AIP-1 ameliorates beta-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model.

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.

The beta amyloid peptide can act as a modular aggregation domain.

Although there is compelling evidence that the beta amyloid peptide (Abeta) can be centrally involved in Alzheimer's disease, the natural role (if any) of this peptide remains unclear. Here we use green fluorescent protein (GFP) fusions to demonstrate that the Abeta sequence, like prion domains, can act as a modular aggregation domain when terminally appended to a normally soluble protein. We find that a single amino acid substitution (Leu(17) to Pro) in the beta peptide sequence can abolish this cis capacity to induce aggregation. Introduction of this substitution into full-length APP (i.e., a Leu(613)Pro substitution in APP695) alters the processing of APP leading to the accumulation of the C99 C-terminal fragment (CTF). We suggest that in at least some aggregation disease-related proteins the presence of an aggregation domain is not "accidental", but reflects a selected role of these domains in modulating the trafficking or metabolism of the parental protein.

Transcriptome analysis of genetically matched human induced pluripotent stem cells disomic or trisomic for chromosome 21.

Trisomy of chromosome 21, the genetic cause of Down syndrome, has the potential to alter expression of genes on chromosome 21, as well as other locations throughout the genome. These transcriptome changes are likely to underlie the Down syndrome clinical phenotypes. We have employed RNA-seq to undertake an in-depth analysis of transcriptome changes resulting from trisomy of chromosome 21, using induced pluripotent stem cells (iPSCs) derived from a single individual with Down syndrome. These cells were originally derived by Li et al, who genetically targeted chromosome 21 in trisomic iPSCs, allowing selection of disomic sibling iPSC clones. Analyses were conducted on trisomic/disomic cell pairs maintained as iPSCs or differentiated into cortical neuronal cultures. In addition to characterization of gene expression levels, we have also investigated patterns of RNA adenosine-to-inosine editing, alternative splicing, and repetitive element expression, aspects of the transcriptome that have not been significantly characterized in the context of Down syndrome. We identified significant changes in transcript accumulation associated with chromosome 21 trisomy, as well as changes in alternative splicing and repetitive element transcripts. Unexpectedly, the trisomic iPSCs we characterized expressed higher levels of neuronal transcripts than control disomic iPSCs, and readily differentiated into cortical neurons, in contrast to another reported study. Comparison of our transcriptome data with similar studies of trisomic iPSCs suggests that trisomy of chromosome 21 may not intrinsically limit neuronal differentiation, but instead may interfere with the maintenance of pluripotency.

A glycine zipper motif mediates the formation of toxic ß-amyloid oligomers in vitro and in vivo.

BACKGROUND: The ß-amyloid peptide (Aß) 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 Aß oligomers. We have tested this hypothesis by examining the toxicity of Aß variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model. RESULTS: We found that a Gly37Leu substitution dramatically reduced Aß toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that Aß Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic Aß oligomers. To test this model rigorously, we engineered second site substitutions in Aß 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 Aßtoxicity, further supporting the glycine zipper model. CONCLUSIONS: Our structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of Aß plays an important role in the formation of toxic Aß oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential.

Suppression of in vivo beta-amyloid peptide toxicity by overexpression of the HSP-16.2 small chaperone protein.

Expression of the human beta-amyloid peptide (Abeta) in a transgenic Caenorhabditis elegans Alzheimer disease model leads to the induction of HSP-16 proteins, a family of small heat shock-inducible proteins homologous to vertebrate alphaB crystallin. These proteins also co-localize and co-immunoprecipitate with Abeta in this model (Fonte, V., Kapulkin, V., Taft, A., Fluet, A., Friedman, D., and Link, C. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9439-9444). To investigate the molecular basis and biological function of this interaction between HSP-16 and Abeta, we generated transgenic C. elegans animals with high level, constitutive expression of HSP-16.2. We find that constitutive expression of wild type, but not mutant, HSP-16.2 partially suppresses Abeta toxicity. Wild type Abeta-(1-42), but not Abeta single chain dimer, was observed to become sequestered in HSP-16.2-containing inclusions, indicating a conformation-dependent interaction between HSP-16.2 and Abeta in vivo. Constitutive expression of HSP-16.2 could reduce amyloid fibril formation, but it did not reduce the overall accumulation of Abeta peptide or alter the pattern of the predominant oligomeric species. Studies with recombinant HSP-16.2 demonstrated that HSP-16.2 can bind directly to Abeta in vitro, with a preferential affinity for oligomeric Abeta species. This interaction between Abeta and HSP-16.2 also influences the formation of Abeta oligomers in in vitro assays. These studies are consistent with a model in which small chaperone proteins reduce Abeta toxicity by interacting directly with the Abeta peptide and altering its oligomerization pathways, thereby reducing the formation of a minor toxic species.

Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.

A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). 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, alpha-synuclein, and the beta-amyloid peptide.

Interaction of intracellular beta amyloid peptide with chaperone proteins.

Expression of the human beta amyloid peptide (A beta) 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 A beta in vivo. Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with A beta has identified six likely chaperone proteins: two members of the HSP70 family, three alpha B-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 A beta expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular A beta in this model. Transgenic animals expressing a nonaggregating A beta variant, a single-chain A beta 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 A beta expression. These results suggest that chaperone function can play a role in modulating intracellular A beta metabolism and toxicity.