Transgenic Caenorhabditis Elegans Strain Research Articles

Synthesis, biological evaluation and molecular dynamic simulations of novel Benzofuran-tetrazole derivatives as potential agents against Alzheimer’s disease

A series of novel Benzofuran-tetrazole derivatives were successfully synthesised by integrating multicomponent Ugi-azide reaction with the molecular hybridization approach. Interestingly, a number of synthesized derivatives (5c, 5d, 5i, 5l, 5q and 5s) exhibited significant reduction of aggregation of “human” amyloid beta peptide, expressing on transgenic Caenorhabditis elegans (C. elegans) strain CL4176. Further, in silico docking results have evidenced the exquisite interaction of active compounds with the help of TcAChE–E2020 complex. These findings underscore the potential of these hybrids as lead molecules against Alzheimers’s disease.

Betaine reduces \u03b2-amyloid-induced paralysis through activation of cystathionine-\u03b2-synthase in an Alzheimer model of Caenorhabditis elegans

BackgroundThe neurodegenerative disorder Alzheimer’s disease is caused by the accumulation of toxic aggregates of β-amyloid in the human brain. On the one hand, hyperhomocysteinemia has been shown to be a risk factor for cognitive decline in Alzheimer’s disease. On the other hand, betaine has been demonstrated to attenuate Alzheimer-like pathological changes induced by homocysteine. It is reasonable to conclude that this is due to triggering the remethylation pathway mediated by betaine-homocysteine-methyltransferase. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, to test whether betaine is able to reduce β-amyloid-induced paralysis in C. elegans. This model expresses human β-amyloid 1–42 under control of a muscle-specific promoter that leads to progressive, age-dependent paralysis in the nematodes.ResultsBetaine at a concentration of 100 μM was able to reduce homocysteine levels in the presence and absence of 1 mM homocysteine. Simultaneously, betaine both reduced normal paralysis rates in the absence of homocysteine and increased paralysis rates triggered by addition of homocysteine. Knockdown of cystathionine-β-synthase using RNA interference both increased homocysteine levels and paralysis. Additionally, it prevented the reducing effects of betaine on homocysteine levels and paralysis.ConclusionOur studies show that betaine is able to reduce homocysteine levels and β-amyloid-induced toxicity in a C. elegans model for Alzheimer’s disease. This effect is independent of the remethylation pathway but requires the transsulfuration pathway mediated by cystathionine-β-synthase.

Multifunctional Analogs of Kynurenic Acid for the Treatment of Alzheimer's Disease: Synthesis, Pharmacology, and Molecular Modeling Studies.

We report the synthesis and pharmacological investigation of analogs of the endogenous molecule kynurenic acid (KYNA) as multifunctional agents for the treatment of Alzheimer's disease (AD). Synthesized KYNA analogs were tested for their N-methyl-d-aspartate (NMDA) receptor binding, mGluR5 binding and function, acetylcholinesterase (AChE) inhibition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, interference with the amyloid β peptide (Aβ) fibrillation process, and protection against Aβ-induced toxicity in transgenic Caenorhabditis elegans strain GMC101 expressing full-length Aβ42. Molecular modeling studies were also performed to predict the binding modes of most active compounds with NMDAR, mGluR5, and Aβ42. Among the synthesized analogs, 3c, 5b, and 5c emerged as multifunctional compounds that act via multiple anti-AD mechanisms including AChE inhibition, free radical scavenging, NMDA receptor binding, mGluR5 binding, inhibition of Aβ42 fibril formation, and disassembly of preformed Aβ42 fibrils. Interestingly, 5c showed protection against Aβ42-induced toxicity in transgenic C. elegans strain GMC101. Moreover, 5b and 5c displayed high permeability in an MDR1-MDCKII cell-based model of the blood-brain barrier (BBB). Compound 3b emerged with specific activity as a micromolar AChE inhibitor, however it had low permeability in the BBB model. This study highlights the opportunities that exist to develop analogs of endogenous molecules from the kynurenine pathway for therapeutic uses.

The protection of novel 2-arylethenylquinoline derivatives against impairment of associative learning memory induced by neural Aβ in C. elegans Alzheimer's disease model.

Cerebral deposition of amyloid β-peptide (Aβ), a fundamental feature of Alzheimer's disease (AD), damages the neurocytes and impairs the cognition functions and associative learning memory of AD patients. A series of novel 2-arylethenylquinoline derivatives were synthesized and evaluated in our previous study, which inhibited Aβ aggregation in vitro effectively at the concentration of 20μmol/L and exhibited high antioxidant activity. In order to verify the capacity of anti-AD in vivo, the transgenic Caenorhabditis elegans (C. elegans) strain CL2355 expressing neural Aβ was employed as the AD model to investigate the neuroprotective activity of seven high-potential compounds (4a1, 4a2, 4b1, 4b2, 4c1, 4c2, 4c3) selected from those derivatives. Learning memory associated chemotaxis assay was performed to evaluate the neural repairment capacity. The underlying mechanism was investigated by mRNA analysis of Aβ gene and heat shock protein genes (hsp-16.1 and hsp-16.2) and Western blot of Aβ. Our data indicated that among seven tested compound, 4b1 and 4c2 reduced Aβ-induced stress, suppressed the expression of neural Aβ monomers and toxic oligomers, and recovered the damaged associative learning memory in C. elegans AD model. These findings further confirmed their potentials to become valuable agents for AD therapy.

Epigallocatechin-3-gallate and related phenol compounds redirect the amyloidogenic aggregation pathway of ataxin-3 towards non-toxic aggregates and prevent toxicity in neural cells and Caenorhabditis elegans animal model.

The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e. (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analysed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compounds prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.

Inhibition of Aβ Amyloid Growth and Toxicity by Silybins: The Crucial Role of Stereochemistry.

The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.

Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins

The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: β2-microglobulin and Aβ peptide3–42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm’s life cycle (LC50 survival, 0.9 µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer’s disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a “fragile population”, where an underlying pathologic state may amplify their toxicity.

The protein arginine methyltransferase PRMT5 regulates Aβ-induced toxicity in human cells and Caenorhabditis elegans models of Alzheimer's disease.

The protein arginine methyltransferase 5 (PRMT5) controls cell growth and apoptosis by catalyzing mono and symmetric dimethylation of arginine residues. In human brain tissue, PRMT5 is predominantly expressed in neuronal cells. There is evidence that PRMT5 provides protection against cell death, but the impact of PRMT5 on neuronal apoptosis during the evolution of Alzheimer's disease has not been tested. In the present study, we show that PRMT5 is down-regulated by β-amyloid (Aβ) in primary neurons and SH-SY5Y cells, and this is associated with the up-regulation of the PRMT5 target protein E2F-1. Furthermore, knockdown of PRMT5 in SH-SY5Y cells over-expressing the Swedish mutant form of human amyloid-β precursor protein caused activation of E2F-1/p53/Bax, NF-κB, and GSK-3β pathways, which coincided with increased apoptosis. Co-depletion of E2F-1 reduced the activation of p53/Bax, NF-κB, and GSK-3β, and limited cell apoptosis. In addition, inhibiting NF-κB and GSK-3β activity by specific inhibitors also attenuated cell apoptosis, suggesting that E2F-1/NF-κB/GSK-3β pathways mediate for apoptosis induced by PRMT5 depletion. More importantly, knockdown of PRMT5 resulted in more paralysis in a transgenic Caenorhabditis elegans strain CL2006, indicating that PRMT5 provides protection against Aβ toxicity in vivo. Collectively, our findings identify PRMT5 as a novel regulator of Aβ toxicity and suggest that strategies aimed at activating PRMT5 in the neuron may represent a potential therapeutic approach for the prevention of Alzheimer's disease. We propose the following cascade for protein arginine methyltransferase 5 (PRMT5)-mediated neuronal death: amyloid beta (Aβ) deposition decreases PRMT5 expression in neurons, which increases E2F-1 expression - a PRMT5 target protein - and subsequently activates GSK-3β and NF-κB to induce caspase-3-dependent neuronal apoptosis. These findings might provide a strategy for the treatment of Alzheimer's disease.

Resveratrol reduces amyloid-beta (Aβ₁₋₄₂)-induced paralysis through targeting proteostasis in an Alzheimer model of Caenorhabditis elegans.

Resveratrol is a polyphenol present in red wine for which the capability of directly interfering with the hallmark of Alzheimer's disease (AD), i.e. toxic β-amyloid protein (Aβ) aggregation, has been shown recently. Since the stimulation of proteostasis could explain reduced Aβ-aggregation, we searched for proteostasis targets of resveratrol. The transgenic Caenorhabditis elegans strain CL2006, expressing Aβ1-42 under control of a muscle-specific promoter and responding to Aβ-toxicity with paralysis, was used as a model. Target identification was accomplished through specific knockdowns of proteostasis genes by RNA interference. Effects of resveratrol on protein aggregation were identified using ProteoStat(®) Detection Reagent, and activation of proteasomal degradation by resveratrol was finally proven using a specific fluorogenic peptide substrate. Resveratrol at a concentration of 100 µM caused a 40 % decrease in paralysis. UBL-5 involved in unfolded protein response (UPR) in mitochondria proved to be necessary for the prevention of Aβ-toxicity by resveratrol. Also XBP-1, which represents an endoplasmic reticulum-resident factor involved in UPR, was identified to be necessary for the effects of resveratrol. Regarding protein degradation pathways, the inhibition of macroautophagy and chaperone-mediated autophagy prevented resveratrol from reducing paralysis as did the inhibition of proteasomal degradation. Finally, resveratrol reduced the amount of lysosomes, suggesting increased flux of proteins through the autophagy pathways and activated proteasomal degradation. Resveratrol reduces the Aβ-induced toxicity in a C. elegans model of AD by targeting specific proteins involved in proteostasis and thereby reduces the amount of aggregated Aβ.

Epigallocatechin-3-gallate and tetracycline differently affect ataxin-3 fibrillogenesis and reduce toxicity in spinocerebellar ataxia type 3 model.

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid β-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in β-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

Amyloid-beta (Aβ1–42)-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply

Alzheimer’ disease is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid β (Aβ). This process is influenced through supply of cholesterol via apolipoproteins to neurons. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, which expresses Aβ1–42 under control of a muscle-specific promoter, to test the effects of the apolipoprotein B homologue vitellogenin-6 on paralysis. Knockdown of vitellogenin-6 using RNA-interference (RNAi) recently was shown to significantly reduce cholesterol absorption in C. elegans, and both, RNAi for vitellogenin-6 or lowering the cholesterol concentration in the medium was associated with reduced Aβ-aggregation and paralysis in the nematodes. The effects of both interventions are mediated through the inhibition of the steroidal-signaling pathway since knockdown of its key factors DAF-9 or DAF-12 reduced paralysis independent of the cholesterol concentration and without additive effects by vitellogenin-6 RNAi. Double-RNAi for daf-12 and the downstream target of insulin-signaling, the foxo transcription factor daf-16, revealed that the paralysis-triggering effects of daf-16 RNAi were dominant over the preventive effects of daf-12 RNAi. Identical observations were made when the transcriptional co-activators of DAF-16, ftt-2 or par-5 were knocked down instead of daf-16. In conclusion, interactions between the steroidal and insulin-signaling pathways were identified in Aβ1–42 expressing CL2006, where cholesterol deprivation inhibits steroidal-signaling and thereby activates DAF-16-signaling. Those effects were associated with a reduced Alzheimer phenotype in the nematodes, i.e. reduced protein aggregation and paralysis.

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

A Cocoa Peptide Protects Caenorhabditis elegans from Oxidative Stress and β-Amyloid Peptide Toxicity

BackgroundCocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases.Methodology/Principal FindingsA bioactive peptide, 13L (DNYDNSAGKWWVT), was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP) was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ1–42 peptide as a pre-clinical in vivo model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL) showed the highest antioxidant activity (P≤0.001) in the wild-type strain (N2). Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24–47 h period after Aβ1–42 peptide induction (P≤0.0001). This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide.Conclusions/SignificanceThese findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans

Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.

A novel actin binding site of myosin required for effective muscle contraction

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Swietenia macrophylla extract promotes the ability of Caenorhabditis elegans to survive Pseudomonas aeruginosa infection

Ethnopharmacological relevanceSwietenia macrophylla or commonly known as big leaf mahogany, has been traditionally used as an antibacterial and antifungal agent. Aim of the studyThe unwanted problem of antibiotic resistance in many bacterial species advocates the need for the discovery of the new anti-infective drugs. Here, we investigated the anti-infective properties of Swietenia macrophylla with an assay involving lethal infection of Caenorhabditis elegans with the opportunistic human pathogen Pseudomonas aeruginosa. Materials and methodsUsing a slow killing assay, Caenorhabditis elegans was challenged with an infective strain of Pseudomonas aeruginosa (PA14). The ability of Swietenia macrophylla seed ethyl acetate extract to promote the survival of infected worms was assessed by comparing the percentage of survival between extract treated and non-treated worm populations. The effect of Swietenia macrophylla towards PA14 growth, Caenorhabditis elegans feeding rate and degree of PA14 colonization in the worm gut was also evaluated. Lastly, using a fluorescent transgenic Caenorhabditis elegans strain and real time PCR, the effect of Swietenia macrophylla on the expression of lys-7, an immune response gene was also investigated. ResultsOur results demonstrate the ability of Swietenia macrophylla seed ethyl acetate extract in rescuing Caenorhabditis elegans from fatal PA14 infection. Consequently, we showed that the extract promotes the survival without exhibiting any bactericidal effect or perturbation of Caenorhabditis elegans feeding rate. We also showed that Swietenia macrophylla was able to restore the initially repressed lys-7 level in PA14 infected Caenorhabditis elegans. ConclusionSwietenia macrophylla extract is able to enhance the ability of Caenorhabditis elegans to survive PA14 infection without directly killing the pathogen. We further showed that the extract boosted the expression of a gene pivotal for innate immunity in Caenorhabditis elegans. Collectively, these findings strongly suggest the presence of compounds within Swietenia macrophylla seed that either reduces Pseudomonas aeruginosa virulence and/or enhance host resistance.

