The search for evolutionary developmental origins of aging in zebrafish: a novel intersection of developmental and senescence biology in the zebrafish model system

Senescence may be considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena during the process of aging. We investigated whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We conducted experiments to isolate zebrafish mutants expressing an apparent senescence phenotype during embryogenesis (embryonic senescence). Some of the genes we thereby identified had already been associated with cellular senescence and chronological aging in other organisms, but many had not yet been linked to these processes. Complete loss-of-function of developmentally essential genes induce embryonic (or larval) lethality, whereas it seems like their partial loss-of-function (i.e., decrease-of-function by heterozygote or hypomorphic mutations) still remains sufficient to go through the early developmental process because of its adaptive plasticity or rather heterozygote advantage. However, in some cases, such partial loss-of-function of genes compromise normal homeostasis due to haploinsufficiency later in adult life having many environmental stress challenges. By contrast, any heterozygote-advantageous genes might gain a certain benefit(s) (much more fitness) by such partial loss-of-function later in life. Physiological senescence may evolutionarily arise from both genetic and epigenetic drifts as well as from losing adaptive developmental plasticity in face of stress signals from the external environment that interacts with functions of multiple genes rather than effects of only a single gene mutation or defect. Previously uncharacterized developmental genes may thus mediate the aging process and play a pivotal role in senescence. Moreover, unexpected senescence-related genes might also be involved in the early developmental process and regulation. We wish to ascertain whether we can identify such genes promptly in a comprehensive manner. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype and thereby facilitates searching for the evolutionary and developmental origins of aging in vertebrates.     

Loss-of-function mutations identified in the Helical domain of the G protein alpha-subunit, G alpha2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of alpha-, beta-, and gamma-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the alpha-subunit. The alpha-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known alpha-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of alpha-subunit function, we performed a screen for loss-of-function mutations, using the G alpha2-subunit of Dictyostelium. G alpha2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of G alpha2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to G alpha2 function. A library of random point mutations in the g alpha2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a g alpha2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the g alpha2 insert. To date, sixteen single amino acids changes have been identified in G alpha2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the alpha-subunit. These loss-of-function mutations in the alpha-subunit Helical domain may provide important insight into its function.     

Mimicking small G-proteins: an emerging theme from the bacterial virulence arsenal

The identification of the Ras superfamily of small molecular weight GTPases (G-proteins) has opened up new fields in cancer biology, immunity and infectious disease research. Because of their ubiquitous role in cellular homeostasis, small G-proteins are common targets for several pathogens, including bacteria. It is well known that pathogenic bacteria have evolved virulence factors that chemically modify GTPases or directly mimic the activities of key regulatory proteins. However, recent studies now suggest that bacterial 'effector' proteins can also mimic the activities of Ras small G-proteins despite their lack of guanine nucleotide binding or GTPase enzymatic activity. The study of these unique pathogenic strategies continues to reveal novel mechanistic insights into host cellular communication networks and the role of small G-protein signalling during human infectious disease.     

Mechanisms of Granulin Deficiency: Lessons from Cellular and Animal Models

The identification of causative mutations in the (pro)granulin gene (GRN) has been a major breakthrough in the research on frontotemporal dementia (FTD). So far, all FTD-associated GRN mutations are leading to neurodegeneration through a “loss-of-function” mechanism, encouraging researchers to develop a growing number of cellular and animal models for GRN deficiency. GRN is a multifunctional secreted growth factor, and loss of its function can affect different cellular processes. Besides loss-of-function (i.e., mostly premature termination codons) mutations, which cause GRN haploinsufficiency through reduction of GRN expression, FTD-associated GRN missense mutations have also been identified. Several of these missense mutations are predicted to increase the risk of developing neurodegenerative diseases through altering various key biological properties of GRN-like protein secretion, proteolytic processing, and neurite outgrowth. With the use of cellular and animal models for GRN deficiency, the portfolio of GRN functions has recently been extended to include functions in important biological processes like energy and protein homeostasis, inflammation as well as neuronal survival, neurite outgrowth, and branching. Furthermore, GRN-deficient animal models have been established and they are believed to be promising disease models as they show accelerated aging and recapitulate at least some neuropathological features of FTD. In this review, we summarize the current knowledge on the molecular mechanisms leading to GRN deficiency and the lessons we learned from the established cellular and animal models. Furthermore, we discuss how these insights might help in developing therapeutic strategies for GRN-associated FTD.     

Cyclin D involvement demarcates a late transition in C. elegans embryogenesis

During development, progression through the cell cycle must be coordinately regulated with cellular differentiation. Despite significant progress in identifying genes required independently for each of these processes, the molecules which facilitate this cross talk have for the most part been elusive. Using the six macrophage-like coelomocytes of the nematode Caenorhabditis elegans as a model system to gain insight into the mesodermal differentiation pathway, we have isolated a set of mutants that alter coelomocyte numbers. One of these mutations, cc600, apparently results from a partial loss-of-function in the C. elegans cyclin D gene, cyd-1. The mutant has coelomocyte-specific defects without changes in other lineages. The mutants show that cell growth, terminal differentiation and cellular function proceed in the absence of cyd-1 activity and cell division. The results suggest that certain mesodermal lineages may be uniquely affected by changes in cyd-1 activity.     

Stoichiometric design of metabolic networks: multifunctionality,clusters, optimization,weak and strong robustness

Starting from a limited set of reactions describing changes in the carbon skeleton of biochemical compounds complete sets of metabolic networks are constructed. The networks are characterized by the number and types of participating reactions. Elementary networks are defined by the condition that a specific chemical conversion can be performed by a set of given reactions and that this ability will be lost by elimination of any of these reactions. Groups of networks are identified with respect to their ability to perform a certain number of metabolic conversions in an elementary way which are called the network’s functions. The number of the network functions defines the degree of multifunctionality. Transitions between networks and mutations of networks are defined by exchanges of single reactions. Different mutations exist such as gain or loss of function mutations and neutral mutations. Based on these mutations neighbourhood relations between networks are established which are described in a graph theoretical way. Basic properties of these graphs are determined such as diameter, connectedness, distance distribution of pairs of vertices. A concept is developed to quantify the robustness of networks against changes in their stoichiometry where we distinguish between strong and weak robustness. Evolutionary algorithms are applied to study the development of network populations under constant and time dependent environmental conditions. It is shown that the populations evolve toward clusters of networks performing a common function and which are closely neighboured. Under changing environmental conditions multifunctional networks prove to be optimal and will be selected.     

A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs(sg RNAs).Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing(HTS).We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.     

Periodic oscillation in delayed gene networks with SUM regulatory logic and small perturbations

In this paper, we derive new criteria for evaluating the global stability of periodic oscillation in delayed gene networks with SUM regulatory logic and small perturbation, which appear in many biological systems at biomolecular or cellular levels due to the weak coupling and signal diffusion (or transport) process. Our results rely on the Lipschtiz conditions of Hill function, topology of gene networks and delay kernels. In particular, Our method based on the proposed model transforms the original network into matrix analysis problem, thereby not only significantly reducing the computational complexity but also making analysis of periodic oscillation tractable for even large-scale nonlinear networks.     

Arrhythmogenic mechanisms in ryanodine receptor channelopathies

Ryanodine receptors(Ry Rs) are the calcium release channels of sarcoplasmic reticulum(SR) that provide the majority of calcium ions(Ca2+) necessary to induce contraction of cardiac and skeletal muscle cells.In their intracellular environment,Ry R channels are regulated by a variety of cytosolic and luminal factors so that their output signal(Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity(ventricular arrhythmias) or cellular remodeling.The importance of Ry R dysfunction has been recently highlighted with the demonstration that point mutations in RYR2,the gene encoding for the cardiac isoform of the Ry R(Ry R2),are associated with catecholaminergic polymorphic ventricular tachycardia(CPVT),an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart.Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by Ry R2 channels harboring mutations linked to CPVT.Most CPVT mutations characterized to date endow Ry R2 channels with a gain of function,resulting in hyperactive channels that release Ca2+ spontaneously,especially during diastole.The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger,which depolarizes the external membrane(delayed afterdepolarization or DAD) and may trigger untimely action potentials.However,a rare set of CPVT mutations yield Ry R2 channels that are intrinsically hypo-active and hypo-responsive to stimuli,and it is unclear whether these channels release Ca2+ spontaneously during diastole.We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to Ry R2 loss-of-function mutations.     

Loss-of-function mutations identified in the Helical domain of the G protein α-subunit,Gα2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of α-, β-, and γ-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the α-subunit. The α-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known α-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of α-subunit function, we performed a screen for loss-of-function mutations, using the Gα2-subunit of Dictyostelium. Gα2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of Gα2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to Gα2 function. A library of random point mutations in the gα2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a gα2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the gα2 insert. To date, sixteen single amino acids changes have been identified in Gα2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the α-subunit. These loss-of-function mutations in the α-subunit Helical domain may provide important insight into its function.     

Evolutionary change in the functional specificity of genes.

Species throughout the animal kingdom share not only housekeeping but also many key regulatory genes. Nonetheless, species differ from one another developmentally and thus, also morphologically. One of the general aims of comparative developmental genetics is to understand how similar molecules can generate the known diversity of biological form. Here, we argue that gene function can change in different ways during the evolution of developmental processes. Genes can be recruited to serve completely new functions in a new regulatory linkage (co-option), they can change their molecular specificity while remaining in the original (homologous) developmental program and can, at the same time, retain other functions. We describe evidence for such evolutionary patterns based on the comparison of loss-of-function mutations of homologous genes of the two free-living nematodes Caenorhabditis elegans and Pristionchus pacificus. Ultimately, it is the interplay of conservation and change of the specificity of genes and genetic networks that generates developmental novelty over evolutionary time.     

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.     

The role of cellular objectives and selective pressures in metabolic pathway evolution

Evolution results from molecular-level changes in an organism, thereby producing novel phenotypes and, eventually novel species. However, changes in a single gene can lead to significant changes in biomolecular networks through the gain and loss of many molecular interactions. Thus, significant insights into microbial evolution have been gained through the analysis and comparison of reconstructed metabolic networks. However, challenges remain from reconstruction incompleteness and the inability to experiment with evolution on the timescale necessary for new species to arise. Despite these challenges, experimental laboratory evolution of microbes has provided some insights into the cellular objectives underlying evolution, under the constraints of nutrient availability and the use of mechanisms that protect cells from extreme conditions.     

When kinases meet mathematics: the systems biology of MAPK signalling

The mitogen activated protein kinase/extracellular signal regulated kinase pathway regulates fundamental cellular function such as cell proliferation, survival, differentiation and motility, raising the question how these diverse functions are specified and coordinated. They are encoded through the activation kinetics of the pathway, a multitude of feedback loops, scaffold proteins, subcellular compartmentalisation, and crosstalk with other pathways. These regulatory motifs alone or in combination can generate a multitude of complex behaviour. Systems biology tries to decode this complexity through mathematical modelling and prediction in order to gain a deeper insight into the inner works of signalling networks.     

Loss-of-function presenilin mutations in Alzheimer disease. Talking Point on the role of presenilin mutations in Alzheimer disease

Presenilin mutations are the main cause of familial Alzheimer disease. From a genetic point of view, these mutations seem to result in a gain of toxic function; however, biochemically, they result in a partial loss of function in the gamma-secretase complex, which affects several downstream signalling pathways. Consequently, the current genetic terminology is misleading. In fact, the available data indicate that several clinical presenilin mutations also lead to a decrease in amyloid precursor protein-derived amyloid beta-peptide generation, further implying that presenilin mutations are indeed loss-of-function mutations. The loss of function of presenilin causes incomplete digestion of the amyloid beta-peptide and might contribute to an increased vulnerability of the brain, thereby explaining the early onset of the inherited form of Alzheimer disease. In this review, I evaluate the implications of this model for the amyloid-cascade hypothesis and for the efficacy of presenilin/gamma-secretase as a drug target.