Aleister J. Saunders

Role of common and rare APP DNA sequence variants in Alzheimer disease

Objectives: More than 30 different rare mutations, including copy number variants (CNVs), in the amyloid precursor protein gene (APP) cause early-onset familial Alzheimer disease (EOFAD), whereas the contribution of common APP variants to disease risk remains controversial. In this study we systematically assessed the role of both rare and common APP DNA variants in Alzheimer disease (AD) families. Methods: Families with EOFAD genetically linked to the APP region were screened for missense mutations and locus duplications of APP. Further, using genome-wide DNA microarray data, we examined the APP locus for CNVs in a total of 797 additional early- and late-onset AD pedigrees. Finally, 423 single nucleotide polymorphisms (SNPs) in the APP locus, including 2 promoter polymorphisms previously associated with AD risk, were tested in up to 4,200 individuals from multiplex AD families. Results: Analyses of 8 21q21-linked families revealed one family carrying a nonsynonymous mutation in exon 17 (Val717Leu) and another family with a partially penetrant 3.5-Mb locus duplication encompassing APP. CNV analysis in the APP locus revealed an additional family carrying a fully penetrant 380-kb duplication, merely spanning APP. Last, contrary to previous reports, association analyses of more than 400 different SNPs in or near APP failed to show significant effects on AD risk. Conclusion: Our study shows that APP mutations and locus duplications are a very rare cause of EOFAD and that the contribution of common APP variants to AD susceptibility is insignificant. Furthermore, duplications of APP may not be fully penetrant, possibly indicating the existence of hitherto unknown protective genetic factors.

Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

Transgenic models of Alzheimers 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 Aβ40 and Aβ42, 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.

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 Alzheimers 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 Alzheimers disease in recent years.

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

The biggest risk factor for developing Alzheimers 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 β-amyloid (Aβ) 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 (β-site APP cleaving enzyme BACE) specifically in the flys central nervous system. Advantages of our model include the onset of behavioral and neuropathological symptoms later in the flys lifespan due to a gradual accrual of Aβ within the central nervous system (CNS), making age the key factor in the behavioral and neuroanatomical phenotypes that we observe in this model.

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.

Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation

Background: Sterols can alter APP metabolism. Results: Cyclopamine, a phytosterol, alters APP-CTF degradation rate, decreases APP-CTF bioavailability for γ-secretase cleavage, and reduces Aβ and AICD generation. Conclusion: Cyclopamine decreases Aβ and AICD production by altering APP-CTF retrograde trafficking. Significance: Cyclopamine is a novel modulator of APP metabolism and trafficking, which can illuminate new avenues for Alzheimer disease treatment. Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-β (Aβ), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aβ 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 Aβ 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 Aβ 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. Developmental Dynamics 241:553–562, 2012.

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.

Genetic risk factors: their function and comorbidities in Alzheimer's disease

Alzheimers disease (AD) is an epidemiologically complex disorder, in which both genetic and environmental factors play important roles contributing to disease susceptibility. Identification of these risk factors is crucial as they may provide new avenues for the identification of novel disease biomarkers as well as for the design of intervention approaches. Several novel AD susceptibility genes with small risk effects have been recently identified by employing genetic association analyses, in particular by those using a genome-wide approach. For most of the newly identified AD risk factors, however, the biological mechanisms driving these associations remain elusive, emphasizing the need for comprehensive functional characterization of these genes and for determining their relevance for AD pathogenesis. In addition, epidemiological and clinical studies have revealed that certain comorbidities often precede or cooccur with AD. These are often correlated with modifiable life-style factors potentially providing promising alternative routes to be exploited in treatment studies. For this special issue of the International Journal of Alzheimers Disease, we invited investigators to contribute original research and review articles that stimulate efforts to identify novel molecular targets involved in AD pathogenesis. Eventually, we selected to include eight articles on the topic, which we believe to be of particular interest to the readers of the journal. n nThe first set of four studies in this special issue elucidates a number of different genetic aspects of AD. The study by G. Hamilton et al. assessed the impact of recently identified AD genome-wide association signals on cognitive functioning in two birth cohorts from Scotland. Their strongest results implicate a haplotype at the TRAPPC6A locus in individuals lacking the APOEe4 allele. Less-pronounced effects on cognition are also observed for genetic variants in APP and BIN1. The study by L. Polito et al. investigated the potential role of SLC6A4, a serotonin transporter highly expressed in the brain, in contributing to AD risk. While they found nominally significant risk effects in their own case-control sample from Italy, combining these data with those from other groups yields a more ambiguous answer. The study by R. Dominici et al. followed a similar approach investigating the potential effects of DNA sequence variants in G protein-coupled receptor 3 (GPR3) in AD cases and controls from Italy. In agreement with recent genome-wide association studies, they found no evidence that GPR3 is involved in AD epidemiology. Finally,the paper by E. Blom et al. chose a more functionally orientated approach to AD genetics. The authors performed a genome-wide gene expression study in transgenic mouse models of AD, which suggested differences in expression patterns in genes of the Wnt pathway. Validation experiments in human brain samples confirm these findings and suggest that TCF7L2 and MYC show the largest expression differences in AD versus control subjects. n nThe second set of studies focus on comorbidities and life-style factors that are associated with AD. In the paper by V. Leinonen et al., investigators assessed the well-known AD-related biomarkers, such as Aβ42, tau protein, and inflammatory components from the cerebrospinal fluid samples obtained from the patients of idiopathic normal pressure hydrocephalus (iNPH). As AD is the most important differential diagnosis for iNPH, brain biopsy samples obtained from NPH patients provide valuable information on pathogenic events taking place in early phase of AD. The paper by G. Pasinetti et al. summarizes the effects of caloric intake, dietary life-style and macronutrient composition on the risk of AD. More specifically, the investigators discuss how certain cardiovascular and diabetic conditions can induce an increased susceptibility for AD and provide potentialmechanisms through which this may occur. The paper by E. Tuminello et al. provides a comprehensive review related to the hypothesis of apolipoprotein E (protein: apoE; gene: APOE) antagonistic pleiotropy. The leading hypothesis is that the APOEe4 allele may be beneficial in earlier ages, while it leads to cognitive decline later in life. Finally, the last paper by V. Leduc et al. highlights the pleiotropic roles and the structure-function relationship of apoE particularly in the lipid homeostasis-related events in AD and cardiovascular diseases. n nTaken together, we believe that the articles included in this special issue of the International Journal of Alzheimers Disease shed new light on a number of different aspects of AD pathogenesis. In concert with the work from hundreds of other AD laboratories worldwide, these exciting new data will hopefully translate into the identification of novel biomarkers and the development of new therapeutic strategies to efficiently diagnose and treat this devastating disorder. n n nMikko Hiltunen n nLars Bertram n nAleister J. Saunders