γ-secretase inhibitor L-685,458 suppresses the behavioral defects as well as the pre- and postsynaptic defects. We suggest that this model will be useful to assess and model the synaptic dysfunction normally associated with AD, and will also serve as a powerful in vivo tool for rapid testing of potential therapeutics for AD.

Invertebrate models of Alzheimer's disease.

A majority of the genes linked to human disease belong to evolutionarily conserved pathways found in simpler organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The genes and pathways of these simple organisms can be genetically and pharmacologically manipulated to better understand the function of their orthologs in vivo, and how these genes are involved in the pathogenesis of different diseases. Often these manipulations can be performed much more rapidly in flies and worms than in mammals, and can generate high quality in vivo data that is translatable to mammalian systems. Other qualities also make these organisms particularly well suited to the study of human disease. For example, developing in vivo disease models can help illuminate the basic mechanisms underlying disease, as in vitro studies do not always provide the natural physiological complexity associated with many diseases. Invertebrate models are relatively inexpensive, easy to work with, have short lifespans, and often have very well characterized and stereotypical development and behavior. This is particularly true for the two invertebrate model organisms that this review will focus on: Caenorhabditis elegans and Drosophila melanogaster. In this review, we will first describe an overview of modeling Alzheimer's disease in flies and worms, and will then highlight some of the more recent advances that these "simple" animals have contributed to our understanding of Alzheimer's disease in recent years.

Automated analysis of courtship suppression learning and memory in Drosophila melanogaster.

Study of the fruit fly, Drosophila melanogaster, has yielded important insights into the underlying molecular mechanisms of learning and memory. Courtship conditioning is a well-established behavioral assay used to study Drosophila learning and memory. Here, we describe the development of software to analyze courtship suppression assay data that correctly identifies normal or abnormal learning and memory traits of individual flies. Development of this automated analysis software will significantly enhance our ability to use this assay in large-scale genetic screens and disease modeling. The software increases the consistency, objectivity, and types of data generated.

TrkB Isoforms Differentially Affect AICD Production through Their Intracellular Functional Domains.

We report that NTRK2, the gene encoding for the TrkB receptor, can regulate APP metabolism, specifically AICD levels. Using the human neuroblastoma cell line SH-SY5Y, we characterized the effect of three TrkB isoforms (FL, SHC, T) on APP metabolism by knockdown and overexpression. We found that TrkB FL increases AICD-mediated transcription and APP levels while it decreases sAPP levels. These effects were mainly mediated by the tyrosine kinase activity of the receptor and partially by the PLC-γ- and SHC-binding sites. The TrkB T truncated isoform did not have significant effects on APP metabolism when transfected by itself, while the TrkB SHC decreased AICD-mediated transcription. TrkB T abolished TrkB FL effects on APP metabolism when cotransfected with it while TrkB SHC cotransfected with TrkB FL still showed increased APP levels. In conclusion, we demonstrated that TrkB isoforms have differential effects on APP metabolism.

Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects.

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aß(40) and Aß(42), the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions.

A streamlined sub-cloning procedure to transfer shRNA from a pSM2 vector to a pGIPZ lentiviral vector.

RNA interference (RNAi) is a widely used molecular biology technique to investigate the importance of specific genes in molecular pathways. Since mammalian cells are equipped with endogenous RNAi processing machinery, it has become common practice to transfect constructs that encode for short hairpin RNAs that are then cleaved to form the active RNAi sequences that bind to target mRNAs. Given the profit potential of this research approach, companies have developed retroviral libraries of shRNA constructs targeting the majority of the human genes. Recent technologic advances have allowed the rapid improvement of the vectors carrying the shRNA constructs while the silencing sequences remain the same. Therefore, sub-cloning of shRNA sequences from more obsolete vectors to newer vectors is a straightforward way to take advantage of newer delivery technologies. We describe here a streamlined procedure to transfer shRNA sequences from the pSM2 retroviral vector to a newer pGIPZ vector that is more stable, contains a GFP cassette and allows the preparation of high titer viral particles for transduction of cells and in vivo use. We demonstrate that our protocol provides a cost-effective and fast method to successfully sub-clone shRNA from a pSM2 retroviral vector to a pGIPZ lentiviral vector making it a useful tool for the investigators that have purchased pSM2 vectors in the past and wish now to upgrade their constructs by inserting them in more versatile vectors.

An emerging role for Ubiquilin 1 in regulating protein quality control system and in disease pathogenesis.

The process of refolding or degrading misfolded proteins is the most important function of protein quality control (PQC) system. An imbalance between the capacity of PQC system and the quantity and severity of misfolded proteins may result in protein aggregate accumulation, which can ultimately contribute to a class of diseases referred to as conformational disorders. Numerous lines of evidence suggest that Ubiquilin 1 is an important component in PQC. Ubiquilin 1 has been indicated to be involved in the pathophysiology of neurodegenerative diseases and cancer. Here we review the evidence that Ubiquilin 1 is an important component of the PQC system and also review the role of Ubiquilin 1 in human diseases.

MicroRNAs can regulate human APP levels.

A number of studies have shown that increased APP levels, resulting from either a genomic locus duplication or alteration in APP regulatory sequences, can lead to development of early-onset dementias, including Alzheimer's disease (AD). Therefore, understanding how APP levels are regulated could provide valuable insight into the genetic basis of AD and illuminate novel therapeutic avenues for AD. Here we test the hypothesis that APP protein levels can be regulated by miRNAs, evolutionarily conserved small noncoding RNA molecules that play an important role in regulating gene expression. Utilizing human cell lines, we demonstrate that miRNAs hsa-mir-106a and hsa-mir-520c bind to their predicted target sequences in the APP 3'UTR and negatively regulate reporter gene expression. Over-expression of these miRNAs, but not control miRNAs, results in translational repression of APP mRNA and significantly reduces APP protein levels. These results are the first to demonstrate that levels of human APP can be regulated by miRNAs.

Therapeutic targeting of the alpha-secretase pathway to treat Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and degenerative disorder pathophysiologically characterized by the accumulation of beta-amyloid peptides (A-beta) in the brain. A-beta is indicated to be the primary agent in the pathogenesis of AD. A-beta is generated from the amyloid precursor protein (APP) via two proteolytic enzymes, beta- and gamma-secretases. Alpha-secretase conducts an alternative proteolytic cleavage that prevents A-beta production and accumulation. Elevating levels of alpha-secretase cleavage, therefore, is a potential therapeutic strategy to treat AD.

In vivo selection for metastasis promoting genes in the mouse.

Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and beta-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositide 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and beta-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.