A Caenorhabditis elegans model of tau hyperphosphorylation: Induction of developmental defects by transgenic overexpression of Alzheimer's disease-like modified tau

The microtubule-associated tau proteins become functionally and structurally altered in Alzheimer's disease (AD). To analyze tau modification and its role in a non-vertebrate animal model, we produced transgenic Caenorhabditis elegans strains with a panneuronal expression of human tau and a pseudohyperphosphorylated (PHP) tau construct that mimics AD-relevant tau modification. We show that human tau in C. elegans becomes highly phosphorylated and exhibits conformational changes similar to PHP tau and human PHF tau. Both, wt tau and PHP tau induced a progressive age-dependent development of a phenotype of uncoordinated locomotion (unc) in the absence of neuronal degeneration. However, only PHP tau induced a defective pattern of motor neuron development as indicated by the presence of gaps in the dorsal cord, commissures on the wrong side and local broadening of axons. The data indicate that C. elegans is capable of highly phosphorylating human tau to an AD-like state whereas only stable disease-like tau modification induce developmental defects suggesting a specific interference of pathologic tau with intracellular mechanisms of axonal outgrowth and pathfinding.

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation.

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.

Construction and evaluation of a transgenic hsp16‐GFP‐lacZ Caenorhabditis elegans strain for environmental monitoring

A novel integrated transgenic Caenorhabditis elegans strain (PC161) incorporates a double reporter construct with green fluorescent protein (GFP) and lacZ genes fused in-frame into the second exon of the hsp16-1 gene. This construct also includes the Simian Virus 40 (SV40) nuclear localization signal such that the fusion protein accumulates in the nuclei of expressing cells. The PC161 strain was used to monitor the effects of several known stressors, including heat, cadmium, and microwave radiation. The time course of induction was similar for both reporters but was strongly influenced by pretreatment conditions. The PC161 worms kept at 15 degrees C beforehand showed a steady increase in reporter expression (up to at least 16 h) when heated to 30 degrees C. However, if washed on ice prior to heat stress at 30 degrees C, PC161 worms showed a much steeper rise in reporter expression, reaching a maximum after 2.5 h and then plateauing. Heat shock induced strong expression of both reporter genes in all tissues apart from the germ line and early embryos. A highly significant linear dose-response relationship was observed for both transgenes with increasing cadmium concentrations (5-100 microg/ml). Prolonged exposure to microwave radiation (750 MHz and 0.5 W for 16 h) also induced expression of both transgenes at 25 and (to some extent) 27 degrees C, but only beta-galactosidase activity was detectable at 23 degrees C, and neither reporter was detectably expressed at 21 degrees C. Throughout all exposures, the lacZ reporter product was more readily detectable than coexpressed GFP. However, the GFP reporter affords opportunities to monitor the stress response in living worms.

Oxidative stress causes abnormal accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in transgenic Caenorhabditis elegans.

Mutations in the Cu/Zn superoxide dismutase (SOD1) genes are present in approximately 20% of families suffering from familial amyotrophic lateral sclerosis (FALS). Results from several transgenic studies in which FALS-related SOD1 mutations have been expressed have suggested that mutant SOD1 proteins induce cytotoxicity through a toxic gain of function, although the specific mechanism of this has not been fully clarified. To investigate the mechanism of toxicity induced by the mutant SOD1 associated with FALS, we generated transgenic Caenorhabditis elegans strains that contain wild-type and mutant human A4V, G37R and G93A SOD1 recombinant plasmids. The transgenic strains expressing mutant human SOD1 showed greater vulnerability to oxidative stress induced by 0.2 mM paraquat than a control that contained the wild-type human SOD1. In the absence of oxidative stress, mutant human SOD1 proteins were degraded more rapidly than the wild-type human SOD1 protein in C.elegans. In the presence of oxidative stress, however, this rapid degradation was inhibited, and the transgenic C.elegans co-expressing mutant human SOD1 and green fluorescent proteins (GFPs) in muscle tissues demonstrated discrete aggregates in the adult stage. These results suggest that oxidative damage inhibits the degradation of FALS-related mutant human SOD1 proteins, resulting in an aberrant accumulation of mutant proteins that might contribute to the cytotoxicity.