Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

Protein misfolding neurodegenerative diseases arisethrough neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding--induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion--like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion--induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease.     

Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila

Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.     

Dietary restriction in Drosophila

The fruit fly Drosophila is a useful organism for the investigation of the mechanisms by which dietary restriction (DR) extends lifespan. Its relatively short generation time, well-characterised molecular biology, genetics and physiology and ease of handling for demographic analysis are all major strengths. Lifespan has been extended by DR applied to adult Drosophila, by restriction of the availability of live yeast or by co-ordinate dilution of the whole food medium. Lifespan increases to a maximum through DR with a progressive dilution of the food and then decreases through starvation as the food is diluted further. Daily and lifetime fecundities of females are reduced by food dilution throughout the DR and starvation range. Standard Drosophila food ingredients differ greatly between laboratories and fly stocks can differ in their responses to food dilution, and a full range of food concentrations should therefore be investigated when examining the response to DR. Flies do not alter the time that they spend feeding in response to DR. Both mean and maximum lifespan are extended by DR. The nutrients critical for the response to DR in Drosophila require definition. The extension of lifespan in response to DR is very much greater in females than in males. Two nutrient-sensing pathways, the insulin/IGF-like and TOR pathways, have been implicated in mediating this response of lifespan to DR in Drosophila, as have two protein deacetylases, dSir2 and Rpd3, although the precise nature of this interaction remain to be characterised. Although female fecundity is reduced by DR, the response of lifespan to DR appears normal in sterile females, possibly implying that reduced fecundity is not necessary for extension of lifespan by DR. There is no reduction in metabolic rate or in the rate of generation of superoxide and hydrogen peroxide from isolated mitochondria in response to DR. DR acts acutely and rapidly (within 48 h) to reduce the mortality of flies that are fully fed to the level found in animals exposed to DR throughout life. This rapid mortality rate recovery provides a powerful framework within which to further investigate the mechanisms by which DR extends lifespan.     

V180I mutation of the prion protein gene associated with atypical PrP glycosylation

A valine to isoleucine mutation at residue 180 was identified in a French patient with Creutzfeldt-Jakob disease (CJD). The mutation is located in the close vicinity of one of the two N-glycosylation sites of the cellular prion protein (PrP^C). Western blot analysis revealed accumulation in the brain of the pathogenic proteinase K-resistant PrP (PrP^Sc) isoform with the notable absence of the diglycosylated band. The mutant protein expressed in CHO cells was correctly glycosylated, suggesting that the atypical glycosylation pattern of PrP^Sc was not due to the mutation at position 180. These results suggest that the diglycosylated form of the mutant PrP^180I prevents its conversion into the pathogenic mutant form PrP^Sc180I, supporting a central role of N-linked glycan chains in the PrP conversion process.     

PrPSc level and incubation time in a transgenic mouse model expressing Borna disease virus phosphoprotein after intracerebral prion infection

Our previous studies have shown that the persistent expression of Borna disease virus phosphoprotein (BDV P) in mice leads to behavioral abnormalities resembling those in BDV-infected animals. In this study, we investigated whether the neurobehavioral abnormalities genetically induced by BDV P influence experimental prion disease. The effect of the phosphoprotein on prion diseases was evaluated based on the incubation time and survival curve, as well as the abnormal isoform of prion protein (PrP(Sc)) levels in brains of BDV P Tg mice treated with proteinase K (PK) treatment and subjected to western blotting. Increased expression of the BDV P transgene had no effect on the PrP(Sc) level, incubation time, or survival curve. The abnormalities induced by BDV P are different from those induced by prion diseases, indicating that the signaling cascades induced by the phosphoprotein differ from those induced by prion diseases.     

Restriction of amino acids extends lifespan in Drosophila melanogaster

Dietary restriction extends adult Drosophila melanogaster life span when the concentration of dietary yeast is diluted in a media with abundant carbohydrates. Here we vary the concentration of casein as a source of amino acids in adult diet to uncover a quality of nutrient yeast responsible for longevity control. Longevity is maximized upon diet with intermediary levels of casein. Differences in survival are not caused by elevated age-independent mortality; the longevity maximum at intermediate casein does not arise because casein is non-specifically harmful at higher concentrations. Furthermore, fecundity increases when the level of dietary casein is elevated. The demographic phenotypes of adult Drosophila maintained on intermediate levels of casein resemble their response to limited dietary yeast. Dietary restriction through dilution of yeast may extend longevity because this limits the intake of amino acids.     

