Summary: The great majority of lifespan‐augmenting mutations were discovered in the nematode Caenorhabditis elegans. In particular, genetic disruption of insulin‐like signaling extends longevity 1.5‐ to 3‐fold in the nematode, and to lesser degrees in other taxa, including fruit flies and mice. C. elegans strains bearing homozygous nonsense mutations in the age‐1 gene, which encodes the class‐I phosphatidylinositol 3‐kinase catalytic subunit (PI3K_CS), produce progeny that were thought to undergo obligatory developmental arrest. We now find that, after prolonged developmental times at 15–20 °C, they mature into extremely long‐lived adults with near‐normal feeding rates and motility. They survive to a median of 145–190 days at 20 °C, with nearly 10‐fold extension of both median and maximum adult lifespan relative to N2DRM, a long‐lived wild‐type stock into which the null mutant was outcrossed. PI3K‐null adults, although a little less thermotolerant, are considerably more resistant to oxidative and electrophilic stresses than worms bearing normal or less long‐lived alleles. Their unprecedented factorial gains in survival, under both normal and toxic environments, are attributed to elimination of residual and maternally contributed PI3K_CS or its products, and consequent modification of kinase signaling cascades.

Many lifespan-modulating genes are involved in either generation of oxidative substrates and end-products, or their detoxification and removal. Among such metabolites, only lipoperoxides have the ability to produce free-radical chain reactions. For this study, fatty-acid profiles were compared across a panel of C. elegans mutants that span a tenfold range of longevities in a uniform genetic background. Two lipid structural properties correlated extremely well with lifespan in these worms: fatty-acid chain length and susceptibility to oxidation both decreased sharply in the longest-lived mutants (affecting the insulinlike-signaling pathway). This suggested a functional model in which longevity benefits from a reduction in lipid peroxidation substrates, offset by a coordinate decline in fatty-acid chain length to maintain membrane fluidity. This model was tested by disrupting the underlying steps in lipid biosynthesis, using RNAi knockdown to deplete transcripts of genes involved in fatty-acid metabolism. These interventions produced effects on longevity that were fully consistent with the functions and abundances of their products. Most knockdowns also produced concordant effects on survival of hydrogen peroxide stress, which can trigger lipoperoxide chain reactions.

Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease-specific 'seed' proteins; however, roles of other co-aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control-subject pools using magnetic-bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high-resolution proteomics. Although total detergent-insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or Aβ1-42 pulldown, the protein constituents of Alzheimer-derived aggregates were more abundant, diverse, and post-translationally modified than those from controls. Tau- and Aβ-containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer-specific protein enrichment in tau-containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co-aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co-localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer-specific enrichment in tau-containing aggregates, were assessed for aggregation-promoting roles in C. elegans by RNA-interference 'knockdown'. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle Aβ, and 12 (57%) rescued chemotaxis disrupted by neuronal Aβ expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by Aβ1-42 . These observations imply shared mechanisms driving both types of aggregation, and/or aggregate-mediated cross-talk between tau and Aβ. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.

Summary: Age‐dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP‐43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation‐prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc‐54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody‐coupled magnetic beads, were characterized by high‐resolution mass spectrometry. Three Q40::YFP‐associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan‐neuronal Aβ_1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine‐rich, favoring hydrophobic interactions with other random‐coil domains. A particularly potent modulator of aggregation, CRAM‐1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72‒86% (P < 10⁻⁶). In worms expressing Aβ_1–42, knockdown of cram‐1 reduced β‐amyloid 60% (P < 0.002) and slowed age‐dependent paralysis > 30% (P < 10⁻⁶). In wild‐type worms, cram‐1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM‐1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM‐1 (or human orthologs) with ubiquitin, and we verified that CRAM‐1 reduces degradation of a tagged‐ubiquitin reporter. We propose that CRAM‐1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II–induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each P <0.05. One fifth of these proteins were previously associated with age-progressive neurodegenerative or cardiovascular diseases, or both (eg, ApoE, ApoJ, ApoAIV, clusterin, complement C3, and others involved in stress-response and protein-homeostasis pathways). Because fibrosis is a characteristic of both aged and hypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual.

We previously reported high expression of RAD51 and increased homologous recombination (HR) rates in multiple myeloma (MM) cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX) is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did not produce significant synergy against normal human CD19+ B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ≥5 foci rose 4-fold, whereas γH2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas γH2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy.