Identifying Aß-specific pathogenic mechanisms using a nematode model of Alzheimer's disease.

Multiple gene expression alterations have been linked to Alzheimer's disease (AD), implicating multiple metabolic pathways in its pathogenesis. However, a clear distinction between AD-specific gene expression changes and those resulting from nonspecific responses to toxic aggregating proteins has not been made. We investigated alterations in gene expression induced by human beta-amyloid peptide (Aß) in a Caenorhabditis elegans AD model. Aß-induced gene expression alterations were compared with those caused by a synthetic aggregating protein to identify Aß-specific effects. Both Aß-specific and nonspecific alterations were observed. Among Aß-specific genes were those involved in aging, proteasome function, and mitochondrial function. An intriguing observation was the significant overlap between gene expression changes induced by Aß and those induced by Cry5B, a bacterial pore-forming toxin. This led us to hypothesize that Aß exerts its toxic effect, at least in part, by causing damage to biological membranes. We provide in vivo evidence consistent with this hypothesis. This study distinguishes between Aß-specific and nonspecific mechanisms and provides potential targets for therapeutics discovery.

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.

Quantitative trait Loci specifying the response of body temperature to dietary restriction.

Dietary restriction (DR) retards aging and mortality across a variety of taxa. In homeotherms, one of the hallmarks of DR is lower mean body temperature (T(b)), which might be directly responsible for some aspects of DR-mediated life extension. We conducted a quantitative trait locus (QTL) analysis of the response of T(b) to DR in mice using a panel of 22 LSXSS recombinant inbred strains, tested in two cohorts. T(b) in response to DR had a significant genetic component, explaining approximately 35% of the phenotypic variation. We mapped a statistically significant QTL to chromosome 9 and a provisional QTL to chromosome 17, which together accounted for about two thirds of the genetic variation. Such QTLs could be used to critically test whether the response of T(b) to DR also affects the response of life extension. In addition, this study demonstrates the feasibility of trying to map QTLs that affect other physiological responses to DR, including the life extension response. Importantly, the genes underlying such QTLs would be causal factors affecting these responses and could be identified by positional cloning.

Strain variation in the response of body temperature to dietary restriction.

Dietary restriction (DR, also referred to as calorie restriction, energy restriction, and food restriction) retards senescence and increases longevity in mammals. DR also lowers mean body temperature (T(b)), and thus mean T(b) might be useful as a covariate of DR-induced life extension. Indeed, lower T(b) could itself underlie some of the beneficial life-extension effects that occur during DR. To assess the relationship between lower T(b) during DR and life extension, we asked whether significant strain variation exists in the T(b) response of mice being fed 60% ad libitum (AL). Individually-housed, female mice from 28 strains, representing a genealogically diverse sample of the classical inbred strains, were directly compared. The mean T(b)s in response to DR exhibited highly significant strain variation, ranging from 1.5 degrees C below normal to a phenomenal 5 degrees C below normal. This variation was not explained by differences in loss of thermoregulation, AL adiposity, sensitivity to a nonadaptive hypothermia, motor activity, thermal arousal, absolute food intake, or efficacy of nutrient extraction. The variation in strain mean T(b) was also present in the absence of torpor. This strain variation could be used to critically test whether lower T(b) is a covariate of life extension during DR.