A fruitful fly forward : the role of the fly in drug discovery for neurodegeneration

AD, Alzheimer’s disease; APP, amyloid precursor protein; BBB, blood brain barrier; GFP, green fluorescent protein; HTS, high-throughput screening; HD, Huntington’s disease; LB, Lewy bodies; PD, Parkinson’s disease; PolyQ, Polyglutamine; RNAi, RNA interference; SNCA, α-synuclein gene; UAS, Upstream Activating Sequence.peer-reviewe

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

Background: Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information.Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels. Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.Fil: Fustiñana, María Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Ariel, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Federman, Maria Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Freudenthal, Ramiro A. M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Romano, Arturo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Apolipoprotein E-Mimetics Inhibit Neurodegeneration and Restore Cognitive Functions in a Transgenic Drosophila Model of Alzheimer's Disease

BACKGROUND: Mutations of the amyloid precursor protein gene (APP) are found in familial forms of Alzheimer's disease (AD) and some lead to the elevated production of amyloid-beta-protein (Abeta). While Abeta has been implicated in the causation of AD, the exact role played by Abeta and its APP precursor are still unclear. PRINCIPAL FINDINGS: In our study, Drosophila melanogaster transgenics were established as a model to analyze AD-like pathology caused by APP overexpression. We demonstrated that age related changes in the levels and pattern of synaptic proteins accompanied progressive neurodegeneration and impairment of cognitive functions in APP transgenic flies, but that these changes may be independent from the generation of Abeta. Using novel peptide mimetics of Apolipoprotein-E, COG112 or COG133 proved to be neuroprotective and significantly improved the learning and memory of APP transgenic flies. CONCLUSIONS: The development of neurodegeneration and cognitive deficits was corrected by injections of COG112 or COG133, novel mimetics of apolipoprotein-E (apoE) with neuroprotective activities

Investigating the spread and toxicity of glycine-alanine dipeptides in C9orf72 ALS/FTD using Drosophila melanogaster

Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The role of repeat size in disease onset and severity in humans remains controversial. Transcripts of the expansions are translated into five dipeptide repeat (DPR) proteins. Most preclinical studies have used relatively short and tagged poly-DPR constructs to investigate DPR-mediated toxicity, and shown that poly-GR, poly-PR and, to a lesser extent, poly-GA DPRs are neurotoxic. Consequently, a major emphasis has been placed on understanding poly-GR- and poly-PR-mediated toxicity. However, poly-GA is the most abundant DPR in patient tissue. Transmission of protein aggregates may be a major driver of toxicity in neurodegeneration. In this study, I show for the first time that only poly-GA DPRs can spread trans-neuronally in vivo using the adult fly brain. Repeat length and tissue age modulate this phenomenon, and exosomes and synaptic vesicles are relevant in the extracellular release of GA DPRs. I also compared the toxicity, aggregation and cellular responses of GA100 DPRs carrying or not commonly used tags. Expression of tagged GA100 was markedly less toxic. GA100 tagged with GFP and mCherry exhibited aggregation differences and failed to cause DNA damage or proteostasis stress compared to untagged GA100 and GA100FLAG. These findings highlight the need to use untagged DPRs as controls when investigating their pathobiology. Finally, I tested the role of repeat size in modulating GA toxicity, subcellular localization, aggregation and cellular responses by comparing these in flies expressing untagged GA100, GA200 and GA400 DPRs. While aggregation propensity and proteostasis stress hold a positive correlation with repeat length, and GA400 was markedly more toxic than GA100, the latter was in turn more toxic than GA200. This highlights a non-linear correlation between repeat length and toxicity. GA100 and GA200 formed numerous puncta-like aggregates both in the soma and axons of neurons and, especially GA200, exhibited spreading, whereas GA400 resided only in somata and did not spread. Surprisingly, GA200 caused more DNA damage than GA100, but this effect was not observed upon GA400 expression. Collectively, I show that GA DPRs have a unique ability to spread in vivo, and their toxicity may have been previously underestimated by the use of short and tagged constructs. Therefore, my data support the further characterization of GA DPRs of a clinically relevant composition to develop strategies with therapeutic potential for C9orf72 mutation carriers

Parkinson Phenotype in Aged PINK1-Deficient Mice Is Accompanied by Progressive Mitochondrial Dysfunction in Absence of Neurodegeneration

Background Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1 -/- mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death