Long-lived genotypes for studies of life extension in Drosophila melanogaster

Numerous single-gene mutations obtained by insertion of P elements in white (w) genetic backgrounds have been reported to extend the life span of Drosophila melanogaster, but life extension is sometimes observed only in relatively short-lived backgrounds. The objective of this study was to develop long- and short-lived, high and low fertility backgrounds in which to test the reproducibility and possible additivity of effects of prospective life-extending treatments. Flies previously reported to be long- or short-lived, following artificial selection for early or delayed reproduction and inbreeding, were rendered essentially isogenic, and a w visible marker was introduced. Isogeny adversely affected both life span and fertility, but w had little or no effect on either trait. Unexpectedly, none of these lines or a stock under uninterrupted selection for delayed reproduction lived any longer than an unselected, highly fertile y w strain used in earlier studies of longevity. Strains derived from one artificial selection experiment were found to contain functional P elements, as did the two longest-lived genotypes in this study, which were inbred without artificial selection. The y w background appears to be at least equally as long-lived as any other currently available for tests of life extension by P{w⁺} mutations.     

L-arginine enhances immunity to parasitoids in Drosophila melanogaster and increases NO production in lamellocytes

Drosophila melanogaster was used as a model system to explore the link between nutrition and immunity, and to investigate the role of nitric oxide (NO) in enhancing immunity following dietary enhancement with l-arginine. First, we show that adding l-arginine to the food medium increases the ability of D. melanogaster larvae to encapsulate the eggs of the parasitoid Asobara tabida. Secondly, we show that the increase in immunity is specific to l-arginine, and not to an enhanced calorific content, and that immunity decreases when larvae are fed food with added l-NAME, an inhibitor of nitric oxide synthase. Finally, we show that parasitised larvae fed l-arginine have increased haemocyte numbers, and that the lamellocytes (haemocytes which play a key role in encapsulation) show evidence of an increased production of NO. These results suggest that NO plays a key role in immunity and that the effect of NO is mostly targeted via the lamellocytes.     

c-Abl tyrosine kinase modulates tau pathology and Cdk5 phosphorylation in AD transgenic mice

The c-Abl tyrosine kinase is an important link in signal transduction pathways that promote cytoskeletal rearrangement and apoptotic signalling. We have previously shown that amyloid-β-peptide (Aβ) activates c-Abl. Herein we show that c-Abl participates in Aβ-induced tau phosphorylation through Cdk5 activation. We found that intraperitoneal administration of STI571, a specific inhibitor for c-Abl kinase, decreased tau phosphorylation in the APPswe/PSEN1ΔE9 transgenic mouse brain. In addition, when neurons were treated with Aβ we observed: (i) an increase in active c-Abl and tau phosphorylation, (ii) the prevention of tau phosphorylation by STI571 and (iii) the inhibition of c-Abl expression by shRNA, as well as the expression of a c-Abl kinase death mutant, decreased AT8 and PHF1 signals. Furthermore, the increase of c-Abl was associated with Tyr15 phosphorylation of Cdk5 and its association with c-Abl. Brains from APPswe/PSEN1ΔE9 mice showed higher levels of c-Abl and phospho-Cdk5 than wild-type mice. Moreover, STI571 treatment decreased the phospho-Cdk5 levels. Together, the evidence suggests that activation of c-Abl by Aβ promotes tau phosphorylation through Tyr15 phosphorylation-mediated Cdk5 activation.     

Structural and functional characterization of pseudopodocyte, a shaggy immune cell produced by two Drosophila species of the obscura group

We recently reported that most of the Drosophila species of the obscura group were unable to mount cellular capsules and no lamellocyte was ever found in the hemolymph of any of the tested species. Only three species were able to encapsulate, despite lacking lamellocytes. Their encapsulation ability was always associated with the presence of an unpreviously described kind of capsule-forming immunocytes designated as “atypical hemocytes”. Here, we describe the ultrastructural and functional characteristics of this type of hemocyte. We show that these cells share many ultrastructural and morphological features with Drosophila melanogaster plasmatocytes, although they are involved in the formation of the external layers of the cellular capsule, a functional property exhibited by lamellocytes in D. melanogaster. Due to the high number of pseudopodes in these cells, we suggest to name them “pseudopodocytes”. After structural and functional characterization of these atypical hemocytes, their ambiguous status between plasmatocytes and lamellocytes is discussed.     

Regulatory sequences of the PRNP gene influence susceptibility to sporadic Creutzfeldt-Jakob disease

The prion diseases are fatal neurodegenerative disorders that afflict both humans and animals. They comprise kuru, Creutzfeldt-Jakob disease (CJD), Gerstmman-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). Both GSS, FFI and approximately 10% of CJD cases are genetically linked disorders, whereas 90% of CJD cases are not associated with mutations in the PRNP coding region, therefore other factors must be involved in pathogenesis of these forms of CJD. There is strong evidence that in transgenic mice the level of PrP gene expression influences the initiation and progression of the prion diseases. Moreover, in in vitro experiments demonstrated that mutations in the regulatory region of PRNP gene altered gene expression, therefore it may be expected that PrP expression level influences the susceptibility to CJD. In order to investigate whether single nucleotide polymorphisms within regulatory region of PRNP may modulate genetic susceptibility to sporadic CJD we examined an association of the C/G polymorphism at position -101 with the sCJD. In our study -101G polymorphism is over-represented among sCJD PRNP codon 129M/V cases compared with the control group. Our data suggest that polymorphism at position -101 in the regulatory region of PRNP may be a risk factor for sCJD among codon 129 heterozygotes.     

The effects of larval density on adult life-history traits in three species of Drosophila

There is evidence that longevity and starvation resistance are determined by a common genetic mechanism. Starvation resistance in Drosophila strongly correlates with both fat content and longevity, and is affected by density during rearing. In this study, we examine how three species, Drosophila melanogaster, Drosophila ananassae and Drosophila willistoni, respond to three larval density treatments. Starvation resistance after adult eclosion, and after 2 days of feeding, and longevity were examined in each sex. D. willistoni reacted differently to larval density than the other two species. This species showed an effect of density on longevity whilst D. ananassae and D. melanogaster showed no such effects. The results also indicate that starvation resistance is not solely determined by fat content. Resistance to starvation at two time points after eclosion differed among species. This may reflect differences in resource acquisition and allocation, and we discuss our findings in relation to how selection may operate in the different species.     

The effects of exogenous antioxidants on lifespan and oxidative stress resistance in Drosophila melanogaster

We used the fruit fly Drosophila melanogaster to test the effects of feeding the superoxide dismutase (SOD) mimetic drugs Euk-8 and -134 and the mitochondria-targeted mitoquinone (MitoQ) on lifespan and oxidative stress resistance of wild type and SOD-deficient flies. Our results reaffirm the findings by other workers that exogenous antioxidant can rescue pathology associated with compromised defences to oxidative stress, but fail to extend the lifespan of normal, wild type animals. All three drugs showed a dose-dependent increase in toxicity in wild type flies, an effect that was exacerbated in the presence of the redox-cycling drug paraquat. However, important findings from this study were that in SOD-deficient flies, where the antioxidant drugs increased lifespan, the effects were sex-specific and, for either sex, the effects were also variable depending on (1) the stage of development from which the drugs were given, and (2) the magnitude of the dose. These findings place significant constraints on the role of oxidative stress in normal ageing.     

Misexpression screen delineates novel genes controlling Drosophila lifespan

In an initial preliminary screen we identified factors associated with controlling Drosophila aging by examining longevity in adults where EP elements induced over-expression or antisense-RNA at genes adjacent to each insertion. Here, we study 45 EP lines that initially showed at least 10% longer mean lifespan than controls. These 45 lines and a daughterless (da)-Gal4 stock were isogenized into a CS10 wild-type background. Sixteen EP lines corresponding to 15 genes significantly extended lifespan when their target genes were driven by da-Gal4. In each case, the target genes were seen to be over-expressed. Independently derived UAS-gene transgenic stocks were available or made for two candidates: ImpL2 which is ecdysone-inducible gene L2, and CG33138, 1,4-alpha-glucan branching enzyme. With both, adult lifespan was increased upon over-expression via the GeneSwitch inducible Gal4 driver system. Several genes in this set of 15 correspond to previously discovered longevity assurance systems such as insulin/IGF-1 signaling, gene silencing, and autophagy; others suggest new potential mechanisms for the control of aging including mRNA synthesis and maturation, intracellular vesicle trafficking, and neuroendocrine regulation.     

Systemic and oral immunogenicity of hemagglutinin protein of rinderpest virus expressed by transgenic peanut plants in a mouse model

Rinderpest causes a devastating disease, often fatal, in wild and domestic ruminants. It has been eradicated successfully using a live, attenuated vaccine from most part of the world leaving a few foci of disease in parts of Africa, the Middle East, and South Asia. We have developed transgenic peanut (Arachis hypogaea L.) plants expressing hemagglutinin (H) protein of rinderpest virus (RPV), which is antigenically authentic. In this work, we have evaluated the immunogenicity of peanut-expressed H protein using mouse model, administered parenterally as well as orally. Intraperitoneal immunization of mice with the transgenic peanut extract elicited antibody response specific to H. These antibodies neutralized virus infectivity in vitro. Oral immunization of mice with transgenic peanut induced H-specific serum IgG and IgA antibodies. The systemic and oral immunogenicity of plant-derived H in absence of any adjuvant indicates the potential of edible vaccine for rinderpest.     

Effects of resveratrol on lifespan in Drosophila melanogaster and Caenorhabditis elegans

It was recently reported that the plant polyphenol resveratrol, found, e.g., in grape berry skins, extended lifespan in the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans. This lifespan extension was dependent on an NAD(+)-dependent histone deacetylase, Sir2 in Drosophila and SIR-2.1 in C. elegans. The extension of lifespan appeared to occur through a mechanism related to dietary restriction (DR), the reduction of available nutrients without causing malnutrition, an intervention that extends lifespan in diverse organisms from yeast to mammals. In Drosophila, lifespan extension by DR is associated with a reduction in fecundity. However, a slight increase in fecundity was reported upon treatment with resveratrol, suggesting a mode of action at least partially distinct from that of DR. To probe this mechanism further, we initiated a new study of the effects of resveratrol on Drosophila. We saw no significant effects on lifespan in seven independent trials. We analysed our resveratrol and found that its structure was normal, with no oxidative modifications. We therefore re-tested the effects of resveratrol in C. elegans, in both wild-type and sir-2.1 mutant worms. The results were variable, with resveratrol treatment resulting in slight increases in lifespan in some trials but not others, in both wild type and sir-2.1 mutant animals. We postulate that the effect of resveratrol upon lifespan in C. elegans could reflect induction of phase 2 drug detoxification or activation of AMP kinase.     

Genetic variation for life span, resistance to paraquat, and spontaneous activity in unselected populations of Drosophila melanogaster: implications for transgenic rescue of life span

Genetic variation in adult life span, resistance to paraquat, resistance to DDT, and spontaneous flying activity were measured in 138 recombinant inbred lines of Drosophila melanogaster. We find that the phenotypic correlation between life span and resistance to an exogenous oxidizing agent is positive, though weak, and that there is little correlation between the two traits at the level of quantitative trait loci (QTLs). The sign of the life span-resistance correlation is haplotype-specific, suggesting a high degree of statistical interaction and dependence on genetic background. Because of the genotype-specificity in the relationship between life span and resistance phenotypes, interventions to extend life span by overexpression of antioxidant enzymes are likely to produce strain-specific results. These observations are in general agreement with the "genetic rescue" hypothesis of Sohal et al. [Sohal, R.S., Mockett, R.J., Orr, W.C., 2002. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic. Biol. Med. 33, 575-586.], though we emphasize that such statistical interaction is a normal feature of standing genetic variation, and does not imply that some genotypes are pathological. Ad hoc observation of spontaneous flying activity 5 days after emergence proved to be a much better predictor of life span than resistance to an exogenous oxidant in these populations.     

Infection with the wMel and wMelPop strains of Wolbachia leads to higher levels of melanization in the hemolymph of Drosophila melanogaster, Drosophila simulans and Aedes aegypti

Introduction of the life-shortening strain of Wolbachia pipientis, wMelPop, into the key dengue vector, Aedes aegypti, and the anti-pathogen effects in Wolbachia-infected hosts highlights the need for more research into its interactions with its original host, Drosophila melanogaster, and the novel mosquito host. The visual difference in darkness between the eggs of wMelPop Wolbachia-infected and uninfected mosquito hosts after egg deposition led to further investigation into melanization levels of the insects. Both D. melanogaster and A. aegypti infected with wMelPop showed increased levels of melanization, especially in females. This result was also seen in D. melanogaster and Drosophila simulans infected with the closely related wMel strain. D. simulans infected with other strains of Wolbachia did not display this difference. HPLC analysis of hemolymph from mosquitoes showed that this difference was not due to dopamine levels in the host as they were no different in wMelPop-infected and control mosquitoes before or after blood feeding.