BackgroundThe definitive indicator of Alzheimer’s disease (AD) pathology is the profuse accumulation of amyloid-ß (Aß) within the brain. Various in vitro and cell-based models have been proposed for high throughput drug screening for potential therapeutic benefit in diseases of protein misfolding. Caenorhabditis elegans offers a convenient in vivo system for examination of Aß accumulation and toxicity in a complex multicellular organism. Ease of culturing and a short life cycle make this animal model well suited to rapid screening of candidate compounds.ResultsWe have generated a new transgenic strain of C. elegans that expresses full length Aß1-42. This strain differs from existing Aß models that predominantly express amino-truncated Aß3-42. The Aß1-42 is expressed in body wall muscle cells, where it oligomerizes, aggregates and results in severe, and fully penetrant, age progressive-paralysis. The in vivo accumulation of Aß1-42 also stains positive for amyloid dyes, consistent with in vivo fibril formation. The utility of this model for identification of potential protective compounds was examined using the investigational Alzheimer’s therapeutic PBT2, shown to be neuroprotective in mouse models of AD and significantly improve cognition in AD patients. We observed that treatment with PBT2 provided rapid and significant protection against the Aß-induced toxicity in C. elegans.ConclusionThis C. elegans model of full length Aß1-42 expression can now be adopted for use in screens to rapidly identify and assist in development of potential therapeutics and to study underlying toxic mechanism(s) of Aß.

RNA-binding protein TDP-43 has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have engineered pan-neuronal expression of human TDP-43 protein in Caenorhabditis elegans, with the goal of generating a convenient in vivo model of TDP-43 function and neurotoxicity. Transgenic worms with the neuronal expression of human TDP-43 exhibit an 'uncoordinated' phenotype and have abnormal motorneuron synapses. Caenorhabditis elegans contains a single putative ortholog of TDP-43, designated TDP-1, which we show can support alternative splicing of CFTR in a cell-based assay. Neuronal overexpression of TDP-1 also results in an uncoordinated phenotype, while genetic deletion of the tdp-1 gene does not affect movement or alter motorneuron synapses. By using the uncoordinated phenotype as a read-out of TDP-43 overexpression neurotoxicty, we have investigated the contribution of specific TDP-43 domains and subcellular localization to toxicity. Full-length (wild-type) human TDP-43 expressed in C. elegans is localized to the nucleus. Deletion of either RNA recognition domain (RRM1 or RRM2) completely blocks neurotoxicity, as does deletion of the C-terminal region. These deleted TDP-43 variants still accumulate in the nucleus, although their subnuclear distribution is altered. Interestingly, fusion of TDP-1 C-terminal sequences to TDP-43 missing its C-terminal domain restores normal subnuclear localization and toxicity in C. elegans and CFTR splicing in cell-based assays. Overexpression of wild-type, full-length TDP-43 in mammalian cells (differentiated M17 cells) can also result in cell toxicity. Our results demonstrate that in vivo TDP-43 neurotoxicity can result from nuclear activity of overexpressed full-length protein.

Expression of the human ␤-amyloid peptide (A␤) in a transgenic Caenorhabditis elegans Alzheimer disease model leads to the induction of HSP-16 proteins, a family of small heat shockinducible proteins homologous to vertebrate ␣B crystallin. These proteins also co-localize and co-immunoprecipitate with A␤ in this model (Fonte, V., Kapulkin, V., Taft Accumulation of the ␤-amyloid (A␤)2 peptide in the brain has been proposed to be causally linked to Alzheimer disease (the "Amyloid Cascade" hypothesis (1)), even though the specific mechanisms by which the A␤ peptide induces AD pathology have not been resolved. Intracellular A␤ accumulation has also been proposed to underlie the muscle pathology observed in inclusion body myositis (2). To investigate A␤ toxicity in a genetically tractable model, we have engineered Caenorhabditis elegans nematodes to express the human A␤-(1-42) peptide in either body wall muscle (3) or neurons (4).In C. elegans transgenic models with muscle expression of A␤, the peptide accumulates in intracellular cytoplasmic deposits (5) despite the inclusion of a signal peptide in the transgene construct. The appropriate removal of the signal peptide and the association of Abeta with hsp-3, an ER chaperone homologous to mammalian GRP78/BiP (6), have led us to propose that Abeta is routed to the secretory pathway in this model but is retrotranslocated out of the ER because it is recognized as an abnormal protein (4). We have also demonstrated a role for autophagosomes and lysosomes in the clearance of Abeta in this model, suggesting that Abeta may also exist in these subcellular compartments (8). Intracellular Abeta is observed in the muscles of IBM patients or in transgenic mouse models of IBM (9, 10), although the subcellular distribution of Abeta has not been determined. Intracellular A␤ has also been observed in human brain neurons (11), and the relevance of intracellular A␤ in Alzheimer disease has been supported by studies with the LaFerla 3ϫ transgenic AD mouse model, where accumulation of intracellular A␤ precedes neurofibrillary tangle formation (12). A number of neurodegenerative diseases (Parkinson, Huntington, amyotrophic lateral sclerosis, etc.) are characterized by intracellular cytoplasmic accumulation of proteins causally associated with theses diseases, and thus the C. elegans transgenic model described in this study may be generally relevant to the proteotoxicity underlying neurodegenerative diseases. In this context, a transgenic C. elegans strain expressing human A␤ has been used recently to investigate the roles of insulin-like signaling and heat shock factor in proteotoxicity (13).A robust finding in these transgenic C. elegans models is the induction of the HSP-16 family of small chaperone proteins by A␤ expression (14, 15). HSP-16 proteins readily co-immunoprecipitate with A␤ in transgenic C. elegans worms and closely associate with intracellular A␤ deposits as observed by immunohistochemistry (16). The HSP-16 family proteins are homologous to ␣B crystallin and have been show...

Epidemiological studies have reported that coffee and/or caffeine consumption may reduce Alzheimer's disease (AD) risk. We found that coffee extracts can similarly protect against b-amyloid peptide (Ab) toxicity in a transgenic Caenorhabditis elegans Alzheimer's disease model. The primary protective component(s) in this model is not caffeine, although caffeine by itself can show moderate protection. Coffee exposure did not decrease Ab transgene expression and did not need to be present during Ab induction to convey protection, suggesting that coffee exposure protection might act by activating a protective pathway. By screening the effects of coffee on a series of transgenic C. elegans stress reporter strains, we identified activation of the skn-1 (Nrf2 in mammals) transcription factor as a potential mechanism of coffee extract protection. Inactivation of skn-1 genetically or by RNAi strongly blocked the protective effects of coffee extract, indicating that activation of the skn-1 pathway was the primary mechanism of coffee protection. Coffee also protected against toxicity resulting from an aggregating form of green fluorescent protein (GFP) in a skn-1-dependent manner. These results suggest that the reported protective effects of coffee in multiple neurodegenerative diseases may result from a general activation of the Nrf2 phase II detoxification pathway.

This instrument has demonstrated sufficient convergent and discriminant validity and reliability to be used in subsequent research, although the validation process should continue and it is hoped that the instrument will be adapted for use in other contexts.

Caenorhabditis elegans mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. Nevertheless, transcriptome sequencing revealed that many RNAs were altered in accumulation and/or processing in the mutant. Analysis of these transcriptional abnormalities demonstrates that a primary function of TDP-1 is to limit formation or stability of double-stranded RNA. Specifically, we found that deletion of tdp-1: (1) preferentially alters the accumulation of RNAs with inherent double-stranded structure (dsRNA); (2) increases the accumulation of nuclear dsRNA foci; (3) enhances the frequency of adenosine-to-inosine RNA editing; and (4) dramatically increases the amount of transcripts immunoprecipitable with a dsRNA-specific antibody, including intronic sequences, RNAs with antisense overlap to another transcript, and transposons. We also show that TDP-43 knockdown in human cells results in accumulation of dsRNA, indicating that suppression of dsRNA is a conserved function of TDP-43 in mammals. Altered accumulation of structured RNA may account for some of the previously described molecular phenotypes (e.g., altered splicing) resulting from reduction of TDP-43 function.

BackgroundThe β-amyloid peptide (Aβ) contains a Gly-XXX-Gly-XXX-Gly motif in its C-terminal region that has been proposed to form a "glycine zipper" that drives the formation of toxic Aβ oligomers. We have tested this hypothesis by examining the toxicity of Aβ variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model.ResultsWe found that a Gly37Leu substitution dramatically reduced Aβ toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that Aβ Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic Aβ oligomers. To test this model rigorously, we engineered second site substitutions in Aβ predicted by the glycine zipper model to compensate for the Gly37Leu substitution and expressed these in C. elegans. We show that these second site substitutions restore in vivo Aβtoxicity, further supporting the glycine zipper model.ConclusionsOur structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of Aβ plays an important role in the formation of toxic Aβ oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential.

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