Expression of the human β amyloid peptide (Aβ) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular Aβ in vivo . Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with Aβ has identified six likely chaperone proteins: two members of the HSP70 family, three αB-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription–PCR analysis shows that the small heat shock proteins are also transcriptionally induced by Aβ expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular Aβ in this model. Transgenic animals expressing a nonaggregating Aβ variant, a single-chain Aβ dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with Aβ expression. These results suggest that chaperone function can play a role in modulating intracellular Aβ metabolism and toxicity.

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...

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We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, ␣-synuclein, and the ␤-amyloid peptide.Aggregating proteins or peptides have been associated with numerous neurodegenerative diseases (1), although the molecular mechanisms remain unclear. The apparent toxicity of these protein aggregates has been demonstrated in cell culture and in many transgenic mouse and invertebrate models (reviewed in Refs. 2-4). We have shown previously that transgenic expression of the human ␤-amyloid peptide (A␤) 2 in Caenorhabditis elegans leads to the formation of intracellular aggregates and associated toxicity (5). Similar observations have been made for transgenic C. elegans animals expressing polyglutamine repeat proteins (6 -8), ␣-synuclein (9), or tau (10). One unanswered question for the C. elegans (as well as Drosophila and mammalian) disease models is the specificity of the observed toxicity, i.e. would any aggregating protein have the same effect? In theory, this question could be addressed by control experiments in which transgenic animals are constructed in parallel that express a nondisease-associated, "generic" aggregating protein. We show here that GFP can be converted into such a control aggregating protein and that expression of this aggregating GFP variant results in in vivo toxicity grossly similar to that observed for diseaseassociated aggregating proteins.In a search for random peptides that would confer instability on proteins expressed in yeast, Gilon et al. (11) identified a non-natural 16-residue peptide (CL1) that conferred apparent ubiquitin-dependent degradation on the Ura3 protein. This short C-terminal "degron" peptide was subseq...

Cells of the mouse line Balb/3T3 as well as three virus-induced transformants and two spontaneous transformants grown in vitro have been studied for their topography by scanning electron microscopy . The parent cell in confluent culture closely resembles an endothelial cell in its form and in the structure of its association with adjacent cells . The tumorigenic transformants produced by SV40, murine sarcoma virus, or polyoma viruses are fusiform to pleomorphic and distinctly different from the cell of origin . They show relatively smooth surfaces except for blebs and marginal microvilli . Perhaps most surprising is the similarity they bear to one another. This is made the more singular by the very different form shown by the tumorigenic transformants of spontaneous origin . One of these, S2-4, possesses a thickened rather than the lamellar form of the parent A31 cell and is covered by long microvilli and many spherical blebs . The other, TuT3, more closely resembles the cell of origin but shows extensive ruffling at its margins . All transformants grow without evidence of contact inhibition .The significance of the surface morphologies and the factors influencing cell form are discussed .

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Multiple neurodegenerative diseases are causally linked to aggregation-prone proteins. Cellular mechanisms involving protein turnover may be key defense mechanisms against aggregating protein disorders. We have used a transgenic Caenorhabditis elegans Alzheimer's disease model to identify cellular responses to proteotoxicity resulting from expression of the human beta amyloid peptide (Abeta). We show up-regulation of aip-1 in Abeta-expressing animals. Mammalian homologues of AIP-1 have been shown to associate with, and regulate the function of, the 26S proteasome, leading us to hypothesize that induction of AIP-1 may be a protective cellular response directed toward modulating proteasomal function in response to toxic protein aggregation. Using our transgenic model, we show that overexpression of AIP-1 protected against, while RNAi knockdown of AIP-1 exacerbated, Abeta toxicity. AIP-1 overexpression also reduced accumulation of Abeta in this model, which is consistent with AIP-1 enhancing protein degradation. Transgenic expression of one of the two human aip-1 homologues (AIRAPL), but not the other (AIRAP), suppressed Abeta toxicity in C. elegans, which advocates the biological relevance of the data to human biology. Interestingly, AIRAPL and AIP-1 contain a predicted farnesylation site, which is absent from AIRAP. This farnesylation site was shown by others to be essential for an AIP-1 prolongevity function. Consistent with this, we show that an AIP-1 mutant lacking the predicted farnesylation site failed to protect against Abeta toxicity. Our results implicate AIP-1 in the regulation of protein turnover and protection against Abeta toxicity and point at AIRAPL as the functional mammalian homologue of AIP-1.

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.