A novel inhibitor of the insulin/IGF signaling pathway protects from age‐onset,neurodegeneration‐linked proteotoxicity

Aging manipulation is an emerging strategy aimed to postpone the manifestation of late‐onset neurodegenerative disorders such as Alzheimer's (AD) and Huntington's diseases (HD) and to slow their progression once emerged. Reducing the activity of the insulin/IGF signaling cascade (IIS), a prominent aging‐regulating pathway, protects worms from proteotoxicity of various aggregative proteins, including the AD‐associated peptide, Aβ‐ and the HD‐linked peptide, polyQ40. Similarly, IGF1 signaling reduction protects mice from AD‐like disease. These discoveries suggest that IIS inhibitors can serve as new drugs for the treatment of neurodegenerative maladies including AD and HD. Here, we report that NT219, a novel IIS inhibitor, mediates a long‐lasting, highly efficient inhibition of this signaling cascade by a dual mechanism; it reduces the autophosphorylation of the IGF1 receptor and directs the insulin receptor substrates 1 and 2 (IRS 1/2) for degradation. NT219 treatment promotes stress resistance and protects nematodes from AD‐ and HD‐associated proteotoxicity without affecting lifespan. Our discoveries strengthen the theme that IIS inhibition has a therapeutic potential as a cure for neurodegenerative maladies and point at NT219 as a promising compound for the treatment of these disorders through a selective manipulation of aging.     

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome‐blocking complexes

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.     

The Role of Autophagy During Development in Higher Eukaryotes

Autophagy is a lysosome‐mediated degradation pathway used by eukaryotes to recycle cytosolic components in both basal and stress conditions. Several genes have been described as regulators of autophagy, many of them being evolutionarily conserved from yeast to mammals. The study of autophagy‐defective model systems has made it possible to highlight the importance of correctly functioning autophagic machinery in the development of invertebrates as, for example, during the complex events of fly and worm metamorphosis. In vertebrates, on the other hand, autophagy defects can be lethal for the animal if the mutated gene is involved in the early stages of development, or can lead to severe phenotypes if the mutation affects later stages. However, in both lower and higher eukaryotes, autophagy seems to be crucial during embryogenesis by acting in tissue remodeling in parallel with apoptosis. An increase of autophagic cells is, in fact, observed in the embryonic stages characterized by massive cell elimination. Moreover, autophagic processes probably protect cells during metabolic stress and nutrient paucity that occur during tissue remodeling. In light of such evidence, it can be concluded that there is a close interplay between autophagy and the processes of cell death, proliferation and differentiation that determine the development of higher eukaryotes.     

Age‐related degeneration of the egg‐laying system promotes matricidal hatching in Caenorhabditis elegans

The identification and characterization of age‐related degenerative changes is a critical goal because it can elucidate mechanisms of aging biology and contribute to understanding interventions that promote longevity. Here, we document a novel, age‐related degenerative change in C. elegans hermaphrodites, an important model system for the genetic analysis of longevity. Matricidal hatching—intra‐uterine hatching of progeny that causes maternal death—displayed an age‐related increase in frequency and affected ~70% of mated, wild‐type hermaphrodites. The timing and incidence of matricidal hatching were largely independent of the levels of early and total progeny production and the duration of male exposure. Thus, matricidal hatching appears to reflect intrinsic age‐related degeneration of the egg‐laying system rather than use‐dependent damage accumulation. Consistent with this model, mutations that extend longevity by causing dietary restriction significantly delayed matricidal hatching, indicating age‐related degeneration of the egg‐laying system is controlled by nutrient availability. To identify the underlying tissue defect, we analyzed serotonin signaling that triggers vulval muscle contractions. Mated hermaphrodites displayed an age‐related decline in the ability to lay eggs in response to exogenous serotonin, indicating that vulval muscles and/or a further downstream function that is necessary for egg laying degenerate in an age‐related manner. By characterizing a new, age‐related degenerative event displayed by C. elegans hermaphrodites, these studies contribute to understanding a frequent cause of death in mated hermaphrodites and establish a model of age‐related reproductive complications that may be relevant to the birthing process in other animals such as humans.     

WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis

Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed ‘WormFarm’, an integrated microfluidic device for culturing nematodes. Cohorts of 30–50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof‐of‐principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate‐based assay for both RNAi‐mediated and dietary‐induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age‐associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo‐3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.     

The evolution of plasticity of dauer larva developmental arrest in the nematode Caenorhabditis elegans

Organisms can end up in unfavourable conditions and to survive this they have evolved various strategies. Some organisms, including nematodes, survive unfavourable conditions by undergoing developmental arrest. The model nematode Caenorhabditis elegans has a developmental choice between two larval forms, and it chooses to develop into the arrested dauer larva form in unfavourable conditions (specifically, a lack of food and high population density, indicated by the concentration of a pheromone). Wild C. elegans isolates vary extensively in their dauer larva arrest phenotypes, and this prompts the question of what selective pressures maintain such phenotypic diversity? To investigate this we grew C. elegans in four different environments, consisting of different combinations of cues that can induce dauer larva development: two combinations of food concentration (high and low) in the presence or absence of a dauer larva-inducing pheromone. Five generations of artificial selection of dauer larvae resulted in an overall increase in dauer larva formation in most selection regimes. The presence of pheromone in the environment selected for twice the number of dauer larvae, compared with environments not containing pheromone. Further, only a high food concentration environment containing pheromone increased the plasticity of dauer larva formation. These evolutionary responses also affected the timing of the worms’ reproduction. Overall, these results give an insight into the environments that can select for different plasticities of C. elegans dauer larva arrest phenotypes, suggesting that different combinations of environmental cues can select for the diversity of phenotypically plastic responses seen in C. elegans.     

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5‐trisphosphate (IP₃) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP₃ signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up‐regulating IP₃ signalling decreases sensitivity. Tissue‐specific rescue experiments suggest IP₃ functions in the intestine. We also exploit IP₃ signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.