Genetic analysis of dopaminergic neuron survival

Pathological changes in the dopaminergic system account for a number of devastating illnesses including schizophrenia, psychosis, depression, addiction, obsessive compulsive disorder or the most well known Parkinson’s disease (PD). The nigrostriatal pathway is an important component of the dopaminergic (DA) system mediating voluntary movement and originates in the ventral midbrain from where substantia nigra pars compacta (SN) neurons send their axons to the dorsal striatum. Massive loss of SN neurons as seen in PD leads to postural imbalance, rigidity, tremor and bradykinesia, however, the precise mechanisms involved in the maintenance and the demise of SN neurons are poorly understood. Endogenous neurotrophic factors such as the Glial cell line-derived neurotrophic factor (GDNF; signaling via the Ret receptor tyrosine kinase) and Brain-derived neurotrophic factor (BDNF; signaling via the TrkB receptor tyrosine kinase) were reported to have protective and rescuing properties on DA neurons; however, their physiological roles in SN neurons remained unknown. Inactivation of the oxidative stress suppressor DJ-1 causes PD; remarkably, mice lacking DJ-1 function do not display overt SN degeneration, suggesting that additional DJ-1 interactors compensate for loss of DJ-1 function. To begin characterizing the cellular and molecular networks mediating SN neuron survival, I used mouse genetics to investigate the roles and the interaction between GDNF/BDNF-mediated trophic signaling and the DJ-1-mediated stress response in SN neurons. While mice lacking TrkB function specifically in SN neurons display a normal complement of SN neurons up to 24-months, loss of Ret function in DA neurons causes adult-onset and progressive SN degeneration, suggesting that GDNF/Ret signaling is required for long-term maintenance of SN neurons. I then generated and aged mice lacking Ret and DJ-1 and found remarkably that they display an enhanced SN degeneration relative to mice lacking Ret. Thus, DJ-1 promotes survival of Ret-deprived SN neurons. Interestingly, the survival requirement for Ret and DJ-1 is restricted to those SN neurons which express the ion channel GIRK2, project exclusively to the striatum and specifically degenerate in PD. This is the first in vivo evidence for a pro-survival role of DJ-1. To understand how DJ-1 interacts molecularly with Ret signaling, I performed epistasis analysis in Drosophila melanogaster. Although DJ-1 orthologs DJ-1A and DJ-1B are dispensable for fly development, the developmental defects induced by targeting constitutively active Ret to the retina were suppressed in a background of reduced DJ-1A/B function. Moreover, DJ-1A/B interacted genetically with Ras/ERK, but not PI3K/Akt signaling to regulate photoreceptor neuron development. Flies with reduced ERK activity and lacking DJ-1B function had more severe defects in photoreceptor neuron and wing development than flies with reduced ERK function. These observations establish, for the first time, a physiological role for DJ-1B in the intact Drosophila. Our findings suggest that the triple interaction between aging, trophic insufficiency and cellular stress may cause Parkinsonism. Because Ret and DJ-1 show convergence of their pro-survival activities, we predict that striatal delivery of GDNF might be most effective in PD patients carrying DJ-1 mutations. A better understanding of the molecular connections between trophic signaling, cellular stress and aging will accelerate the process of drug development in PD

Nanoparticle toxicity in Drosophila melanogaster: a case study with nickel, nickel oxide, and iron-nickel alloy nanoparticles

With an increase use of nanomaterials, growing concerns have risen about their potential exposure to environment and the risk of side effects on human health. My research investigates the ecological and mechanistic insights of in vivo nanoparticle toxicology via oral exposure, specifically metallic nickel, nickel-iron alloy, and nickel oxide particles, using the Drosophila melanogaster model system. In order to understand the physical and chemical behavior of the nickel-based nanoparticles that were used in this study, I characterized the particle size, morphology, aqueous aggregation state, and hydrodynamic zeta potential of these nanomaterials. I found that these nanomaterials displayed a distinct set of physicochemical properties and that these properties appear to have a significant role in the toxicological effects that I observed. Metallic nickel nanoparticles were toxic to the larvae D. melanogaster, having a dose-dependent mortality, a development delay in pupariation, inhibition of tissue growth, reduction of wing size, as well as the induction of the stress protein Hsp70 and ROS production. I also found unique fluorescent mineral depositions form in the Malpighian tubules after oral exposure to nickel nanoparticles. Energy-dispersive X-ray spectroscopy reveals that the chemical composition of these mineral crystals was calcium carbonate. The experimental evidence of the toxic effects of nickel nanoparticle effect via the oral route provides valuable information of risk and biohazard to the community